中国药理学与毒理学杂志

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2015, 29(S1): 0-10.

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药物表型组学与传统中医药

段大跃

2015, 29(S1): 1-2.

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In the post-genome era, different omics methods have been used to establish the relationship between clinical phenotypes and molecular characterizations. The combination of genomics, proteomics, and metabolomics has unraveled the etiology and pathophysiology of various diseases in a big-data fashion and the recent Genome-Wide Association Studies (GWAS) have provided a powerful systematic method to investigate the impact of common genomic variations on human cardiovascular pathophysiology and disease. But, these studies also revealed unmatched relationships between the genomic variability and the much narrower definition of various clinical phenotypes of cardiovascular diseases in individual patients. In the majority of GWAS a single targeted disease or a pre-defined and limited phenotype (trait) was studied and the accrual of such a large number of single gene variant-phenotype associations has led to the serendipitous identification of single loci associated with multiple diseases, or one gene being responsible or affecting more than one phenotypic characteristic (pleiotropy). Clearly, Western medicine is now facing the same challenges as traditional Chinese medicine (TCM) and newer approaches are needed for the redefinition of diseases using the underlying molecular causes and other factors in addition to traditional signs and symptoms. These are the same old questions for TCM for many years. A new method named "Phenome-Wide Association Study (PheWAS)" as an alternative approach that complements GWAS and utilizes phenomics and big-data technologies to analyze all genetic/proteomic variants and all available phenotypic information in the estimation of association between genome-phenome and detection of pleiotropy. Phenomics is a recently developed new transdiscipline that provides a suite of new technologies and platforms for the transition from focused phenotype-genotype study to a systematic phenome-genome approach, which can be used to redefine the clinical phenotypes of diseases. Accordingly, disease will be defined as a clinical phenome that is the sum total of a patient's clinical characteristics or phenomic traits that signify the expression of the whole genome, proteome, and metabolome under specific environmental influence. With the fast advance and development of big-data technology and phenomics, we believe that the application of PheWAS in medicine opens important avenues to enhance systematically-integrated analysis of the genomic basis of human disease and responses to drug therapy and to reform our understanding and clinical treatment of diseases with a new concept of wholism. With well-defined clinical disease phenome, a new transdiscipline termed "pharmacophenomics" has been emerging. As a complement of pharmacogenomics, pharmacoproteomics, and pharmacometabolomics, pharmacophenomics offers a suite of new technologies and platforms for the transition from focused phenotype-genotype study to a systematic phenome-genome approach and refine drug research with systematically-defined drug response and therapeutic targets. Therefore, pharmacophenomics will provide a new paradigm for the study of drug response including effects and toxicities at the level of systems biology and will identify the corresponding therapeutic targets suitable for personalized medicine.

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溶血磷脂受体调节因子芬戈莫德:从传统中药到FAD批准药物

Jerold CHUN

2015, 29(S1): 1-1.

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Lysophospholipids are small, membrane-derived lipids that act through a growing family of G protein-coupled receptors (GPCRs) that account for around 40% of the known lipid GPCRs. They comprise a range of distinct chemical structures and include glycerophospholipids like lysophosphatidic acid (LPA) and sphingoid lipids like sphingosine 1-phosphate (S1P). S1P has five cognate GPCRs, four of which mediate the actions of a current medicine used in the treatment of multiple sclerosis (MS): fingolimod (also known as FTY720 or Gilenya), which was approved by the FDA in 2010. Fingolimod has its origins in Chinese medicine as a derivative of fungal natural products. It's mechanism of action in MS is partly known, through effects on lymphocyte trafficking, however current research has identified direct CNS actions that may represent a particular opportunity area for natural products and their derivatives that can target lysophospholipid receptors. The history of lysophospholipid receptors and fingolimod will be discussed, along with mechanistic aspects of receptor-ligand interactions, particularly those with disease relevance.

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开发天然产物药物的尝试与困难

David J. NEWMAN

2015, 29(S1): 2-2.

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The development of natural product based drugs for antitumor therapy is fraught with problems of supply, unless the agent is ato provide enough for early preclinical studies. In this presentation I will give examples from three sources, one of which NCI worked on extensively, the others are examples of work by small companies from an Australian aboriginal usage of a plant, and some very clever work by an academic chemist at Yale University.The story of Picato® is the one from an Australian aboriginal lead and shows how compounds can come from odd places. The second, Kyprolis® is a tour-de-force from an academic laboratory, and the third, Eribulin® is the one that started from a very small amount of material from a Japanese sponge and ultimately led to the most complex totally synthesized drug for any disease. Highlights and "lessons learned" will be discussed for each of these as the overall problems, though similar, were addressed successfully in different ways.

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国际药理学联合会,导向药理学与天然产物

Michael SPEDDING

2015, 29(S1): 3-4.

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The pharmaceutical industry has struggled to provide new drugs for stroke and neurodegenerative disease using classical medicinal chemistry approaches. However there is a disconnect between single molecular targets and such severe diseases. Life style changes, particularly exercise, and some natural products, may extend life span: but what are the molecular targets? IUPHAR has built a database of the molecular targets within the human genome which is freely available, with gold standard ligands: the IUPHAR/BPS GuidetoPHARMACOLOGY (GtoP) database (see GuideToPharmacology.org). The database is unique containing information reviewed by the >90 NC-IUPHAR expert committees with their publications (H-index 72). These expert committees consider features beyond the capability of machine-based data trawling, such as what we know and don't know, variables affecting drug receptor affinity, the crucial challenges of multiple gene products, alternative splicing, epigenetics, allostery, disease and drug ontologies. The database is being actively promoted worldwide by the main pharmacological societies, resulting in a large international user-base. This can now be extended to the molecular targets of natural products. Natural products also markedly affect cellular metabolism and I will also cover how recent human evolution selected certain molecular pathways, allowing metabolomics analysis to develop new directions for the treatment of neurodegenerative disease. IUPHAR can be a partner in developing new therapeutic paradigms, by expert assessmentof complex research areas at clinical and preclinical levels. This is a unique cooperative international initiative.

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中药特征:诱导多靶点效应

张均田

2015, 29(S1): 3-3.

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In the past decades, single target drugs showed less therapeutic action and more side effects in treatment of complicated diseases such as tumor, AIDS, inflammation, diabetes, stroke and neurodegenerative disorders. The reason for this is that complicated diseases have multiple pathogenesis and multiple genes or pathological changes occur in many organs or in different kinds of cells. Therefore, scientists of worldwide hope to develop multi-target drugs recently traditional Chinese medicine (TCM) and their many effective components are characterized by inducing multi-target effects, the following 2 components isolated from TCM were proved in our laboratory to have multi-target effects which benefit complicated diseases. Anti-stroke drug-salvianolic acid B (Sal B): In MCAO rats, Sal B was shown to have many biological activities. Firstly, Sal B could past through blood-brain barrier (BBB) and could repair damage of BBB induced by cerebral ischemia. Secondly, Sal B improved blood flow in ischemic hemisphere with no steal blood and without hypotention, inhibited platelet aggregation a thrombosis but no hemorrhagic risk. More importantly, Sal B could inhibit three risk factors-intracellular Ca²⁺ overload, excessive regeneration of free radicals, excitotoxicity which aggravate ischemic brain injuries. Thirdly, Sal B activated organism protective mechanism such as increasing neurogenesis, angiogenesis, and many anti-oxidative substances and improving energy supply. Taking all these results we believe that sal B is a good anti-stroke agent. Anti-dementia drug-(-) clausenamide: (-) Clausenamide is a novel compound isolated from clausena lansium (Lour) which is the first chiral compound having anti-dementia effect in recent years. As the content in the plant is very low, after long term of effort this compound now been chemically synthesized by our institute and the production are has reached semi-industry scale that satisfies the demand for clinical trial and hereafter therapeutic use. For pharmacology, (-) clausenamide improved cognition and inhibited Aβ pathogenesis including inhibition of Aβ toxicity and tau hyperphosphorylation. According to the new theory "Synaptic loss=AD", a good anti-dementia drug must be able to improve synaptic plasticity and promote synaptogenesis. Fortunately, (-) clausenamide happened to be such compound. As proved in our study that (-) clausenamide increased synaptic plasticity both in efficacy and structure. For latter, (-) clausenamide increased synaptic density and expression of growth associate protein (GAP-43) in the brain significantly. Now (-) clausenamide has been developed to phaseⅡ of clinical trial.

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天然产物促进药物发现

Christina L.L. CHAI

2015, 29(S1): 4-5.

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Natural products or natural product derived drugs comprised 32% of small molecule approved drugs between 1981 and 2010. In the same period of time, 16% of small molecule approved drugs were synthetic or natural mimics based on the study of pharmacophores related to natural products. Indisputably, natural products provide diverse structural diversity and intricate carboskeletal frameworks. As it is believed that nature has evolved optimized biologically active compounds-the secondary metabolites-to ensure survival of the species that produce them, natural products are perceived by some to be more 'drug-like' than totally synthetic compounds. As such, natural products may provide us with the 'best' lead compounds yet for drug discovery, giving rise to natural product inspired drug design. This talk will provide an overview of some of my research in this area. Specifically, I will outline the challenges and some of the lessons learnt in this quest to develop natural products as leads to potential drugs.

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天然来源的赖氨酸乙酰基转移酶小分子调节剂在治疗学中的意义

Tapas K KUNDU

2015, 29(S1): 4-4.

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The altered function of any epigenetic modification also causally affects the physiological homeostasis in different pathophysiological conditions such as cancer, neurodegenerative disorders, diabetes, asthma, COPD etc. Among the different epigenetic enzymes we focus on three important classes: lysine acetyltransferases, arginine methyltransferases and aurora kinases in the context of cancer and neurodegenerative diseases. Our laboratory has discovered several small molecule modulators of these enzymes, which may serve as lead scaffolds to design new generation therapeutics. We have shown that specific as well as broad spectrum inhibitors of lysine acetyltransferases repress the oral, liver as well as prostate cancer progression in the xenografted animal model system. Furthermore, we have shown that one of the p300 specific inhibitors discovered in our laboratory potently inhibit the multiplication of HIV in a cellular system. By using a novel histone acetyltransferase activator molecule, we find that p300/CBP mediated acetylation of histones is an important inducing factor for robust neurogenesis; which presumably contributes to long-term spatial memory. Remarkably, the p300/CBP activator treatment efficiently enhances the memory of Tau mice almost to the normal level. The molecular basis of this phenomenon is being understood.

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中药治疗肝纤维化的药理学研究

黄怡超

2015, 29(S1): 5-5.

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Many patients with chronic viral or alcoholic hepatitis, or non-alcoholic steatohepatitis are in the state of chronic liver injury and inflammation and may lead to hepatic fibrosis. The major culprit of liver fibrosis is activation of hepatic stellate cells (HSCs), characterized by transformation from a quiescent phenotype to a proliferative, contractile and secretory phenotype. In a series of study, we use: (1) in vitro cell assays (HSCs) and (2) in vivo rat models of hepatic fibrosis induced by (a) bile duct ligation, and (b) repeated administration of thioacetamide to investigate the anti-hepato-fibrotic effects of potential herbs or their active compounds and illuminate their mechanisms of action. An immortalized cell line of rat HSCs (HSC-T6) is stimulated by transforming growth factor-1 (TGF-1), tumor necrosis factor-α (TNF-α) or platelet-derived growth factor (PDGF), and to evaluate the inhibitory effects of herbs on activated HSCs. The possibilities of HSC inhibition via signaling cascades and networks of apoptosis, migration, inflammation, or oxidative stress are examined by standard assays. The in vitro and in vivo anti-fibrotic effects of both salvianolic acids A and B (active hydrophilic compounds from Salvia miltiorrhiza) have been demonstrated in thioacetamide-intoxicated rats, with mechanisms related to reduction of oxidative stress via inhibiting NADPH oxidase in HSCs. Of note is that salvianolic acids A and B exerted both "common" and "different" effects in terms of phosphorylations of various kinases. We also demonstrated that tanshinone ⅡA (C₁₉H₁₈O₃, a lipophilic diterpene from S. miltiorrhiza) inhibited lipopolysaccharide-induced HSC chemotaxis and NF-κB activity. The in vivo anti-hepato-fibrotic effects of a new 3-herb formula including S. miltiorrhiza, Ligusticum chuanxiong and Glycyrrhiza glabra in rats. For studies with anti-inflammatory approach, we demonstrated anti-fibrotic effects of triptolide (from Tripterygium wilfordii) and armepavine (from Nelumbo nucifera), respectively, on hepatic stellate cells and rat models of hepatic fibrosis, with both "common" and "different" anti-inflammatory effects in terms of transcription factors and phosphorylations of mitogen-activated protein (MAP) kinases. We have shown de-activating effects on rat HSCs via mechanisms of cell-migration inhibition (rhubarb, Rheum palmatum extracts) or apoptosis induction (curcumin). Moreover, we observed with bioactivity-directed fractionation approach that Bupleurum scorzonerifolium extracts and kaerophyllin (a lignin thereof) inhibited HSC activation and migration after engulfing hepatocyte apoptotic bodies. The protective effects of kaerophyllin against liver fibrogenesis were demonstrated in rats with upregulation of PPAR-γ expression in the liver.

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色谱分离联合在线生物活性测定方法从中草药中发现活性化合物

李绍平, 赵静

2015, 29(S1): 6-6.

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Herbal medicines have been attracting intensive attention with the increasing of health care. The screening and analysis of bioactive components in medicinal plants are very important for ensuring their efficacy, safety as well as quality. A conventional procedure for finding bioactive components is the chemical separation followed by pharmacological screening, or bioassay guided separation. In this presentation, chromatographic methods coupled with online bioassay developed in our lab were introduced for discovery of bioactive components from Chinese herbs.

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小檗碱的故事

蒋建东, 韩燕星, 王璐璐

2015, 29(S1): 6-6.

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Berberine (BBR) is an alkaloid from plants like Coptis chinensis and is for many years an OTC drug in China for bacterial-caused diarrhea. We have identified BBR to be an effective drug in treating hyperlipidemia as well as hyperglycemia. Clinical studies showed that oral administration of BBR caused significant reduction of blood cholesterol, triglyceride as well as glucose in patients with hyperlipidemia and T2D, with no obvious side-effect. Mechanism studies have identified several molecular mechanisms involving in the mode of action of BBR. The cholesterol-lowering effect was associated with the extracellular-signal-regulated kinase (ERK) mediated LDLR mRNA up-regulation; the glucose-lowering effect mainly resulted from the protein kinase D mediated InsR expression and the activation of AMPK. The observed reduction of triglyceride by BBR might reflect its synergistic effect on both sugar and lipid metabolism. The interaction between gut microbiota and BBR explained the molecular mechanism of BBR's intestinal absorption. BBR concentrated in liver after oral administration with a level many times more than that in blood. At least three CYP450 subtypes were responsible for BBR phase-Ⅰ metabolism. Structure-activity relationship of BBR was analyzed, and the clinical advantage of BBR was demonstrated. We consider BBR a new medicine for metabolic disorders.

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姜黄素在缺血和肺癌模型中的抗血管生成作用:计算机模拟、体外和体内试验

范胜军, 李学军

2015, 29(S1): 6-6.

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OBJECTIVE With the rapid development of computer technology, achievements have been gained in the research and application of computer. Network pharmacology combines biological networks and drug network integration, analysis of drug relationship with the node or network module in the network. Since many diseases are caused by complex factors, we try to apply the network pharmacology techniques to study the targets and networks of drugs for complex diseases. METHODS We investigated the angiogenic effects of curcumin on an ischemia and lung cancer model. To induce ischemia combined with lung cancer models, unilateral femoral arteries of C57BL/6 mice were disconnected on one side of the mouse and Lewis lung carcinoma (LLC) cells were xenografted on the opposite side. Angiogenic effects and underlying mechanisms associated with curcumin were investigated. Molecular target(s), signaling cascades and binding affinities were detected by Western blot, two-dimensional gel electrophoresis (2-DE), computer simulations and surface plasmon resonance (SPR) techniques. RESULTSC urcumin promoted post-ischemic blood recirculation and suppressed lung cancer progression in inbred C57BL/6 mice via regulation of the HIF1α/mTOR/VEGF/VEGFR cascade oppositely. Inflammatory stimulation induced by neutrophil elastase (NE) promoted angiogenesis in lung cancer tissues, but these changes were reversed by curcumin through directly reducing NE secretion and stimulating α1-antitrypsin (α1-AT) and insulin receptor substrate-1 (IRS-1) production. Meanwhile, curcumin dose-dependently influenced endothelial cells (EC) tube formation and chicken embryo chorioallantoic membrane (CAM) neovascularization. CONCLUSION Curcumin had opposite effects on blood vessel regeneration under physiological and pathological angiogenesis, which was effected through negative or positive regulation of the HIF1α/mTOR/VEGF/VEGFR cascade. Curcumin had the promise as a new treatment modality for both ischemic conditions and lung cancer simultaneously in the clinic.

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天然产物新靶点:自噬-溶酶体途径

沈汉明

2015, 29(S1): 7-7.

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Autophagy is an evolutionarily well conserved process in which the cellular components including damaged subcellular organelles are engulfed in autophagosome and eventually delivered to lysosome for degradation. It has been well studied that autophagy is closely implicated in many diseases such as cancer and neurodegenerative diseases. Therefore, the autophagy-lysosome pathway becomes an attractive target in developing novel therapeutic approaches. In the past several years, we have studied the effects of several natural products on the autophagy-lysosome pathway: (i) Andrographolide (Andro), a diterpenoid lactone isolated from an herbal plant Andrographispaniculata, is capable of suppressing autophagy and sensitizing cisplatin-mediated apoptosis in human cancer cells, via blockage of autophagosome-lysosome fusion; (ii) (-)-Epigallocatechin-3-gallate (EGCG), an important green tea polyphenol, induces lysosomal membrane permeabilization (LMP) and eventually leads to lysosome-associated cell death; and (iii) Artesunate (ART), an analog of artemisinin, an anti-malaria drug, is able to kill cancer cells via enhancing lysosomal function and induction of lysosomal degradation of ferritin. Collectively, our findings reveal novel insights into the molecular mechanisms underlying the anti-cancer properties of those natural compounds and demonstrate that targeting the autophagy-lysosome pathway could serve as a new strategy in developing anti-cancer therapeutic agents.

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基于六味地黄丸新配方的LW-AFC 对认知缺陷、突触可塑性损伤和神经免疫调节病变APP/PS1和SAMP8小鼠的作用

周文霞, 王健辉, 程斌, 黄晏, 程肖蕊, 张永祥

2015, 29(S1): 8-9.

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OBJECTIVE To investigate the effect of LW-AFC, a new formula of the main active components extracted from Liuwei Dihuang decoction, on treatment of Alzheimer's disease in mouse models. METHODS The APP/PS1 transgenic mice and senescence-accelerated mouse prone 8 (SAMP8) were administered orally by LW-AFC continuously for 3 months. The behavioral tests were conducted by the novel object recognition (recognition memory task), the Morris water maze (spatial memory test), the shuttle box and step-down tests (active and passive avoidance, associative learning task). The synaptic plasticity was evaluated by long-term potentiation (LTP) experiment in anesthetized mice. The cytokine profiles in serum were assayed by multiplex Luminex assay methods. The hormone levels associated with hypothalamo-pituitary-adrenal (HPA) and hypothalamo-pituitary-gonad (HPG) were measured by radioimmunoassay. The levels of Aβ_1-40 and Aβ_1-42 were assayed by AlphaLISA technique. RESULTS The administration of LW-AFC significantly improved the behavioral performances in novel object recognition tasks, spatial memory tasks, and associative learning tasks both in APP/PS1 transgenic mice and SAMP8 mice, and the inductions of LTP were also significantly facilitated. Meanwhile, the concentrations of Aβ_1-40 and Aβ_1-42 in cerebral cortex, hippocampus and serum were decreased in LW-AFC treated groups. The serum concentration of some proinflammatory cytokines, such as IL-1β, IL-2, IL-6, IL-17, IL-23, INF-γ, TNF-α, TNF-β, MCP-1 and MIP-1β were significantly decreased, while the anti-inflammatory cytokines, such as IL-4, IL-10, IL-5 and G-CSF were significantly increased. In addition, LW-AFC could also improved the abnormalities of the corticotropin releasing hormone (CRH) and gonadotropin-releasing hormone (GnRH) levels in hypothalamus, the adrenocorticotropic hormone (ACTH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in pituitary, the corticosterone and testosterone level in serum, respectively. CONCLUSION LW-AFC improves cognitive deficits and synaptic plasticity impairments in AD mouse models, and this may be due to, at least in part, the modulation of neuroimmunomodulation network by LW-AFC, suggesting that LW-AFC might be a promising therapy for AD.

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丹参酮逆转结肠癌细胞多药耐药性应用于治疗的潜能

胡涛, 曹之宪

2015, 29(S1): 8-8.

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Multidrug resistance (MDR) develops during chemotherapy in nearly all colorectal cancerpatients. It is envisaged that reversal of MDR plays a pivotal role in the success of chemotherapy. This study investigated thepotential pharmacological action in reversing MDR in colon cancer cells by the two most potent tanshinones, namely cryptotanshinone and dihydrotanshinone. They targeted two common MDR mechanisms, including overexpression of P-glycoprotein (P-gp) and suppression of apoptosis. Using a bi-directional transport assay, the two tanshinones decreased P-gp-mediated digoxin effluxin Caco-2 cells. They also potentiated the cytotoxicities of doxorubicin and irinotecan in P-gp overexpressing SW620 Ad300 cells via increased intracellular accumulation of both anti-cancer drugs, as a result of down-regulation of P-gp mRNA and protein levels as well as inhibition of P-gp ATPase activity. In addition, the level of apoptosis was also found to be relatively suppressed in SW620 Ad300 cells as compared with the parental SW620 cells. Interestingly, although cryptotanshinone and dihydrotanshinone induced less apoptosis in SW620 Ad300 cells as compared to their parental cells, they produced more autophagic cell death in these MDR cells. In this regard, the drug resistant SW620 Ad300 cells were more prone to cell death in response to the anti-cancer action of thetwo tanshinones. Furthermore, the cytotoxic action of the two tanshinones was shown to be p53-independent, further demonstrated theirunique anti-cancer activities in overcoming drug resistance due to the reduction of p53 expression together with a decrease of apoptosis in colon cancer cells. Taken together, the current findings indicate a great potential for cryptotanshinone and dihydrotanshinone against MDR colon cancer cells,in spite of P-gp overexpression and suppression of apoptosis. They are promising candidates to be developed as therapeutic agents and/or as an adjuvant therapy for colorectal cancer, especially for patients with MDR cancer types.

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中药钩藤中的天然自噬增强因子:分子靶点和神经保护

李敏

2015, 29(S1): 9-9.

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Abstract Macroautophagy (autophagy) is a 'self-eating' machinery used by cells to degrade and recycle long-lived proteins and organelles via lysosomes.Dysregulation of autophagy plays a critical role in the pathogenesis of neurodegenerative disorders. Small molecule autophagy inducers protects against the toxic insults of aggregate-prone proteins by enhancing their clearance, thus leading towards the treatment of neurodegenerative disorders. Uncariarhynchophylla (Miq.) Jacks. (Gouteng in Chinese) is a commonly used Chinese herbal medicine for treating neurological diseases. We identified alkaloidscorynoxine B (Cory B) and corynoxine (Cory) from Gouteng as neuronal autophagy inducers, which promote the degradation of neurodegenerative proteins including alpha-synuclen (SNCA), amyloid precursor protein (APP) and tau, thus protecting neurons. Interestingly, the regulatory mechanisms of Cory B and Cory on autophagy are different. Cory B targets HMGB1-Beclin1 interaction to induce autophagy while Cory enhances autophagy via inhibition of mTOR pathway. The autophagy-enhancing Goutengalkaloids can be developed into therapeutic agents against neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

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抗高血压中草药罗布麻NO释放作用的内皮保护作用和机制

Mohammad Rais MUSTAFA, Lau Yeh-Siang

2015, 29(S1): 10-10.

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OBJECTIVE The extracts of the Apocynum venetum leaves (AVLE), also known as Luobuma, is an antihypertensive medicinal herb used widely in TCM. AVLE has been reported to exert antihypertensive action by dilating the blood vessels in an endothelium-dependent and concentration-dependent manner with optimal effect seen at as low as 10 μg·mL. The present study seeks to further evaluate the free radical scavenging actions and cellular mechanism underlying the nitric oxide (NO)-releasing property of AVLE in rat aorta and human umbilical vein endothelial cells (HUVECs). METHODS Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of N^G-nitro-L-arginine (L-NAME, 100 μmol·L^-1), endothelial NO synthase inhibitor (eNOS), ODQ (1 μmol·L^-1), soluble guanylyl cyclase inhibitor, polyethyleneglycol catalase (PP2, 20 μmol·L^-1), inhibitor of Src kinase and wortmannin (30 nmol·L^-1), and LY294002 (20 μmol·L^-1), PI₃-Kinase inhibitor. Total nitrite and nitrate (NOx) level were measured by Greiss reagent. The cellular effects of AVLE was tested in HUVECs at different concentration with or without inhibitors. The phosphorylation level of Akt and eNOS were assessed by Western blotting. RESULTS In the rat aorta, AVLE (0.3-10 μg·mL^-1) dose-dependently inhibited the contraction to phenylephrine (1 μmol·L^-1) and significantly suppressed the β-NADPH-induced generation of superoxide anion (SOA). Removal of endothelium, treatment with L-NAME or ODQ prevented the vasorelaxant effects of AVLE. Similarly, pre-treatment with PP2, wortmannin and LY294002 reduced the vasorelaxant effects of AVLE. AVLE significantly increased of total NOx level in rat aorta compared to control. It also caused phosphorylation of AKT and eNOS in cultured HUVECs in a dose-dependent manner and which were markedly suppressed by PP2, wortmannin and LY294002. CONCLUSION The present results suggest that the vasorelaxant effect of AVLE is due to its dual ability of releasing NO and protecting it from the scavenging actions of the SOA. Furthermore, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI₃K/Akt dependent NO signalling pathway.

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细胞死亡模式交互作用作为药理学靶点

Marc DIEDERICH

2015, 29(S1): 10-11.

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Cell death plays an essential role in the development of organs, homeostasis, and cancer. Apoptosis and programmed necrosis are two major types of cell death, characterized by different cell morphology and pathways. Accumulating evidence shows autophagy as a new alternative target to treat tumour resistance. Besides its well-known pro-survival role, autophagy can be a physiological cell death process linking apoptosis and programmed necrosis cell death pathways, by various molecular mediators. Here, we summarize the effects of pharmacologically active compounds as modulators of different types of cancer cell death depending on the cellular context. Indeed, current findings show that both natural and synthetic compounds regulate the interplay between apoptosis, autophagy and necroptosis stimulating common molecular mediators and sharing common organelles. In response to specific stimuli, the same death signal can cause cells to switch from one cell death modality to another depending on the cellular setting. The discovery of important interconnections between the different cell death mediators and signalling pathways, regulated by pharmacologically active compounds, presents novel opportunities for the targeted treatment of cancer. The aim of this review is to highlight the potential role of these compounds for context-specific anticancer therapy.

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γ-生育三烯酚可能用做新型胃癌化疗增敏剂

Gautam SETHI

2015, 29(S1): 11-12.

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Gamma-tocotrienol, a member of vitamin E superfamily has attracted great attention of late for its anti-proliferative and anti-carcinogenic potential against different cancers. For example, our group had previously reported that anti-proliferative and chemosensitizing effects of γ-tocotrienol are associated with its ability to suppress activation of signal transducers and activator of transcription 3 (STAT3), a pro-inflammatory transcription factor that plays a pivotal role in the survival, proliferation, angiogenesis and chemoresistance of hepatocellular carcinoma. However, the potential of gamma-tocotrienol to overcome chemoresistance in gastric cancer, which is one of the deadliest cancers in Asia-pacific region, has never been explored before. Hence, we analyzed the efficacy of gamma-tocotrienol in combination with capecitabine to modulate tumor growth and survival in gastric cancer cell lines and xenograft mouse model. Cell proliferation and apoptosis assays indicated that gamma-tocotrienol potentiated capecitabine induced programmed cell death in various gastric cancer cell lines. Gamma-tocotrienol also inhibited expression of Bcl-2, Bcl-xL, cyclin-D1, COX-2, ICAM-1, VEGF, CXCR4, MMP-9 proteins, induced PARP cleavage and inhibited constitutive and capecitabine-induced NF-κB activation in gastric cancer cells. In vivo studies using xenograft model of human gastric cancer demonstrated that gamma-tocotrienol alone suppressed tumor growth and this effect was further potentiated in conjunction with capecitabine. Also the markers of proliferation index Ki-67 and the micro vessel density CD31 were significantly downregulated in tumor tissues by the combination of capecitabine and gamma-tocotrienol. As compared to the vehicle control, gamma-tocotrienol further suppressed the NF-κB activation and expression of cyclin D1, COX-2, ICAM-1, MMP-9 and survivin in tumor tissues obtained from treatment groups. Overall our results suggest for the first time that gamma-tocotrienol can potentiate the effects of capecitabine through modulation of multiple markers of proliferation, invasion, angiogenesis and metastasis in gastric cancer.

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植物药:免疫紊乱解决之道

徐丽芬

2015, 29(S1): 11-11.

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Phytomedicines have been used for treating or preventing diseases throughout human history. We have been conducting in exploration of traditional or folk herbal medicines as an attempt to identify novel phytocompounds candidates for further development into botanical supplements or drugs for inflammation related diseases, including cancer, acute liver hepatitis, and sepsis. Comparative "OMICS" technology platforms in combination with various in vitro and in vivo cell- and gene-based bioassays, murine skin inflammatory and syngeneic and xenograft mammary tumor and melanoma models are employed to validate the pharmacological effects and the underlying mechanistic insights of the identified bioactive phytocompounds. The therapeutic potential of phytoagents, alone or in combination, in sensitizing the chemotherapeutic drug efficacy and/or reduction of its side effects in tumor-bearing mice are investigated. In addition, how phytoagents modulate pro- or anti-inflammatory lipid mediators, such as oxylipins, and related signaling cascades systemically or at organ levels are also addressed for understanding sepsis or cancer pathogenesis and the modes of action of bioactive phytoagents.

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通过饮食多酚进行表观遗传修饰和肿瘤化学预防

Ajay GOEL

2015, 29(S1): 12-12.

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Growing evidence indicates that cancer incidence across the world is not similar, and it is more prevalent in certain populations than others, suggesting the critical role for dietary and lifestyle factors. For instance cancer incidence among people from the Indian subcontinent, where most spices are consumed, is much lower than that in the Western World. Spices have been consumed for centuries for a variety of purposes e.g. as flavoring agents, colorants, and preservatives. However, there is increasing evidence for the importance of plant-based foods in regular diet to lowering the risk of most chronic diseases, so spices are now emerging as more than just flavor aids, but as agents that can not only prevent but may even treat disease. Besides suppressing inflammatory pathways, spice-derived nutraceuticals can suppress survival, proliferation, invasion, and angiogenesis of tumor cells. Increasing evidence indicates that genetic alterations are relatively rare, and epigenetic changes (DNA methylation, histone modifications and expression of noncoding RNAs) plays a bigger role in human cancer, and can be easily influenced by environmental, lifestyle and dietary factors, and some estimates suggest that over two-thirds of the cancer incidence can be accounted for by the environmental and dietary factors alone. Among all these factors, diet is probably the single most important factor which may influence carcinogenesis more comprehensively, because diet is readily modifiable and have the potential to modulate multiple epigenetic processes. Polyphenols in dietary botanicals represent a versatile group of phytochemicals with many potentially beneficial activities in terms of disease prevention. Dietary polyphenols (bioflavanoids) have antioxidant and anti-inflammatory properties that might explain their chemopreventive effects. However, the actual therapeutic potential of these compounds may not have been completely realized due to lack of understanding on the effects of these agents on epigenetic modifications. Recent, but limited evidence indicates that some of the polyphenols, including curcumin (from turmeric), genestein (from soy), EGCG (from green tea), diallyl disulfide (from garlic), sulforaphane (from broccoli) and resveratrol (from grapes) may induce epigenetic changes in various cancer cell lines. This presentation will describe some of the current scientific evidence for the role of epigenetic alterations induced by curcumin and boswellia, in support of their anti-cancer activities, which provides a strong scientific foundation for preclinical and human clinical intervention studies in future.

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副凋亡:肿瘤细胞克服凋亡抵抗的途径

Kyeong Sook CHOI

2015, 29(S1): 13-14.

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Since inherent or acquired cellular resistance to various pro-apoptotic treatments often leads to therapeutic failure, a better understanding of alternative non-apoptotic pathways may facilitate the design of novel therapeutics against malignant cancer cells. Paraptosis is a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum (ER) and mitochondria, without any apoptotic characteristics. We found that curcumin, dimethoxycurcumin (a more stable analog of curcumin), and celastol kill malignant cancer cells via induction of paraptosis as a main cell death mode. Study on the underlying mechanisms of paraptosis revealed that simultaneous proteasomal inhibition and mitochondrial Ca²⁺ overload can effectively induce paraptosis in cancer cells. Mitochondrial Ca²⁺ overload can be achieved not only by inhibition of mitochondrial Na⁺/Ca²⁺ exchanger but also by IP₃ receptor- or ryanodine receptor-mediated release of Ca²⁺ from the ER and its subsequent mitochondrial Ca²⁺ uniporter-mediated Ca²⁺ influx into mitochondria. Since malignant cancer cells are more vulnerable to oxidative stress and ER stress than normal cells, clarification of the molecular basis of paraptosis that targets mitochondria and the ER at the same time may provide a rational therapeutic strategy for effectively killing resistant cancer cells.

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Brahmi研究与开发:一种能改善记忆的药用植物

Kornkanok INGKANINAN, Nanteetip LIMPEANCHOB, Sakchai WITTAYA-AREEKUL, Krongkarn CHOOTIP, Pornnarin TAEPAWARAPRUK, Nuwat TAEPAWARAPRUK, Jintanaporn WATTANATHORN, Seewaboon SIREERATAWONG, Waraporn PUTALUN, Watoo PHROMPITTAYARAT, Hiroyuki TANAKA

2015, 29(S1): 13-13.

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OBJECTIVE Brahmi (Bacopa monnieri Wettst., Schrophulariaceae) has been used in Ayuravedic medicine as a memory enhancer. We aimed at research and development of Brahmi for clinical use as the food supplement for memory improvement. METHODS The standardized extract of Brahmi has been developed. The neuroprotective effect of the extract was tested using in vitro and in vivo assay. A double-blinded randomized control trial of brahmi tablet was conducted in 60 middle-aged elderly volunteers. RESULTS The in vivo studies indicated that the standardized extract had neuroprotective effect at the doses of 40 and 80 mg·kg^-1. The escape latency time of rats in Morris water maze test was reduced. Moreover, the reduction of neurons and cholinergic neuron densities were mitigated. We also tested its protection effect against the beta-amyloid protein and glutamate-induced neurotoxicity in primary cortical cultured neurons. The result demonstrated that Brahmi extract protected neurons from beta-amyloid-induced cell death, but not glutamate-induced excitotoxicity. The standardized extract of Brahmi is formulated as a film-coated tablet. The effect of Brahmi extract on learning and memory was studied in the middle aged and elderly volunteers. The results showed that Brahmi extract (300 and 600 mg·d^-1) could improve quality of life in the elderly by improving the physical fitness via the increase in efficiency of postural balance. It also improved the psychological fitness by increasing alertness and attention resulted in the improvement of learning and memory. In addition, Brahmi extract could decrease depression-like symptom. The dosage used in this study did not produce toxic and side effects. CONCLUSION The in vitro and in vivo studies suggested that brahmi is a potential medicinal plant for memory enhancer. The clinical study showed that brahmi tablet could improve physical fitness and enhance memory in healthy volunteers.

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灵芝多糖对肿瘤逃避免疫监视的拮抗作用

林志彬

2015, 29(S1): 14-15.

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Recent years, our research group focused the effects of G. polysaccharides, which are extracted from fruiting body of Ganoderma lucidum, on tumor evasion from immune surveillance. The immune system in patients with tumor often fails to control tumor growth because of deficient immunogenicity of tumor cells. Deficient major histocompatibility complex (MHC) classⅠ and costimulatory molecules on malignant cells partially results in tumor evasion since antigen bond MHC and costimulatory molecules provide two signals for T cell activation. Therefore, enhancement of MHC-Ⅰ and costimulatory molecules may favor restraint of the evasion. Our study found that G. polysaccharides can increase MHC classⅠ molecules such as H-2Kb and H-2Db as well as the costimulatory molecules B7-1 and B7-2 expression on B16F10 melanoma cells at both mRNA and protein levels, and can promote lymphocyte-mediated cytotoxicity. Tumour cells produce immune suppressive factors such as interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. Our study also found B16F10 cell culture supernatant (B16F10-CS) suppressed lymphocyte proliferation and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin (PHA), as well as lymphocyte proliferation in the mixed lymphocyte reaction. The suppression also associated with elevated levels of immunosuppressive IL-10, TGF-β1 and VEGF in B16F10-CS. Further, the production of IL-10, TGF-β1, and VEGF in B16F10 melanoma cells and lung carcinoma LA795 cells was suppressed by G. polysaccharides at both mRNA and protein levels. On the contrary,the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes was suppressed by B16F10-CF at both the mRNA and protein levels, whereas the suppression was ameliorated by G. polysaccharides. B16F10-CS was suppressive to the viability, phagocytic activity, NO production, TNF-α production and activity in peritoneal macrophages while G. polysaccharides had the antagonistic effects against this suppression. Then subsequently, the plasma of patients with lung cancer suppressed proliferation, CD69 expression, and perforin and granzyme B production in lymphocytes upon activation by PHA. However, these suppressive effects were reversed by G. polysaccharides. In conclusion, G. polysaccharides can improve the nature of B16F10 cells to activate lymphocytes and antagonize immunosuppression induced by B16F10-CS in lymphocytes and macrophages. These findings indicate that G. polysaccharides can restraint tumor evasion from immune surveillance, suggesting this potential value of G. polysaccharide to facilitate cancer immunotherapy.

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淫羊藿提取物对绝经后骨质疏松、乳腺癌和前列腺癌的标准化、临床前和临床评价

Eu-Leong YONG, Ryan ZL LIM, Ee Min TAN, Sun FENG, Li JUN, Inthrani R INDRAN

2015, 29(S1): 15-15.

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Estrogens are critical for bone health and the development of breast and prostate tissues. At menopause, our cohort studies show that naturally occurring estrogens in the body protect against osteoporotic hip fractures, but can also increase the risk of breast cancer. Extracts of Epimedium spp. is widely used to treat osteoporosis and improve general health in post-menopausal women. Prenylated flavonoids from Epimedium spp. exert potent estrogenic effects making it an attractive alternative to estrogen replacement therapy for treatment of osteoporosis. Lead compounds from epimedium such as icaritin impact pro-osteoblastic, anti-osteoclastic signaling pathways in bone cells; and anti-tumour effect on nuclear receptors in breast and prostate cancer animal models. Oral administration of a Epimedium spp. extract in open-label, two-period, randomized, crossover study indicate significant ex-vivo effects on ERα and ERβ-mediated bioactivities and breast cancer cell proliferation. Challenges in the taxonomy, standardization and quality manufacturing of extracts suitable for pharmacokinetic, pharmacodynamics and efficacy studies in humans will be discussed.

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对传统中药药理学研究的思考

He (Henry) SUN

2015, 29(S1): 15-16.

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There are pharmacological aspects that multi-herbal traditional Chinese medicine (TCM) presents unique characters, to which conventional pharmacology research methods are not applicable. Few examples to mention: The generally lower in toxicity profiles for a TCM product makes the PharmTox study methods need to be re-visited for validity, as the maximum tolerance dose (MTD) is very often unreachable by oral feeding, an injectable dose of unpurified herbal extract for toxicology check cannot be used for TCM products as well. Due to the so-called multi-components, multi-target phenomenon, the definition of "Pharmacologicial Effect" is hard to set and should be clearly re-defined for TCM. Plasma drug concentrations are normally low and hard to detect, especially the contribution of each chemical moiety to the apparent clinical effects is unknown, and generally observed concentration-response models (CR) are not applicable due to the mixture of hundreds of chemical moieties; Bioavailability studies from varies manufacture batches are not available but should be determined for drugs with potent active moieties and/or metabolites or has an narrow therapeutic window. However, the beauty is that, in the regulatory world, pre-clinical pharmacology data and the mechanism of action (MOA) information are valuable only if can be used to guide the drug development process. In this presentation, experiences gained during the process of developing a TCM product in the US and interactions with the US FDA are to be shared for the audience reference. Additional scientific proposals and discussion points on alternative methods for pharmacology studies of TCM product are to be presented to stimulate a fruitful discussion.

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将天然产物转化为老年病新药和保健食品

李铭源

2015, 29(S1): 16-16.

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Increasing evidence points towards a strong association between cerebrovascular dysfunction and neurodegenerative diseases in aging population. The global disease projection indicates that the healthcare burden derived from these disease problems will continue to rise. Many traditional Chinese medicines (CMs) have been used to prevent and treat the multi-faceted diseases in China and other Asian countries. These herbs are potential rich sources of new leads that may also reveal previously unidentified mechanisms. Previously, our team has initiated a research program to analyze and characterize the bioactive extracts and pure natural components from the CMs using multipleexperimental models of vascular and neurodegenerative diseases. Some of the natural bioactive compounds have been further chemically modified to series of derivatives using different organic chemistry approaches (e.g. heterodimer and one-pot synthesis) and proven improved potency.The advantages of zebrafish modelfor in vivo high content screening of CMs will be presented. Our resultsprovide scientific rationales for clinical usage of the CMs and also probablylead to develop reproducible, higher potency and lower toxic agents for healthcare in the future.

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日本汉方药中对免疫和神经系统的活性成分和可能作用机制

Haruki YAMADA

2015, 29(S1): 16-17.

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Many of the diseases are multifactorial and still difficult to treat by only modern medicines, however Kampo medicines, which contain multiple ingredients due to component herbs, seem to be suitable for recovery of such complicated symptoms because these ingredients may attack multiple target sites to improve the symptoms caused by disturbance of whole body system. In Japan, 148 standardized Kampo pharmaceutical preparations have been developed, and covered by national health insurance system, and more than 80% practicing physicians have experience of using the Kampo medicines for the treatment depending on patient's clinical situation, either separately or to complement modern western medicines. In present paper, I'd like to introduce few examples of our studies on elucidations of pharmacological actions and active ingredients of Kampo medicines as multi-ingredients drugs. Although Kampo medicines have been taken orally as the decoction, their hot water extracts contain not only low molecular ingredients but also high molecular ingredients like polysaccharides. Our recent studies suggest that Hochuekkito (HET: Bu-Zhong-Yi-Qi-Tang) prevents respiratory infection through potentiation of mucosal immune system in upper respiratory tract, and certain pectic polysaccharides were identified to involve in potentiation of the immune response. It was also clarified that synergic action of two stereoisomers are required for the oral adjuvant activity of Shoseiryuto (SST: Xiao-Qing-Long-Tang). Like examples of the studies of Kososan (KS: Xiao-Su-San) and SST, proteomic analysis of disease model animals using agarose 2-DE is also useful another approach to find target protein candidates. These accumulated comprehensive results may contribute for more evidence-based clinical application.

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探寻药用植物及其临床应用的发展趋势

Ákos MÁTHÉ

2015, 29(S1): 17-18.

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Plant biodiversity has been source of myriad forms of natural substances utilized in similarly multiple ways. The history of medicinal and aromatic plant (MAP) utilization dates back to the beginnings of mankind. Our forefathers used plants they could find in nature, to ease, cure their sufferings, illnesses, or heal their wounds. This type of approach has survived in the traditional medicinal (TM) uses, until today, since nearly 80% of the world population still relies on MAPs in their medications (Akerele, 1992). Especially rich are the traditions in China and India, where TMs have been used for thousands of years. The use of TM, however, goes well beyond these countries. It has been estimated that more than 1.5 billion people all over the world are trusting in the efficacy and safety of Chinese medicine (Wang and Franz, 2014). The renaissance of MAP-use in the high-income countries has brought about different types of use i.e.: in the form of Herbal Medicines (CAM=Complementary and Alternative Medicine). MAPs have become "industrial products" with versatile and innovative new concepts in phytotherapy and veterinary medicine, aromatherapy, nutraceuticals, cosmeceuticals, animal welfare, etc. New, innovative, value added applications include MAP use in functional foods, animal husbandry, as well as plant protection in agriculture. The versatile utilization of essential oils is most promising. MAPs are sourced from both wild-crafting and cultivation, where quality is primarily determined by the genotype, the environment and the conditions during the life cycle of the plant. Processing to preserve/isolate their active principles is an additional precondition for the safety and efficacy in use. Current areas of utilization constitute powerful drivers for the exploitation of these natural resources. Increasing demands, coupled with the already limited availability and potential exhaustion of natural resources, make it necessary to take stock of both our resources and our knowledge regarding research and development, production, trade and utilization, especially in view of sustainability National/international regulatory authorities have elaborated guidelines (GACP, GMP and GLP) to be included in quality assurance systems. The entire production system should observe these practices and this fact should be continuously documented, certified. Modern approaches in production and use have called attention to the importance of quality, safety and efficacy of both MAPs and their produce. There is also a need to ensure the quality of medicinal plant products by using modern sample preparation and control techniques as well as suitable standards.In Europe,this regards also the quality of TCM herbals. Their quality assurance systems should guarantee that they can be freely imported into the European Union and other Western European Countries (Wang and Franz, 2014). A new aspect in quality assurance is the trend for the protection of 'geographical indication'(GI) botanicals in the context of intellectual property rights. MAPs will also maintain their importance in the search for new, valuable sources of drugs and lead compounds. The ultimate aim would be to use MAPs with well-defined and constant composition. There is a need to ensure the quality of medicinal plant products by using modern sample preparation and control techniques. Thepharmacological effects of herbs are caused by the interaction several biologically active principles, therefore, the present clinical testsaimed at single chemicals, are also not really adequate. Todate, the lesson worthily worded by the Chiang Mai Declaration (1988) is more than valid: "Let us save plants that Save Lives".

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靶向K-ras的半合成穿心莲内酯衍生物:发现潜在抗癌药物先导物的有效方法

Johnson STANSLAS, Charng Choon WONG, Hui Chyn WONG, Shun Ying QUAH, Pran Kishore DEB, Sreenivasa Rao SAGINEEDU, Khozirah SHAARI

2015, 29(S1): 17-17.

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We embarked on the discovery of anticancer agents from andrographolide nearly 15 years ago. Thus far, a few lead semisynthetic compounds have been identified, but only recently we managed to pinpoint their potential molecular target. Through in silico and cell-based studies, these lead molecules have been found to bind K-ras oncoprotein and disrupt its function. Further molecular docking analysis suggested the compounds targeted both wild-type and oncogenic mutant K-ras. However, the binding affinity was greater for the oncogenic protein. Low binding energies to wild-type K-ras protein suggested transient binding and inhibition. The compounds showed stronger binding interactions to all three mutant K-ras proteins (G12V, G12C and G12D) with average free energies (ΔG_bind) of -82 kcal·mol^-1 as compared with -61 kcal·mol^-1 for the wild-type protein. It is noteworthy that the binding pocket in wild-type K-ras protein, however, is different from that of the mutant proteins. SRJ23, one of the lead compounds, showed the strongest binding interactions to all three mutant K-ras proteins. Stronger binding to the mutant proteins could lead to more targeted and prolonged inhibition. Investigation into the effect of the compounds on RAS-MAPK pathway showed this pathway was disrupted in colon, breast and prostate cancer cells. In vivo studies revealed the compounds retarded the growth of human colon (HCT-116) and prostate (PC-3) cancer xenografts in mice. All of the above prompted us to synthesise derivatives of the lead compounds for improvement of binding affinity for the oncogenic K-ras. A preliminary in silico exploration found some compounds with such property and these compounds are presently undergoing extensive pharmacological investigations.

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为天然的微生物工厂开发程序化生物功能

张旭

2015, 29(S1): 18-19.

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Synthetic biology aims to engineer genetically modified biological systems that perform novel functions that do not exist in nature, with reusable, standard interchangeable biological parts. The use of these standard biological parts enables the exploitation of common engineering principles such as standardization, decoupling, and abstraction for synthetic biology. With this framework in place, synthetic biology has the potential to make the construction of novel biological systems a predictable, reliable, systematic process. Recent efforts to implement a highly systematic frame work in biological engineering have provided long-awaited evidence that engineering principles can facilitate the construction of novel biological systems. Synthetic biology has so far demonstrated that its framework can be applied to a wide range of areas such as energy, environment, and health care. In this talk, our recent efforts to develop recombinant microbial cells with programmable biological functionalities will be presented. In particular, an emphasis will be placed on our development of auto-regulatory genetic circuits that transformed microbes into cell factories that autonomously monitor biosynthetic activities. http://synbiolab.org/

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应用整合方法发现新的抗癌药物和研究肿瘤转移

Kathy Qian LUO, Afu FU

2015, 29(S1): 19-20.

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Cardiovascular diseases, cancer, diabetes and neurodegenerative diseases are four major life threatening and health damaging diseases that affect a large portion of the world population at present. Although these diseases differ significantly in terms of symptoms and pathogeneses, they have one thing in common-a defect in the process of programmed cell death, mainly apoptosis. For example, resistance to apoptosis results in cancer formation. On the other hand, over-activation of apoptosis in endothelial cells leads to dysfunction of vascular system which can contribute to cardiovascular diseases and complications of diabetes. Therefore, understanding what causes the defects of apoptosis in those disease-related cells will provide new insights in designing target-specific therapies to treat those diseases. In order to develop a method of real-time detection of apoptosis within the target cells, we genetically engineered a fluorescence resonance energy transfer (FRET)-based biosensor. When this biosensor is synthesized in the transfected cells, the sensor protein can detect caspase activation-mediated apoptosis by changing the color from green to blue. We have generated a bank of 32 sensor cell lines consisting of various types of cancer and non-cancerous cells. We also integrated these sensor cells into six model systems including a sensor cell-based high throughput drug screening assay, 3D in vitro tumor model, co-culture system, microfluidic system, zebrafish cancer model and xenograft tumor mouse models. In this talk, I will discuss the applications of these sensor cells and model systems in discovering new anti-cancer agents and elucidating the survival mechanisms of metastatic cancer cells in circulation.

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从天然和传统药物中发展光动力和声动力药物

梁荣能, 许川山

2015, 29(S1): 19-19.

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Malignant tumors and infectious diseases are two main threats to human beings. Photodynamic therapy (PDT) is an effective approach to treat malignancies and infections through the generation of reactive oxygen species (ROS) of a non-toxic photodynamic drug (photosensitizing agent) upon light irradiation. PDT shows promising in treating areas where light can reach, for example, skin and cavities. To treat the problems on deep tissues, much evidence has accumulated in recent years suggesting that sonodynamic therapy (SDT), which eradicates malignant or infectious cells by ROS produced from the interaction of ultrasound energy and sonodynamic drug (sonosensitizing agent), has potential in treating diseases in deep tissues because ultrasound energy has deeper tissue penetration than light energy. Natural products are a valuable source for developing novel drugs. In recent years, we are focusing on developing novel and naturally occurring photodynamic and sonodynamic agents. We have found some active components from natural products, for example, curcumin and hypocrellin, have not only photodynamic activity and also sonodynamic activity in killing bacterial and tumor cells. This talk will briefly introduce two new technologies including photodynamic and sonodynamic therapy, and highlight the recent development in photodynamic and sonodynamic drugs from natural products or traditional Chinese medicines. Chemical modifications of the active compounds have also been attempted to achieve better results.

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富含花青素天然产物延缓血管衰老的潜力:内皮衰老作用

Valérie SSCHINI-KERTH

2015, 29(S1): 20-20.

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Numerous clinical studies have indicated that regular intake of polyphenol-rich products such as red wine and green tea is associated with a reduced risk of cardiovascular diseases. The protective effect has been attributable, in particular, to their high level of polyphenols such as flavanols and anthocyanins. The beneficial effect of polyphenols on the vascular system involves their ability to improve the lipid profile and to prevent platelet activation and inflammatory responses, and possibly also by a direct action at blood vessels. Indeed, grape-derived polyphenols and tea catechins are potent inducers of endothelium-dependent relaxations of isolated blood vessels by causing a sustained stimulation of the endothelial formation of nitric oxide (NO) and endothelium-derived hyperpolarization, two potent vasoprotective mechanisms. Besides grape-derived and tea products, several red and black fruit-derived products induce also potent vasoprotective effects. Intake of polyphenol-rich products has been shown to retard the induction of aging-related endothelial dysfunction and to improve an established aging-related endothelial dysfunction in rats. The beneficial effect is mostly due to the normalization of the high level of oxidative stress in the arterial wall and of the local angiotensin system. Moreover, polyphenol-rich products prevented the induction of replicative senescence at indicated by levels of senescence-associated beta-galactosidase activity and of key cell cycle regulators p53/p21 and p16 in cultured endothelial cells. The induction of endothelial senescence is associated with the down-regulation of endothelial NO synthase expression and the up-regulation of tissue factor expression most likely triggered by NADPH oxidase- and cyclooxygenase-derived oxidative stress, and leads to a reduced ability of endothelial cells to effectively inhibit platelet aggregation. Thus, regular intake of polyphenol-rich products can delay the induction of aging-related endothelial dysfunction most likely by preventing endothelial senescence, and as a consequence help to maintain an optimal protective effect of endothelial cells on the vascular system.

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在替代模型中基于“行为组学间”分析预测抗肿瘤药物联合用药

Ee-Sin CHEN, Thi Thuy Trang Nguyen, Zoey TAY

2015, 29(S1): 21-21.

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Development of drug resistance is a challenge to chemotherapeutic success but the mechanisms remain elusive. To study cellular resistance against doxorubicin, which is a widely employed chemotherapeutic agents in the clinic against solid tumors, lymphomas and leukemias, we employed fission yeast as a model to set up a synthetic lethality workflow to isolate 91 doxorubicin-resistance (DXR) genes. These DXR genes interlock to form a large network to ensure high viability when cells were exposure to doxorubicin. Interestingly, the gene network is, at least in part, conserved in the human cells and the understanding of how DXR genes are connected has enabled us to design a unique drug combination to sensitize in human cervical carcinoma cells to sub-lethal dose of doxorubicin through concomitant targeted inhibition of the multidrug resistance 1 protein and the proton pump vacuolar-ATPase. Further dissection of the DXR network showed a significant degree of overlap with that regulating resistance against other drugs with mechanistically varied modes of action. We will show evidence of how we took advantage of this 'interactomic' platform to target differential overlap in drug resistance networks to destabilize gastric cancer cells in conjunction with an agent that modulates chromatin landscape.

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整合化学和定量蛋白质组学方法用于鉴定药物靶点

林青松, 王继刚

2015, 29(S1): 21-21.

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Most drugs exert pharmacological effects through interaction with their target proteins. Therefore, drug target identification is a crucial step towards the understanding of the mechanism of drug action. It is also imperative to study the pharmacodynamics of a known drug, with an aim to discover the potentially unrevealed actions and thus refine its future clinical applications. Currently, drug-target identification is either through in vitro affinity chromatography-based approaches or in vivo activity-based protein profiling (ABPP) approaches. However, these approaches generally face difficulties discriminating specific drug targets from non-specific ones. To address this issue, we have come up with a strategy by coupling iTRAQTM (isobaric tags for relative and absolute quantitation) quantitative proteomics approach with clickable ABPP, to specifically and compre hensively identify drug targets in live cells. Using this approach, we identified the protein targets of andrographolide, a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. The identified target list not only confirmed the known functions of the drug but also revealed its potential novel application as a tumor metastasis inhibitor. We have also used this strategy, combining with a cleavable probe to identify the protein targets of aspirin and its binding sites. Our results revealed the roles of aspirin ininhibition of protein synthesis and induction of autophagy, which have been functionally validated. Our strategy is widely applicable to the identification of protein targets of covalent drugs.

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2-Methoxystypandrone induces iNOS expression by H₂O₂-dependent JNK activation for multiple cancer cell death

Wen SUN, Jiao-lin BAO, Wei LIN, Wen-wen ZHAO, Jin-jian LU, Qing-wen ZHANG, Xiu-ping CHEN

2015, 29(S1): 22-22.

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OBJECTIVE To investigate the mechanism of anticancer effect of 2-methoxystypandrone(2-MS), a natural naphthoquinone isolated from Polygonum cuspidatum Sieb. et Zucc. METHODS Three types of cancer cells were investigated in the research (A549, MCF7, B16-F10). Flow cytometer was used to determine ROS/RNS generation. Western blotting was used to detect related protein expression. Apoptosis assay, GSH/GSSG (reduced glutathione/ oxidized glutathione) assay were performed using commercial kit. Si-RNA knockdown was used to silence c-jun N-terminal kinase (JNK) and iNOS. High-performance liquid chromatography (HPLC) was used to detect the direct reaction of 2-MS with GSH. RESULTS 2-MS induced cytotoxity towards a panel of cancer cells, with less effect on normal cells. 2-MS induced necroptosis in A549 cell and apoptosis in B16-F10 and MCF7 cells. 2-MS increased phosphorylation of JNK in three types of cancer cells. Inhibition of JNK with SP600125 or silencing JNK attenuated 2-MS-induced cell death. JNK also activated iNOS expression and led to nitric oxide (NO) generation in three cancer cells. NO-induced nitrative stress was responsible for DNA damage and necroptosis in A549 cells. NO also inhibited NF-κB expression and induced intrinsic apoptosis in B16-F10 and MCF7 cells. Both NO scavenger hemoglobin and silencing iNOS can partially reverse 2-MS-induced cell death. Furthermore, we found that all of these were attributed to induction of hydrogen peroxide (H₂O₂), which was caused by glutathione (GSH) depletion through interaction of 2-MS with GSH. The interaction was validated through cell-free HPLC analysis. Both the H₂O₂ scavenger catalase and exogenous GSH can significantly reverse the 2-MS-induced cell death. But catalase did not protect against the decrease in GSH level. In contrast, there showed no clear increase of both H₂O₂ and NO in non-carcinoma liver cell LO2. CONCLUSION Taken together, a medicinal plant-derived 1,4-napthoquinone, induced iNOS expression by H₂O₂-dependent JNK activation, caused nitrative stress, finally led to cancer cell death by necroptosis or apoptosis.

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Determining relaxin-3/RXFP3 activation by inhibition of forskolin induced cAMP assay

Jayakody JPT, Herr DR, Dawe GS

2015, 29(S1): 22-23.

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OBJECTIVE Relaxin-3 is a novel neuropeptide of the relaxin insulin family of peptides. So far, many studies have reported the role of relaxin-3/ RXFP3 system in regulating feeding, stress response and cognition. In previous studies using RXFP3 stable cell lines, inhibition of adenylate cyclase by the activation of Gi/o proteins have been reported. Based on this signaling event, we have developed inhibition of forskolin induced cAMP assay to detect RXFP3 activation or inhibition by its agonists or antagonists. METHODS To detect inhibition of adenylate cyclase up on RXFP3 activation by human relaxin-3 (H3 relaxin), HEK-RXFP3, CHO-RXFP3, SN56 and GT1-7 cells were plated in poly-L-lysine coated 24 well plates. The following day, the cells were starved in serum free cell culture media for 6 h. Next, cells were treated in triplicate with serum free media (control) and H3 relaxin for 5-15 min. Then, the cells were treated with serum free media with DMSO (control) or forskolin (5-100 μmol·L^-1) for 15 min at 37℃ with 5%CO₂. At the end of the incubation, cell culture media was discarded and the cells were lysed with 0.1 mol·L^-1 HCl. The cAMP concentration in each lysate was detected by ELISA (Cayman Chemicals).The data from three experiments were analysed using one way ANOVA followed by Bonferroni post hoc test or Dunnett's post hoc test. RESULTS In CHO-RXFP3 and HEK-RXFP3 cells, 10 nmol·L^-1 of H3 relaxin was able to significantly inhibit the forskolin (5 μmol·L^-1) induced cAMP levels (P<0.05). In SN56 neuronal like cell line endogenously expressing RXFP3, 100 nmol·L^-1 H3 relaxin was able to significantly reduce forskolin (3 μmol·L^-1) induced cAMP (P<0.05). However, in wild type HEK293T and CHO-K1 cells, 10 nmol·L^-1 H3 relaxin was not able to significantly reduce the forskolin 22(5 μmol·L^-1) induced cAMP levels. In GT1-7 mouse hypothalamic cells endogenously expressing RXFP3,100 nmol·L^-1 H3 relaxin and 5 or 3 μmol·L^-1 forskolin, was able toa significantly increase cAMP levels (P<0.05). CONCLUSION Inhibition of forskolin induced cAMP assay can be used to detect G_i/o mediated cAMP inhibition related signaling events due to RXFP3 activation by its agonists in CHO-RXFP3, HEK-RXFP3 and SN56 cell lines.

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Effect of osthole, a Fructus Cnidii-derivednature coumarin, on osteogenesis and bone healing in mice

Zhong-rong ZHANG, Wing Nang LEUNG, Chun Wai CHAN

2015, 29(S1): 23-24.

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OBJECTIVE To investigate the effect of osthole, a natural coumarin isolated from traditional Chinese medicine Fructus Cnidii, on osteogenesis in vitro and bone fracture healing in vivo. METHODS Primary bone marrow mesenchymal stem cells (MSCs) were isolated from 6-week C57/B6 mice, and osteogenic differentiation was assessed by alkaline phosphate (ALP) activity and calcium nodule formation. Adult (12-week) C57 mice were subjected to mid-shaft osteotomy on femur. The mice were oral administrated with osthole (5, 20 or 50 mg·kg^-1) or vehicle solvent daily from post-operational week 1. Radiographic imaging, real time molecular imaging, micro computed tomography (μCT) and histology analysis were performed to evaluate the healing progress. RESULTS Results showed that osthole promoted osteogenesis of bone marrow MSCs by enhancing ALP activity and mineralization dose dependently in the range of 1-100 μmol·L^-1. Plain radiographs showed that administration of osthole at 20 and 50 mg·kg^-1 significantly accelerated fracture healing by reducing the period of reparative phase. Further investigation with μCT and histology showed that osthole-treated group had high proportion of newly-formed woven bone and smaller cartilage island compare to control group at week 2; and treatment group had completed endochondral ossification and started remodeling phase at week 3. Molecular imaging of near-infrared (NIR) fluorescent labeled palmidronate depositing on newly formed bone suggested that osthole treatment (20 mg·kg^-1) augmented callus mineralization process at both postoperative week 2 and week 3 by 80.72% and 25.95% respectively. CONCLUSION Osthole demonstrates significant osteopromotive effect in vitro and anabolic effect on bone formation in fracture repair, which makes it a potential agent for bone regeneration and against osteoporosis.

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Preparation of hypocrellin B nanoparticlesfor photodynamic therapy

Yue JIANG, Xin-na WANG, Albert Wingnang LEUNG, Chuan-shan XU

2015, 29(S1): 24-25.

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OBJECTIVE To prepare hypocrellin B-loaded apoferritin nanoparticles (HB-AFT NPs) for photodynamic therapy on tumor. METHODS HB-AFT NPs were prepared by taking advantage of the reversibleunfolding and refolding character of apoferritin in different pH environments. The photophysical and photobiological properties of hypocrellin B-loaded apoferritin were measured. RESULETS HB molecules were successfully encapsulated within apoferritincavity. HB-AFT-NPs exhibited higher ROS productionthan free HB. Additionally, phototoxicity of HB-AFT NPs to MDA-MB-231 cells was significantly improved as compared to free HB. CONCLUSION Together these results demonstrate that hypocrellin B-loaded apoferritin might be a potential nanoscale photosensitizer.

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Usage of fresh medicinal plants and users' perceptions in Singapore

Yin SIEW, Sogand ZAREISEDEHIZADEH, Guang SEETOH, Ying NEO, Hoon TAN, Ling KOH

2015, 29(S1): 24-24.

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OBJECTIVE To gather information regarding the usage of fresh medicinal plants and users' perceptions via face-to-face interviews. To date, there is no published report of firsthand account of their usage in Singapore. Such important information may be permanently lost if not properly collated in time. METHODS Information on demographic data, plant-use methods and perception of usage was collated. Participants were recruited via the local newspapers, by word of mouth etc. The survey protocol was approved by the NUS Institutional Review Board. Fresh plant samples/photographs were obtained from the users and voucher specimens were kept. RESULTS Two hundredusers who have used a total of 103 species of fresh medicinal plants anytime in the last 5 years participated in the survey. The five most commonly used plants were Clinacanthusnutans(34 users), Strobilanthescrispus(31 users), Pereskiableo(25 users), Aloe vera(18 users), and Zingiberofficinale(16 users). The top 3 most commonly cited medical conditions were diseases of respiratory system (50 users), neoplasm (29 users) anddiseases of circulatory system (20 users). A total of 173 users (86.5%) did not consult any healthcare professional for advice about plant usage, and only one user consulted the pharmacist. Some of the common reasons given for using fresh medicinal plants were recommendation by others (150, 75.0%), efficacy (137, 68.5%), and safety (117, 58.5%). Most users (170, 85.0%) were satisfied or highly satisfied with the outcome of plants used. CONCLUSION Two hundred users of fresh medicinal plants have been successfully interviewed and the information documented systematically in a database. The results suggest that fresh medicinal plants have a role to play in healthcare in modern society. The information collated will serve as a useful resource for identifying promising plants for future drug discovery efforts.

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Anti-malaria drug artesunate protects bronchial epithelium from DNA damage induced by asthma

Tze Khee CHAN, WN Felicia TAN, Bevin P ENGELWARD, WS Fred WONG

2015, 29(S1): 25-26.

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OBJECTIVE To investigate the genome protective effects of anti-malaria drug, artesunate in an experimental allergic asthma model. METHODS Mice were sensitized on day 0 and 7 and challenged on day 14 with 100 μg house dust mite (HDM) via intratracheal administration. Artesunate (30 mg·kg^-1) was administered intra-peritoneally on day 6, 7, 8, 13, 14 and 15. Samples were collected on day 1, 3 and 5 post last HDM-challenge for analysis of air way inflammation and DNA damage. Lung sections were immunofluorescence (IF)-stained for DNA double strand breaks (DSBs) markers, γH2AX and 53BP1. Levels of DNA repair proteins Ku70 and Rad51, which are involved in non-homologous end joining (NHEJ) and homologous recombination (HR) DNA DSB repair pathways respectively, were measured. To quantify cell death in asthmatic lung, TUNEL staining was performed. Comet assay, a single cell gel electrophoresis was employed to detect DNA damage induced by HDM in BEAS-2B human bronchial epithelial cell line, in vitro. RESULTS Artesunate treatment significantly reduces immune cells infiltration in BAL fluid of asthmatic mice, collected on day 3 and 5 post-challenge. Importantly, artesuante is able to protect bronchial epithelium from DNA DSBs induced by asthma, as detected by the reduced level of γH2AX and 53BP1 foci formation in the nucleus. This genome protective effect is evident even on day 1 post-challenge, when immune cells infiltration remained high. This indicates that artesunate confers protection on bronchial epithelium in the presence of inflammation. Additionally, artesunate is also able to reduce cell death in asthmatic lung revealed by TUNEL assay and cleaved caspase 3 level. Interestingly, the levels of DNA repair proteins in artesuante-treated asthmatic mice are unchanged as compared to HDM-only mice, suggesting that artesunate treatment does not augment the level of DNA repair proteins. When human bronchial epithelial BEAS-2B cells were exposed to HDM in vitro, we observed an increase in the levels of DNA damage. Artesunate (60 μmol·L^-1) co-incubated with HDM is not able to prevent direct DNA damage induced by the allergen. Together, these studies suggest that the genome protective effect of artesunate in vivo may be attributed to physiological effects (such as its anti-inflammatory effects) rather than serving to directly prevent DNA damage. CONCULSION This study highlights a novel role for artesunate in protecting bronchial epithelial cells from asthma-induced DNA damage.

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PFMG1 promotes osteoblast differentiation and prevents osteoporotic bone loss

Li-yuan LI, Pan WANG, Zhao WANG

2015, 29(S1): 25-25.

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OBJECTIVE To investigate the effect of Pinctadafucata mantle gene 1 (pfmg1) on osteoporotic bone lossand the role in osteoblast differentiation and matrix mineralization, and to explore the molecular mechanismof how PFMG1 functions through both animal and cellular experiments. METHODSFor animal experiments, female BALB/c mice were subjected to sham-operation (sham) or ovariectomy(ovx) at 5 weeks of age, control and pfmg1 lentiviral particles were packaged and injected through tail vein to ovx mice (2×10⁷ TU/mouse), respectively. Bone mineral density (BMD) was detected 2 months after the surgery, and the proximal tibia was scanned in three dimensions by μCT. For cellular experiments, GST-PFMG1 protein was expressed and purified, then added to MC3T3E1 cell culture medium. MTT, ALP activity and the level of matrix mineralization were detected after the treatment. RESULTS Ectopic expression of pfmg1 gene enhanced the BMD level of ovx mice. μCT images revealed that PFMG1 improvedthe osteoporotic characteristics caused by ovariectomy, including the decreases in trabecular number (Tb.N), trabecular thickness (Tb.Th), and in trabecular bone volume as a percentage of total bone volume (BV/TV); and the increases in trabecular spacing (Tb.Sp) and trabecular bone pattern factor(TBPf). The alkaline phosphatase (ALP) activity and the level of matrix mineralization increased, while the MTT activity decreased after treated with PFMG1 in the osteoblast cell line MC3T3E1. CONCLUSION PFMG1 from the mental of P. fucata could promote osteoblast differentiation and matrix mineralization in vitro, and couldprevent bone loss caused by ovariectomy in vivo. These findings showed the potential of PFMG1 from nacre as a therapeutic drug for osteoporosis.

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Ca²⁺ influx and ROS generation trigger CDDO-Me-induced dilation of the ER and apoptosis in breast cancer cells

In Young KIM, Soo Ah JEONG, A Reum LEE, Mi Jin YOON, Hyeseong CHO, Jong-Soo LEE, Kyeong Sook CHOI

2015, 29(S1): 26-27.

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OBJECTIVE The synthetic triterpenoid 2-cyano-3,12-dioxoolean-1,9(11)-dien-C₂₈-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we investigated the anti-cancer effect of CDDO-Me on breast cancer cells and its underlying mechanisms. METHODS To investigate the effect of CDDO-Me on various breast cancer cells, cell viability assay using calcein-AM and EthD-1 as well as MTT assay was performed. To clarify the origin of CDDO-Me-induced vacuoles, electron microscopy as well as fluorescence microscopy using YFP-ER or YFP-Mito construct was performed. To measure the changes in intracellular Ca²⁺ and ROS levels, flow cytometry using Fluo-3 and H₂DCF-DA was performed. RESULTS CDDO-Me treatment induces progressive ER-derived vacuolation and subsequent apoptosis in various breast cancer cells. CDDO-Me-induced increases in intracellular Ca²⁺ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca²⁺ levels, CDDO-Me markedly increases the generation of reactive oxygen species (ROS). Interestingly, we found that there exists a reciprocal positive-regulatory loop between Ca²⁺ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. CONCLUSION ER-derived vacuolation via Ca²⁺ influx and ROS generation is responsible for the potent anticancer effects of CDDO-Me on breast cancer cells.

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ZYZ451 Protects cardiomyocytes from hypoxia-induced apoptosis by enhancing MnSOD and STAT3 interaction

Shan-shan LUO, Ya-qi SHEN, Li-ming MA, Xian-feng GU, Yi-zhun ZHU

2015, 29(S1): 27-28.

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OBJECTIVE 3,5-Dimethoxy-4-(2-amino-3-prop-2-ynylsulfanyl-propionyl)-benzoic acid 4-guanidino-butyl ester (ZYZ451) showed excellent cardio-protective effects in our previous work. However, its therapeutic potential in vivo and the mechanism remained to be elucidated. Herein, we evaluated cardiac protective role of ZYZ451 in post-myocardial infarction (post-MI) rats, and elucidated the underlying mechanism. METHODS Neonatal rat ventricular cardiomyoctys (NRVCs) were separated and subjected to pre-deoxidized (1%O₂, 5%CO₂), and serum-free medium for 4 h to obtain ischemic model. Mitochondrial ROS, MnSOD activity and cell apoptosis were tested to verify the cardiac protective effects of ZYZ451. Inhibitors and siRNA for Stat3 were used to determine role of Stat3 played in cardio-protective effectes of ZYZ451. Mitochondria were isolated from NRVCs to determine expression of Stat3 and MnSOD. Immunofluorescence and coimmunoprecipitation were conducted to determine the interaction between MnSOD and Stat3. To apply post-MI model in rats, the rats were subjected to ligation of LAD except for control group. The vehicle or ZYZ451 (1, 2 or 5 mg·kg^-1) or mixture of Leonurine and SPRC (15 mg·kg^-1) was administered 7 d before and 3 more days after the operation. Area at risk (AAR), apoptosis in AAR, LDH and MDA levels, and MnSOD activity and expression were detected to evaluate the cardiac injury. Tissue mitochondria were isolated to determine MnSOD and Stat3 expression in ischemia, and coimmunoprecipitaion was performed to verify the interaction between MnSOD and Stat3 in vivo. RESULTS ZYZ451 prevented hypoxia induced NRVCs apoptosis via increasing MnSOD activity and inhibiting mitochondrial ROS production. Interestingly, 5,15-DPP (STAT3 phosphorylation inhibitor) failed to inhibit MnSOD activity, while knockout of STAT3 resulted in significant reduction of MnSOD activity, followed by increased mitochondrial ROS production and cardiomyocytes apoptosis in hypoxia. Moreover, protective effects of ZYZ451 were blunted in Stat3 deficient NRVCs. These results indicated the necessity of Stat3 on MnSOD activity independent of its transcriptional activity. We further found co-localization of STAT3 and MnSOD by immunofluorescence in NRVCs, coimmunoprecipitation verified their interaction, and ZYZ451 enhanced this interaction. Similar results were found on H9C2 cardiomyoctyes and knockout of STAT3 attenuated the interaction. Consistent with the in vitro results, ZYZ451 reduced myocardial infarct size, cell apoptosis, LDH and MDA content in myocardial infarction rats. The benefits relied on increased MnSOD activity and enhanced STAT3 and MnSOD interaction, as observed in dangerous area of the infracted hearts. CONCLUSION We are the first to report that STAT3 is involved in MnSOD activity regulation, and that ZYZ451 exerts its cardio-protective effects by enhancing MnSOD and STAT3 interaction. These findings indicate a new role for STAT3 beyond as a transcriptional factor and suggest that ZYZ451 is an effective cardioprotective agent.

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Allergenicity studies of Naoluotai injection and H3 injection before and after ultrafiltration

Fang WANG, Cun-yu LI, Hong-yang LI, Guo-ping PENG, Yun-feng ZHENG

2015, 29(S1): 27-27.

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OBJECTIVE To explore the influence of ultrafiltration on the allergenicity of Naoluotai injection and H3 injectable powder in guinea pig. METHODS The ultra filtrated and non-ultra filtrated Naoluotai injection was utilized to do the sensitization test of guinea pig. The guinea pig was sensitized three times by subcutaneous injection of test solution every other day. After 16 d of the first sensitization. All guinea pigs were challenged.The serotonin level in guinea pig plasma before and after the challenge was recognized as the evaluation criteria of allergenicity. RESULTS The increasing rate of serotonin in guinea pigs sensitized by ultra filtrated Naoluotai injection was 5.80% while by non-ultra filtrated Naoluotai injection was 37.03%. The increasing rate in ultra filtrated and non-ultra filtrated H3 injection was 23.59% and 61.94%, respectively. CONCLUSION The animal protein in tradition Chinese medicine injection was thought to be allergized. The ultrafiltration technology could reduce the allergenicity of tradition Chinese medicine injection and improve its safety.

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心血管药理学

Components of traditional Chinese medicine inhibit platelet aggregation by blocking ADP receptor subtypes P2Y₁ and P2Y₁₂

Xiao-liang WANG, Yi-ying LI, Hong-yan YANG, Shaofeng XU, Ling WANG

2015, 29(S1): 28-29.

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Platelets aggregation and thrombosis formation are major reasons of cardiovascular and cerebral vascular diseases. To develop new generative, potent and safe agents for inhibiting platelet aggregation and preventing above diseases are urgently required. Some traditional Chinese medicines of "Houxue Huayu" have been shown to inhibit platelet aggregation potently. In the present study the mechanisms and the molecular targets of puerarin, salvianolic acid B and the analogue of 3-n-butylphthalide, dl-PHPB were investigated and compared with ticlopidine. Four platelet aggregation inducers, ADP, arachidonic acid, collagen and thrombin were used in the study. It was found that puerarin and dl-PHPB specifically inhibited ADP induced platelet aggregation like ticlopidine did. However, salvianolic acid B inhibited both ADP and collagen induced platelet aggregations with similar potency. Due to existing two ADP receptor subtypes on platelets, P2Y₁ and P2Y₁₂, we studied the action of above compounds on the receptors and the signaling pathways. It was found that dl-PHPB decreased IP₁ accumulation produced by ADP, but had no effect on IP₁ level induced by m-3M3FBS, an activator of PLC. M-3M3FBS might attenuate the inhibitory effect of dl-PHPB on ADP-induced platelet aggregation. In addition, dl-PHPB did not affect cyclic AMP formation in platelets by ADP, which is different from P2Y₁₂ antagonist ticlopidine. Puerarin showed the similar effects of dl-PHPB. Therefore, the actions of dl-PHPB and puerarin might be through P2Y₁ receptor-PLC-β pathway. Salvianolic acid B did not reduce the IP₁ accumulation stimulated by ADP. It might act on the receptor subtype P2Y₁₂. Our results suggest that components of Chinese herb medicine might be a resource for development of novel anti-platelet drugs.

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心血管药理学

Sesamol inhibits atherogenic LDL-induced endothelial cell senescence in vivo and in vitro

Hong-kai HUANG, Fang-yu CHEN, Kuan-hsiang TING, Chih-chung FENG, Chia-ming CHANG, Chu-huang CHEN, Ming-yi SHEN

2015, 29(S1): 29-29.

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OBJECTIVE Highly electronegative L5 low-density lipoprotein (LDL), an atherogenic LDL, induces endothelial cell (EC) senescence and has been implicated in the progression of atherosclerosis. We examine whether sesamol, a natural organic compound and component of sesame oil, prevents EC senescence induced by electronegative LDL (L5) and to investigate the underlying mechanisms. METHODS Syrian hamsters, which have a LDL profile similar to that of humans, were fed a normal chow diet (control), a high-fat diet (HFD), or a HFD supplemented with the administration of 50 or 100 mg·kg^-1 sesamol via oral gavage (HFD+sesamol) for 16 weeks (n=10 per group). Among these groups, we compared plasma L5 levels and aortic endothelial senescence in the aortic arch. In vitro, we examined the effects of sesamol on human aortic endothelial cell (HAEC) senescence and signaling pathways induced by L5. RESULTS Hamsters in the HFD group had higher plasma L5 levels than did the HFD+sesamol groups or control group. Beta-galactosidase (gal) staining showed that aortic endothelial senescence was markedly increased in the aortic arch of the HFD group but not in that of the HFD+sesamol groups when compared with the control group. In vitro, treatment of HAECs with sesamol (1-3 mol·L^-1) blocked L5-induced EC senescence in a dose-dependent manner. Sesamol also markedly inhibited the L5-induced phosphorylation of p38 MAPK and p53 activation and increased Mdm2 and phosphorylation of Akt. CONCLUSION The critical findings of this study suggest that sesamol may provide protection against atherosclerosis and the development of cardiovascular disease in humans.

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心血管药理学

Improvement of lipid profile and atherogenic index in rat treated with Cymbopogon citratus Stapf water extract

Nuntiya SOMPARN, Suphaket SAENTHAWEEUK, Jarinyaporn NAOWABOOT, Atcharaporn THAEOMOR

2015, 29(S1): 30-30.

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OBJECTIVE To investigate the effects of Cymbopogon citratus Stapf water extract (CCS) on lipid profile, atherogenic index and antioxidant enzymes in Sprague-Dawley rats. METHODSRats aged eight weeks (250±20)g were orally administered with CCS at 250 and 500 mg·kg^-1·d^-1 for one month. RESULTS Administration of CCS extract to rats caused significant decrease of serum total cholesterol, triglycerides and LDL levels, whereas, the HDL level was increased compared with untreated rats (P<0.05). Moreover, the CCS extract showed a significant decreased atherogenic index in comparison with untreated rats (P<0.05). Furthermore, serum activities of superoxide dismutases and catalase were also improved in rats treated with CCS. This was consistent with decrease of serum thiobarbituric acid reactive substance. CONCLUSION The present study provides evidence that CCS water extract provides a benefit effect on serum lipid and atherogenic index and exhibits antioxidant effect in vivo.

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心血管药理学

Effect of Cymbopogon citratus Stapf extract on lipid profile and antioxidant status in streptozotocin-induced diabetic rats

Atcharaporn THAEOMOR, Suphaket SAENTHAWEEUK, Nuntiya SOMPARN

2015, 29(S1): 30-30.

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OBJECTIVE To investigate the effects of Cymbopogon citratus Stapf water extract on lipid profile and antioxidant status in streptozotocin-induced diabetic rats. METHODS Diabetes was induced by injection of streptozotocin (STZ, 50 mg·kg^-1) intraperitoneally. Diabetic rats were divided into 5 groups, consisting of 6 rats. GroupⅠ, reserved as diabetic control, was administered distilled water and groupⅡ, reserved as positive control, was administered glibenclamide (10 mg·kg^-1·d^-1) throughout the duration of the experiment. Those in groupⅢ, Ⅳ and Ⅴ were administered 250, 500 and 1000 mg·kg^-1·d^-1 of the extract, respectively for 28 d. RESULTS Treatment with 500 and 1000 mg·kg^-1·d^-1 of the extract resulted in reduction of serum AST, ALT, serum cholesterol, triglycerides and LDL, whereas HDL was found to be increased compared with diabetic control rats (P<0.05). Moreover, increased serum activities of superoxide dismutases and catalase were found in diabetic rats treated with the extract whereas serum thiobarbituric acid reactive substance was decreased, in comparison with diabetic control rat (P<0.05). CONCLUSION Cymbopogon citratus Stapf water extract provides a benefit effect on serum lipid and antioxidant effect in diabetic rats. Thus, the extract may lower cardiovascular disease risk and others complications related to hyperlipidemia and oxidative stress in diabetic patients.

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心血管药理学

A comprehensive cardiotoxicity assay by combining high-throughput multiple ion channel screening with human cardiomyocyte-based action potential validation

He-ming WEI, Jian-jun WU, Ying Ying CHUNG, Cai Hong KOH, Zhen-feng LIU, Stuart COOK, Winston SHIM, Tan Mei LAN

2015, 29(S1): 31-32.

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OBJECTIVE Cardiotoxicity refers to drug-induced arrhythmia such as Torsades de pointes. Current single ion channel (hERG)-based assay generates -30% false results. The aim is to establish an advanced in vitro cardiotoxicity assay by incorporating high throughput multiple cardiac ion channel screening and human cardiomyocytes-based validation. METHODS Effects of drugs were tested on multiple cell lines expressing hERG, Nav1.5 and Cav1.2 by automated patch clamping. Subsequently, the results were validated with human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) in which ion currents and action potentials were measured by manual patch clamping. RESULTS We have tested the cardiotoxicity of monomers extracted from various medical herbs. Mitragynine is the major bioactive compound isolated from kratom, a therapeutic herb from the rain forest of South East Asia. As a popular stimulant, it has been associated with a number of acute fatal incidences. We observed a typical torsadogenic hazard of mitragynine. It exerted a strong hERG inhibition in hERG-HEK293 cell line (IC₅₀: 5.2 μmol·L^-1) and hPSC-CMs (IC₅₀: 0.91 μmol·L^-1) without affecting other cardiac ion channels. Moreover, it caused a significant prolongation of action potential duration (APD) in hPSC-CMs (a -32.5% increase in APD at 50 and 90% repolarization). On the other hand, deoxylelephantopin, a potential anti-cancer reagent, demonstrated low cardiotoxicity. It exerted a week inhibition on hERG in HEK293 cells with an IC₅₀ of 87.2 μmol·L^-1, while the effective concentrations for suppressing the growth of cancer cells ranges from 2 to 20 μmol·L^-1. At 100 μmol·L^-1, deoxylelephantopin showed no effects on Cav1.2 and Nav1.5 and it failed to alter APD in hPSC-CMs. CONCLUSIONWe have successfully tested a new in vitro cardiotoxicity assay strategy which incorporates multiple cardiac ion channels screening and hPSC-CMs validation. This new strategy could facilitate the effective and efficient evaluation of existing and new drugs/reagents for potential pro-arrhythmic risk.

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心血管药理学

Protective effect of salvianolic acid A against isoproterenol-induced myocardial infarction in mice

Lian-hua FANG, Zi-ran NIU, Tian-yi YUAN, Li Li, Yue-hua Wang, Guan-hua DU

2015, 29(S1): 31-31.

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OBJECTIVE To investigate the protective effect of salvianolic acid A (Sal A) on isoproterenol-induced myocardial infarction in mice and its possible mechanisms. METHODS The mice were subcutaneously injected with isopropranol (ISO 8 mg·kg^-1) to induce myocardial infarction and evaluated the myocardial protective effect of Sal A from mortality rate, electrocardiogram (ECG), heart function, myocardial infarction index, serum myocardial enzymes and explored its possible mechanisms from inflammatory, antioxidant and cells apoptosis. RESULTS Sal A can dose-dependently enhanced the heart function of myocardial infarction mice, reduced the heart index, inhibited the myocardial enzyme leakage, showed obvious myocardial protection effects. ELISA results showed that Sal A can reduce the expression of myocardial inflammatory cytokines such as IL-6, TNF-α. Western blotting confirmed that Sal A can increase the expression of anti-apoptotic proteins Bcl-2, reduce the expression of apoptosis protein Bax, and raise the phosphorylation level of PI₃K and Akt. CONCLUSION Sal A have displayed significant protective effect against isoproterenol-induced myocardial infarction and its mechanism may be related to increasing of PI₃K/Akt signal pathway and inhibition of cell apoptosis and inflammatory reaction.

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心血管药理学

Protective effects of scutellarin on vascular dysfunction caused by hypertension in SHR rats

Qing-qing LI, Zhi-ying WENG, Min ZHANG, Chen CHEN, Xiao-hua DU, Pei-hua PENG, Wen-juan WU, Jun ZHANG, Wei-min YANG

2015, 29(S1): 32-32.

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OBJECTIVE To investigate the effect of scutellarin (SCU), which is the main effective component of Erigeron breviscapus (Vant.) Hand-Mazz native to Yunnan in China, on vascular dysfunction (VD) of cardiac coronary artery(CA) and cerebral basilar artery (BA) caused by hypertension in SHR rats. METHODS 1. BA and CA vesselsrings from 40 weeks of SHR rats were isolated and equilibrated in organ bath with MOPS-PSS buffer and ring tension was recorded, comparing with a normal control of WKY rats.SCU was treated accumulatively after pre-contracted with vasoconstrictor U46619(1 μmol·L^-1). 2. ACH and SNP were treated accumulatively after pre-incubation with SCU (300 μmol·L^-1, 100 μmol·L^-1) and pre-contracted by U46619(1 μmol·L^-1), K⁺ 60 mmol·L^-1, respectively. While U46619 was added accumulatively to BA/CA rings pre-incubated with SCU (300 and 100 μmol·L^-1). ACH-induced relaxation rate was to evaluate endothelium-dependent relaxation, and SNP-induced relaxation rate to evaluate the artery non-endothelium-dependent relaxation. RESULTS SCU significantly dilated both BA and CA rings pre-contracted by U46619 in old rats. EC₅₀ value of SCU in WKY ratswas less than that in SHR (P<0.05), which showing VD of CA and BA in SHR rats. Compared with WKY group, ACH relaxation curve of SHR group shifted to the right, suggesting that hypertension induced VD. When SCU 300 μmol·L^-1 pre-treated CA groups and SCU 100 μmol·L^-1 pre-treated BA groups, EC₅₀ to ACH was significantly lower(P<0.05). Likewise, the vasodilatation of CA/BA rings to SNP was also improved obviously when pre-treated with SCU, and Emax to SNP was decreased significantly(P<0.05). Moreover, EC₅₀ to U46619 was significantly lower when pre-treated by SCU. CONCLUSION In SHR rats, SCU antagonized U46619 on CA/BA rings in a noncompetitive manner. Furthermore, SCU should appear to protect VD induced by hypertension.

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心血管药理学

Effect of ethanolic extract of Physalis minima on lowering blood pressure in DOCA-salts hypertensive rat models

Nur PERMATASARI, Dian NUGRAHENNY, Saifurrohman

2015, 29(S1): 33-33.

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OBJECTIVE To explore the effect of ethanolic extract of Physalis minima on lowering blood pressure in the DOCA-salts hypertensive rats. METHODS To produce hypertension, male rats were induced DOCA at dose of 10 mg·kg^-1 ip. twice weekly and 0.9% NaCl in drinking water ad libitum for six weeks. The ethanolic extract of Physalis minima were given at dose of 500, 1500 and 2500 mg·kg^-1, respectively for 4 weeks. After completion of treatment schedule, systolic blood pressure was evaluated using animal blood pressure analyzer. The serum nitric oxide levels was measured using the ELISA methods. SOD activity and MDA level were determined spectrophotometrically. Endothelial dysfunction was determined by acetylcholine-induced vasorelaxation studies on isolated rat aortas. RESULTS The extract decreased systolic blood pressure significantly (P<0.001) in hypertensive rats. In addition, the extract also suppressed the production of MDA and restored SOD activity to normal level. There was a reduced relaxation response to acetylcholine in isolated aortic rings and serum NO level from hypertensive rats that were reversed by extract. CONCLUSION Physalis minima can lower blood pressure in hypertensive rats and its mechanism may be through the improvement of endothelial function due to the decrease of stress oxidative.

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心血管药理学

Effects of scutellarin on PKG in HCMECs after hypoxia roxygenation

Yang LI, Zhi-ying WENG, Min ZHANG, Chen CHEN, Xiao-hua DU, Pei-hua PENG, Wen-juan WU, Xuan LIU, Wei-min YANG

2015, 29(S1): 33-34.

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OBJECTIVE This study discusses the effects of scutellarin(SCU), which is one of effective component of Erigeron breviscapus (Vant.) Hand-Mazz, on cGMP dependent protein kinase(PKG) in human cardiac microvascular endothelial cells (HCMECs) after hypoxia roxygenation(HR). METHODS Based on a model of HCMECs cells with HR injury, cell viability is examined by MTT assay. Protein expression of PKG is analyzed by Western blotting and its enzyme activity is investigated by ELISA technology. RESULTS The results of MTT assay indicate that SCU(1, 10 μmol·L^-1)could protect HCMECs against HR injury. SCU (0.1, 1 and 10 μmol·L^-1) canenhance PKG-I expression in control and HR injury cells. Furthermore, SCU(1, 10 μmol·L^-1 significantly increase PKG activity in control cells(P<0.01), and also SCU(100 μmol·L^-1) appears similar on enzyme activity in HR injury cells(P<0.05). CONCLUSION SCU appears protective effects on endothelial cells against HR damage. While its mechanisms may be related to the influence of SCU on PKG activity in HCMECs.

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心血管药理学

Novel derivative of Danshensu acts as anti-thrombotic agent for modulation of protein disulfide isomerase family member ERp57

Guo-zhen CUI, Lu-chen SHAN, Lin GUO, Ivan Keung CHU, Guo-hui LI, Quan QUAN, Yun ZHAO, Chenong-meng CHONG, Zaijun ZHANG, Pei YU, Pui-man HOI, Ye-wei SUN, Yu-qiang WANG, Simon ming-yuen LEE

2015, 29(S1): 34-34.

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OBJECTIVE To identify the specific targets of a novel derivative of Danshensu ADTM as the protein disulfide isomerase (PDI) family proteins including ERp57. To further investigate the underlying mechanism of ADTM to modulate ERp57 to regulate platelet function with direct interaction with αⅡbβ3 integrin. METHODS To isolate the protein targets that bound to ADTM, a biotin-conjugated ADTM analogue (BAA) was designed and synthesized. BAA (300 μmol·L^-1) was incubated with rat blood platelet lysates and the BAA-protein complexes were pulled down with NeutrAvidin-agarose followed by protein profiling using LC-MS/MS. To determine platelet aggregation in vitro, rabbit platelets were incubated with the indicated concentrations of compounds and aggregation was induced by ADP (10 μmol·L^-1) or AA (200 μmol·L^-1) and measured using a platelet aggregometer. To determine platelet aggregation-induced by ADP in rat in vivo, ADTM (5-20 mg·kg^-1) in comparison with DSS (10 mg·kg^-1) and clopidogrel (18 mg·kg^-1) were administered daily by i.v. injection for 5 d, respectively. To determine the action of ADTM on the ERp57/αⅡbβ3 interaction, it was examined by immunoprecipitation with anti-αⅡbβ3 antibody, followed by detection of ERp57 immunoreactivity using immunoblotting. RESULTSBAA could bind to various proteins involved in platelet function. In particular, platelet aggregation-associated proteins were identified with >95% protein identification probability including ERp72, ERp57 ERp5 and PDI, which are members of the protein disulfide isomerase (PDI) family related to platelet function and redox homeostasis. ADTM exhibited potent inhibition on the redox activity of ERp57 in a concentration-dependent manner (IC₅₀=100 300 μmol·L^-1). In in vitro studies, ADTM exhibited concentration-dependent inhibition on ADP-induced and AA-induced platelet aggregation with comparable effects to aspirin and clopidogrel. In vivo study showed that ADP-induced platelet aggregation was significantly compromised (>40% reduction) in rats treated with ADTM (20 mg·kg^-1). Similarly, ADTM also exhibited significant anti-thrombotic effect in vivo as shown in the ferric chloride (FeCl₃)-induced venous thrombosis. Immunoprecipitation with anti-αⅡbβ3 antibody, followed by detection of ERp57 immunoreactivity using immunoblotting showed that ADTM disrupted the interaction of ERp57 with αⅡbβ3. CONCLUSIONThese results demonstrated that ADTM exhibited broad-spectrum anti-platelet activities and ERp57 is a potential therapeutic target for anti-platelet therapy.

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心血管药理学

Pinocembrin inhibits angiotensin Ⅱ-induced vasoconstriction in a Ca²⁺-dependent and Ca²⁺-independent manner through blocking AT₁R in the rat aorta

Li LI, Hai-guang YANG, Xiao-bin PANG, Bai-nian CHEN, Li GAO, Le WANG, Shou-bao WANG, Tian-yi YUAN, Su-bo WANG, De-pei LIU, Guan-hua DU

2015, 29(S1): 35-35.

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OBJECTIVE To investigate the vasorelaxant effect of pinocembrin (5,7-dihydroxyflavanone), one of the main flavonoids in propolis, on angiotensinⅡ(AngⅡ) induced vasoconstriction and the molecular mechanism of action. METHODS The isometric vascular tone was measured in thoracic aortic rings from SD rat, and the effects of pinocembrin on the single dose and concentration cumulative response curves of AngⅡ were recorded. The binding of pinocembrin to the angiotensin type 1 receptor (AT₁R) was studied by using molecule docking analysis. Intracellular ([Ca²⁺]([Ca²⁺]_i) was measured with Fura2/AM in VSMCs. The phosphorylation levels of myosin light chain 2 (MLC2) and myosin phosphatase target unit 1 (MYPT1), and protein level of Rho kinase 1(ROCK1) in the rat aortic rings were detected by Western blotting. RESULTS Pinocembrin was observed to inhibit AngⅡ-induced vasoconstriction in rat aortic rings with either intact or denuded endothelium. In endothelium-denuded tissues, pinocembrin (pD'2 4.28±0.15) counteracted the contractions evoked by cumulative concentrations of AngⅡ. In a docking model, pinocembrin showed effective binding at the active site of AT1R. Pinocembrin was shown to inhibit both AngⅡ-induced Ca²⁺ release from internal stores and Ca²⁺ influx. Moreover, the increase in the phosphorylation of MLC2 and MYPT1, and the increased protein level of ROCK1 induced by AngⅡ was blocked by pinocembrin. CONCLUSION Pinocembrin inhibits AngⅡ-induced rat aortic ring contraction in a Ca²⁺-dependent and Ca²⁺-independent manner via blocking AT₁R.

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心血管药理学

Cardioprotective and chemosensitizing effects of novel Danshensu derivatives

Liang WANG, Lu-chen SHAN, Guo-zhen CUI, Judy Yuet-Wa CHAN, Jing-jing LI, Qing-wen ZHANG, Maggie Pui-Man HOI, Yu-qiang WANG, Simon Ming-Yuen LEE

2015, 29(S1): 35-36.

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OBJECTIVE To investigate the cardioprotective effect of novel danshensu derivatives against doxorubicin (Dox) cardiotoxicity and their synergistic anti-tumor effect with Dox on breast cancer cells. METHODS Two new Danshensu derivatives were synthesized by conjugation with tetramethylpyrizine and/or 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid and tested for protective effects against Dox induced cardiotoxicity in cell and zebrafish. H9c2 cardiomyoblasts were co-treated with Dox and Danshensu derivatives for 24 h and then were measured for cell viability and cytotoxicity by MTT and LDH assays. The expression levels of mitochondrial biogenesis related proteins PGC-1α, NRF-1 and Nrf2 were detected by Western blotting and qPCR. Moreover, in a Dox-induced cardiotoxicity model of zebrafish, zebrafish embryos were treated with Dox for 36 h, followed by measurement of numerous ventricular function parameters including heart rate, stroke volume, cardiac output and fractional shortening. In addition, the synergistic anti-tumor effects of the Danshensu derivatives and Dox had been studied in MCF-7 breast cancer cells. The effects of the Danshensu derivatives on the cell death and metabolism of MCF-7 cells were measured using apoptosis assay and Seahorse Metabolic Analyzers respectively. RESULTS Our results showed that the Danshensu derivatives were more potent than the parental compounds in ameliorating Dox-induced cytotoxicity in H9c2 cells and significantly preserving stroke volume of heart function in Dox-treated zebrafish. Further mechanistic studies identified that the danshensu derivatives increased mitochondrial copy numbers and protein expressions of PGC-1α, NRF-1 and Nrf2 in H9c2 cells. In addition, the Danshensu derivatives enhanced Dox-induced apoptosis, and decreased glycolysis and mitochondrial function in MCF-7 tumor cells. CONCLUSION Our results revealed that two new Danshensu derivatives displayed promising cardioprotective effects against Dox induced cardiotoxicity both in vivo and in vitro, at least partially through activating mitochondrial biogenesis. Also, the new Danshensu derivatives potentiated the anticancer effects of Dox in breast tumor cells involving induction of glycolytic inhibition and mitochondrial dysfunction.

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心血管药理学

Antithrombotic effect of a novel tissue plasminogen activator from the venom of Gloydius brevicaudus viper

Yun-lu XU, Li-li LIN, Jin-hua ZHANG, Jian-ji CHEN, Zhi-qiang WU, Guo-tu WU, Jian-zhong LIN

2015, 29(S1): 36-36.

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OBJECTIVE To investigate the thrombolytic and antiplatelet effects of a novel plasminogen activator from the venom of Gloydius brevicaudus viper (GBV-PA) in vitro and in vivo. METHODS Thrombolytic experiments were performed in rabbit models of ear vein thrombosis and carotid artery thrombosis, and in dog model of acute cerebral infarction. Inhibition of thrombus formation was evaluated in rat inferior vena cava thrombosis model and ferric chloride-induced arterial thrombosis. In vitro, we assayed the antithrombotic effect of GBV-PA on rabbit blood clots, euglobulin lysis time (ELT) of rabbit plasma, and on ADP-induced platelet aggregation. RESULTS GBV-PA intravenous administ ration significantly reduced vascular recanalization times of rabbit ear veins thrombosis and thrombus weight of rabbit carotid artery thrombosis. The arterial recanalization rates were dose- and time-dependently improved after administration of GBV-PA in canine acute cerebral infarction model. Thrombus length and weight was significantly reduced by GBV-PA both in rat inferior vena cava and ferric chloride-induced arterial thrombosis models. Thrombus formation of blood of rabbits they were administrated with GBV-PA was also inhibited. GBV-PA radically reduced plasma ELT of rabbit's blood clots. ADP-induced platelet aggregation was inhibited by GBV-PA in a dose-dependent manner with a half-maximal inhibitory concentration of 19.9 μg·mL^-1. CONCLUSION This study demonstrates that GBV-PA is a thrombolytic and antiplatelet agent. It has significant antithrombotic effects on various in vitro and in vivo experimental models of thrombosis. The mechanisms that underline its antithrombotic effects were related to GBV-PA's capabilities of increasing fibrinolytic activities and inhibition of platelet aggregation.

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心血管药理学

Effect of Scurrula atropurpurea on blood pressure and angiotensin Ⅱ of deoxycorticosterone acetate-salt hypertensive rats

Setyawati SOEHARTO, Nour ATHIROH

2015, 29(S1): 37-37.

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OBJECTIVE To investigate the effect of methanolic extract of Scurrula atropurpurea on systolic blood pressure and serum angiotensinⅡ concentration in deoxycorticosterone acetate-salt (DOCA-salt) hypertensive rats. METHODS Twenty-five male Wistar rats were divided into normal control group and four hypertensive groups by DOCA-salt for 6 weeks. Further, three hypertensive groups were orally administered with methanolic extract of Scurrula atropurpurea at a doses of 50, 100 and 200 mg·kg^-1, respectively for 6 weeks and one hypertensive group without extract as positive control. At the end of research systolic blood pressure were measured by tail cuff methods. Serum angiotensinⅡ were measured by ELISA. RESULTS The systolic blood pressure increased significantly in the positive control hypertensive group compared to the normal control group (P<0.05). Administration of methanolic extract of Scurrula atropurpurea 50, 100 and 200 mg·kg^-1 significantly decreased systolic blood pressure, respectively compared to the positive control hypertensive group (P<0.05). There was no different angiotensinⅡ consentration between positive control hypertensive group and normal group, while methanolic extract of Scurrula atropurpurea 50 mg·kg^-1 caused decreased angiotensinⅡ conscentration significantly compared to positive control hypertensive group (P<0.05). CONCLUSION Methanolic extract of Scurrula atropurpurea produced antihypertensive effect and decreasing angiotensinⅡ concentration at 50 mg·kg^-1 in hypertensive rat. Beside this, the concentration of angiotensinⅡ couldn't increase in DOCA-salt hypertensive rats.

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心血管药理学

Composition analysis and hpyerglycemic activity of the fruit of Phyllanthus emblica L.

Chun-peng WAN

2015, 29(S1): 37-38.

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OBJECTIVE To investigate the hpyerglycemic activity and elucidate the active composition of the fruit of Phyllanthus emblica L., an edible and medicinal plant used for traditional Chinese medicine and Tibetan medicine to cure the blood disease, bacon disease, liver disease, heart disease and hypertension. METHODS The LO2 normal liver cell lines were used to investigate the hpyerglycemic activity, The LO2 cells were treated with 100, 200 and 400 μg·mL^-1 extracts of P. emblica L.; real-Time PCR was used to detect the expression of glucose transporter type 2 (GLUT-2) and peroxisome proliferator-activated receptor-γ (PPARγ) mRNA. The PPRE (peroxisome proliferator response element) and NF-κB activities were detected by luciferase method. The chemical compositions were analyzed by HPLC-MS. RESULTS The expression of GLUT-2 and PPARγ mRNA were significantly increased treated with 400 μg·mL^-1 extracts of P. emblica L. (P<0.05) and in a dose-dependant manner; PPRE activity was significantly increased in a dose-dependant manner (P<0.05); however, NF-κB activity induced by LPS was inhibited also in a dose-dependant manner (P<0.05). The chemical composition analyzed by HPLC-MS showed that the gallic acid was the major component in P. emblica L. CONCLUSION P. emblica L. showed promising hpyerglycemic activity and the gallic acid was the main constituent.

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心血管药理学

Sesamin enhances nitric oxide bioactivity in aortas of spontaneously hypertensive rats

Jie-ren YANG, Xiang KONG, Wei LI

2015, 29(S1): 38-38.

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OBJECTIVE To explore the underlying mechanisms involved in the effect of sesamin on aortic NO bioactivity in spontaneously hypertensive rat (SHR). METHODS Sesamin was orally administered for consecutive 8 weeks in SHR. Systolic blood pressure (SBP) was measured using the tail-cuff method. The aortas were isolated and in vitro vascular reactivity studies were performed. Superoxide anion production in carotid arteries was assessed by dihydroethidium fluorescence staining. The protein expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (P-eNOS), dihydrofolate reductase (DHFR), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47phox and copper, zinc-superoxide dismutase (Cu/Zn-SOD) in aortas was detected by Western blotting. The dimeric form of eNOS in aortas was determined by low-temperature SDS-PAGE. Aortic level of nitrotyrosine and activities of antioxidant enzymes, namely, total SOD (T-SOD), glutathione peroxidase (GPx) and catalase were also detected. RESULTS In SHR, sesamin treatment reduced SBP, improved vascular relaxation induced by acetylcholine and enhanced aortic NO bioactivity. Sesamin treatment enhanced NO biosynthesis in SHR aortas was due to upregulated P-eNOS and suppressed eNOS uncoupling, and the latter effect might be attributed to decreased nitrotyrosine and upregulated DHFR. Sesamin also reducd the NO oxidative inactivation and decreased the superoxide anion production through downregulation of p47phox and amelioration of eNOS uncoupling. In addition, sesamin treatment did not alter the levels of GPx and catalase activity but obviously reduced the compensatory elevated T-SOD activity and Cu/Zn-SOD protein expression. CONCLUSION Chronic treatment with sesamin could reduce hypertension and improve endothelial dysfunction through enhancement of NO bioactivity in SHRs aortas.

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心血管药理学

Combined antihypertensive effect of GSY and metoprolol, based on endothelium-dependent vasodilatation function

Bo LI, Jie SU, Shan-shan LEI, Zheng-biao YANG, Ze-wu JIN, Hui-ming HU, En-wei ZHU, Gui-yuan LYU, Su-hong CHEN

2015, 29(S1): 38-39.

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OBJECTIVE To investigate the synergistic effect on dilating blood vessels and anti-hypertension of GYS combined with metoprolol. METHODS ① Spontaneously hypertensive rats(SHR) were administered orally with the vehicle, GSY, metoprolol or GSY combined with metoprolol for 4 weeks. Blood pressure, which included SBP, DBP and MBP was measured by a noninvasive method every week. At the end of 4 weeks, blood was drawn from the ophthalmic venous plexus to determine blood fat levels(serum TC, TG, LDL-c, HDL-c), liver function(serum ALT, AST), and kidney function(serum BUN, UA and Cr) by the ACCUTE(TBA-40FR) automatic. ② The aortae of normal SD rats were prepared and cleaned from periadventitial fat and surrounding connective tissue and cut transversely into 4-mm width rings. To observe different concentration of GYS, metoprolol or GSY combined with metoprolol causing relaxation of the isolated aortic rings precontracted until a stable plateau by noradrenaline(NA) directly or in the presence of eNOS inhibitor L-NAME and cyclooxygenase inhibitor indomethacin(INDO) respectively. ③ The concentrations of plasma GSY was determined by the HPLC after rats administered orally with GSY or GSY combined with metoprolol for single-dose. DAS data processing software calculated the pharmacokinetic parameters of GSY. RESULTS There was a significant synergism between GYS and metoprolol in lowering blood pressure and the concentrations of serum TC and LDL-c of SHR. The relaxant effect of GYS combined with metoprolol on the aortic rings precontracted by NA could be attenuated by L-NAME or INDO. The AUC_0-t of GSY significantly increased after in conjunction with metoprolol. CONCLUSION GYS combined with metoprolol increases the concentrations of plasma GSY and synergistically lowers blood pressure based on endothelium-dependent vasodilatation function(EDVF).

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心血管药理学

Study on establishing hypertension animal models by adverse lifestyles and the antihypertensive effect of traditional Chinese medicine

Gui-yuan LYU, Bo LI, Su Jie, Na SHI, Chao-qun XIA, Qi CHEN, Su-hong CHEN

2015, 29(S1): 39-40.

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OBJECTIVE Nowadays, with the living habits and diet structure changes, the morbidity and mortality of hypertension has been rising yearly, which induced by adverse lifestyles, excessive alcohol and poor diet. However, the animal models of hypertension resulted from adverse lifestyles have rarely been reported. It is essential to set up some more systematic models to study the characters of hypertension and evaluate the efficacy of traditional Chinese medicine(TCM). METHODS To build the rat model of human life-habitual hypertension,SD rats had free access to alcohol with high-sucrose-fat diet (AHSFD), alcohol with high-fat diet (AHFD), high-sucrose-fat diet (HSFD), high-fat diet (HFD) and alcohol etc. Various of indicators were detected systematically to explore the features of hypertension models which included blood pressure (BP), liver and kidney functions, the lipid profiles and indexes quantifying TCM symptoms. Then we choose one of the stable and sustained animal models, studying the effects of RPA.E(which extracted from a TCM) on modulating the level of hypertension and the preliminary mechanism in this abstract. RESULTS ① The BP level, serum TC, TG, LDL-C of AHSFD-induced and AHFD-induced rats increased significantly, while the HDL-C reduced in the 4th week. ② The BP level HSFD-induced rats increased significantly in the 6th week, serum TC, TG increased in 10th week. ③ The BP level of alcohol-induced rats increased significantly in the 9th week, serum TC, TG increased in 24th week. ④ The level of serum ALT, AST, UA and Cr of all model rats increased significantly after 12 weeks. Meanwhile the microcirculation increased significantly after 8 weeks. ⑤ After 4-week administration, RPA.E could significantly reduce BP of AHFD-induced hypertensive rats, and could reduce the serum TC, LDL-C, HDL-C, ALT, AST level after 8-week. CONCLUSION The rats induced by AHSFD, AHFD, HSFD and alcohol can raise BP obviously with underlying hepatic and kidney damage, the lipid profiles disorder and TCM symptoms change, while the AH(S)FD-induced hypertension models are earlier and more stable. RPA.E could mildly reduce BP level, and improve the lipid profiles disorders, hepatic damage, which reflects the characteristics of TCM on antihypertensive effects. The above models have their own characteristics, can be used to study the causes and pathogenesis of hypertension complicating metabolic disorder and the related treatment drug screening.

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神经科学

Traditional Chinese herbal medicine for vascular dementia

Dianne BAUTISTA, Ya-nan ZHU, Yong YOU, Wen-yun LI, Edwin SY CHAN, Christopher CHEN

2015, 29(S1): 40-41.

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OBJECTIVE To systematically review the clinical efficacy and safety on TCHMs that are used for vascular dementia (VaD). METHODS To identify studies for systematical review, electronic searches were performed through several databases-ALOIS, CNKI, CBM, Weipu, Wanfang, etc. Only randomized control trials (RCTs) or controlled clinical trials (CCTs) were included. Patients were diagnosed with VaD by diagnostic criteria (DSM, NINDS-AIREN, ICD or HIS) as well as imaging technique (CT, MRI or functional imaging, etc). Eligible TCHMs must be recognized in the Chinese Pharmacopeia or the National Essential Drug List of the People's Republic of China. Included studies were appraised using the Cochrane Collaboration risk-of-bias criteria. Efficacy and safety outcomes were evaluated by meta-analysis. Efficacy outcomes include cognition, daily function, global performance and behaviour; safety was assessed by the number of adverse events and number of subjects experiencing adverse events. Assessment of heterogeneity, subgroup analysis and sensitivity analysis were also performed. RESULTS A total of 46 trials on 29 TCHMs (3522 patients) were included. 45 studies were RCTs and 1 was CCT. In these 45 RCTs, only 2 were appraised as adequately randomised. 5 of 46 trials were appraised as having low risk of bias in blinding. Sample sizes were generally small ranging from 26 to 216 with a median of 68. All trials were conducted in China from 1997 to 2013. All 46 studies assessed cognition using one or a combination of the following scales: MMSE (n=40 studies; 3096 patients), HDS (n=22; 1664 patients), ADAS-Cog (n=4; 241 patients), CDT (n=1; 60 patients) and CCSE (n=1; 26 patients). Half of the studies assessed daily function using either the ADL (n=22; 1743 patients) or IADL (n=2; 203 patients). Only 6 studies measured behaviour using the FAQ (n=3; 226 subjects), BBS (n=1; 48 patients), NPI (n=1; 100 subjects) or Neurological Deficits Function Scale (n=1; 91 patients). 30 studies measured global performance. 31 of 46 studies made conclusions regarding the safety of the TCHMs. Despite the problems of methodology and reporting, we can identify three TCHMs-NaoXinTong, Shenfu and Tongxinluo as having relatively stronger evidence of efficacy. There is weak evidence for the safety of TCHMs for VaD. CONCLUSION There is weak evidence for the efficacy and safety of TCHMs for VaD because of the poor methodology, short duration of follow-up and inadequate reporting. However the agents appear to be relatively free of severe short-term AEs, hence we encourage further better designed and reported trials.

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神经科学

Effects of DL0410 on the learning and memory deficit in APP/PS1 transgenic Alzheimer's disease model mice

Rong YAN, Ran-yao YANG, Ai-lin LIU, Guan-hua DU

2015, 29(S1): 41-41.

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OBJECTIVE DL0410, one novel compound discovered inhigh throughput screening (HTS), was found to be a potent inhibitor for AChE and BuChE. Memory deficit mice model induced by scopolamine have been conducted to verify its effects on the improvement of memory deficit. In this study, the effects of DL0410 on inhibiting β-amyloid (Aβ) aggregation and attenuating cognition and memory impairment of APP/PS1 mice were further investigated. METHODS Th-T binding test was used to determinethe effect of DL0410 on Aβ_1-42 aggregation. In addition, locomotor test, object recognition test, step-down test, and Morris Water maze were performedto investigate the effect of DL0410 on the cognition and memoryfunctions of APP/PS1 mice. RESULTS In vitro results showed that DL0410(10 and 30 μmol·L^-1) could inhibit significantly the monomer Aβ_1-42 from aggregation, when incubated together with monomer Aβ_1-42 for 24 h(P<0.01). Several behavioral tests demonstrated that DL0410 (10 and 30 mg·kg^-1) could shortened latency time innavigation test (P<0.01), increased platform crossing-times in space probe test (P<0.05), and reduced the error times in step-down test (P<0.01). CONCLUSIONDL0410 could inhibit Aβ aggregation in vitro and alleviate cognition and memory impairment of APP/PS1 mice, which make DL0410 a promising candidate for Alzheimer's disease treatment.

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神经科学

Chinese herbal medicine formula SYM prevents brain calcification in a murine model of IBGC

Cheng WANG, Xuan XU, Jun-yu LUO, Jing-yu LIU

2015, 29(S1): 41-42.

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OBJECTIVE To establish Idiopathic basal ganglia calcification (IBGC's) disease model in mouse, and investigate the effect of Chinese herbal medicine formula SYM to prevent brain calcification caused by SLC20A2 mutations. METHODS In order to apply the IBGC model in mice, we introduced S602W point mutation into the mouse Slc20a2. Mice (3 months old) with homozygous mutation developed brain calcification as similar as IBGC in human. The SYM decoction was diluted and added into the drinking water of 2-week-old homozygous Slc20a2 KI mice for 5 months. Another group of homozygous Slc20a2 KI mice were set as control and received no treatment. After 5 months, to analyze the effect of the SYM decoction on brain calcification, pathological sections with the brain of Slc20a2 KI mice were made and stained. RESULTS Calcified nodules were not seen in the brain of Slc20a2 KI mice that received SYM decoction treatment, while the control group developed multiple calcifications in the thalamus region. CONCLUSION SYM decoction produced preventive effect on the formation of calcified nodules in IBGC model mice, which might be useful for the treatment of IBGC caused by SLC20A2 mutations.

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神经科学

Effects of oxymatrine on calcium channels and GABA release in mice under neuropathic pain condition

Li YANG, Yang-ou DENG, Xiao-qiang LYU, Shi-xing WU, Yong-gang LIU

2015, 29(S1): 42-42.

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OBJECTIVE To investigate effects of oxymatrine, an alkaloid from Sophora flavescens Ait., on high-voltage dependent calcium channel and inhibitory neurotransmitter GABA under neuropathic pain condition. METHODS The partial sciatic nerve ligation (PSNL) was executed on C57/BL6 mice to produce neuropathic pain. Oxymatrine (150 mg·kg^-1) was administrated intraperitoneally to PSNL mice. Mechanical hindpaw withdral threshold(MWT) was measured under Von-Frey filament stimulation with up-and-down method. In brain tissue, GABA concentration was measured with ELISA. Change of GABA_A receptor protein expression, N-type calcium channel (Cav2.2) and L-type calcium channel (Cav1.3) protein expressions were detected with Western-blot; intracellular calcium concentration was measured in cultured cortical neurons with Fluo-3/AM fluorescent probe. RESULTS Compared to saline, oxymatrine significantly increased ED₅₀ of MWT on PSNL mice (P<0.05). GABA concentration and GABA_A receptor protein level in brain tissue were decreased in PSNL mice, while administration of oxymatrine increased both GABA concentration and GABA_A receptor expression. Intracellular calcium concentration was increased in cultured cortical neurons by oxymatrine treatment, but this phenomenon was not seen under calcium-free condition. Protein expression of Cav2.2, but not Cav1.3, was found to be decreased in the brains of PSNL mice and to be restored to a normal level with oxymatrine administration. CONCLUSIONOxymatrine has analgesic effect on PSNL-induced neuropathic pain in mice. This phenominon relates to the increase of GABA release, GABA_A receptor expression, and also the restoration of expression level of Cav2.2 but not Cav1.3 in brain tissues, which suggesting that Ca²⁺ flow through Cav2.2 calcium channel may be the key point underlying oxymatrine analgesia.

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神经科学

Morroniside antagonizes tau hyperphosphorylation against neurodegeneration: a cell culture study

Xue-xian KUAI, Cui-cui YANG, Wen-bin GAO, Lin LI, Lan ZHANG

2015, 29(S1): 42-43.

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OBJECTIVE Protein phosphatase 2A (PP2A), a major protein phosphatase, have been reported to be involved in the microtubule-associated protein tau hyperphosphorylation and aggregation in Alzheime disease (AD). Morroniside (MOR) is the isolated component from Cornus officinalis Sieb. et Zucc. The present study is to investigate the inhibitory effect of MOR on tau hyperphosphorylation and the underlying mechanisms. METHODS SK-N-SH cells were pretreated with MOR 50-200 μmol·L^-1 for 24 h and then treated with okadaic acid (OA) (20 nmol·L^-1) for 6 h to induce tau hyperphosphorylation by inhibiting PP2A activity. To determine whether the inhibitory effect of MOR on tau hyperphosphorylation was dependent on PP2A directly, we transfected PP2Ac siRNA into HEK293 cells. Cell morphology was visualized under contrast microscope. Western blotting was used to measure the expressions of phosphorylated tau, total tau, Protein phosphatase-2A (PP2A), phosphorylated PP2A at Tyr307 (P-PP2A), demethylated PP2A at Leu309 (DM-PP2A), protein phosphatase methylesterase 1 (PME-1), Leucine carboxyl methyltransferase 1 (LCMT-1), phosphorylated Src at Tyr416 and Tyr529, total Src, glycogen synthase kinase-3β (GSK-3β) and phospho-GSK3β (Ser9). The activity of PP2A was measured by a protein phosphatases activity assay kit. RESULTS Compared with the control, the OA-treated cells became retracted and rounded up and their tau phosphorylation levels at pSer199/202, pT205, pT212, pS214, pT217 markedly increased. Pretreatment with MOR improved the cellular morphology and reduced OA-induced tau hyperphosphorylation. In addition, MOR treatment increased PP2A activity accompanied by a decrease of DM-PP2A and P-PP2A expression. MOR decreased PME-1 expression and the ratio of PME/LCMT-1. Furthermore, MOR treatment altered the level of Src phosphorylated at Tyr416, which can regulate phosphorylation of PP2A. PP2Ac siRNA could inhibit PP2Ac expression and induce tau hyperphosphorylation. MOR had no effect on PP2Ac expression, correspondingly, didn't affect tau hyperphosphorylation in PP2Ac siRNA transfected HEK293 cells. CONCLUSION Morroniside attenuates OA-induced tau hyperphosphorylation through regulating PP2Ac posttranslational modification. MOR is a potential protein phosphatase 2A activator which might be a therapeutic drug for AD and other tau pathology-related degenerative diseases.

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神经科学

Fuzi promoted the formation of myelin sheath in Schwann cells induced by high glucose

Tian-tian LYU, Jing HAN, Hong-liang WANG, Yan WU, Gai-mei HAO, Jie HE, Wei WANG

2015, 29(S1): 43-44.

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OBJECTIVE Diabetic peripheral neuropathy (DPN) is the cause of considerable morbidity and mortality indiabetic patients.The loss of nerve fibersis the main pathological characteristics of the DPN and the pathway of Oct6-Krox20plays an important role in the formation of myelin sheath.In our previous study, we found that Fuzi(Radix aconite lateral ispreparata) could significantly improve the nerve conduction deficits and thermal hypoalgesia deficits in the diabetic rats, but the underlying molecular mechanisms have not been established. The aim of this study is to investigate the expression of Oct6,Krox20, myelin basic protein(Mbp) and myelin protein zero (Mpz) in Schwann cells and analyze the effect of Fuzi in the formation of myelin sheath. METHODS There were six groups in the study. In the control group, thecells weresupplemented with normal cell culture medium. In the mannitol group, the cells were fed with normal glucose plus mannitol. In the model group, the cells were supplemented with high glucose medium(75 mmol·L^-1). In the other group, the cells weretreated with high glucose medium(75 mmol·L^-1) plus different concentrations of Fuzi (0.1, 1.0 and 10.0 μg·mL^-1). After three days, real-time PCR was used to detect gene expression. RESULTS Compared with the control group, the model group showed lowerexpression of Oct6, Krox20, Mbp and Mpz. In comparison to the model group, Fuzi(0.1, 1.0 and 10.0 μg·mL^-1) increased the expression of Oct6, Krox20, Mbp and Mpz. CONCLUSION These results demonstrate that Fuzi enhances the protein of myelin sheath through impacting the pathway of Oct6-Krox20.

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神经科学

Multi-targets antidepressant mechanism of the bis-benzylisoquinoline 7-O-ethylfangchinoline: Involvement of noradrenergic, dopaminergic and AMPAergic systems

Zhao-fu SHENG, Xiang-yu CUI, Su-ying CUI, Bin YU, Xue-qiong ZHANG, Sheng-jie LI, Qing CAO, Yuan-li HUANG, Ya-ping XU, Jin-zhi SONG, Hui DING, Zhi-ge LIN, Guang YANG, Yong-he ZHANG

2015, 29(S1): 44-44.

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OBJECTIVE Bisbenzylisoquinoline (BBI) alkaloids have extensive pharmacological functions. The aim of this study was to investigate the mechanisms underlying the antidepressant-like action of 7-O-ethylfangchinoline (YH-200) in mice. METHODS Male ICR mice were used in the forced swimming (FST) and tail suspension tests (TST). RESULTS YH-200 (60 mg·kg^-1, ig) decreased the immobility time in FST and TST, and prolonged the latency to immobility in FST. YH-200 revealed more potent anti-immobility activity than its BBI derivative tetrandrine. In addition, the pretreatment of mice with prazosin (1 mg·kg^-1, ip, an α₁-adrenoceptor antagonist), propranolol (2 mg·kg^-1, ip, a nonselective β-adrenoceptor antagonist), SCH23390 (0.05 mg·kg^-1, ip, a dopamine D₁/D₅ receptor antagonist), haloperidol (0.2 mg·kg^-1, ip, a dopamine D₂/D₃ receptor antagonist) and NBQX (10 mg·kg^-1, ip, an AMPA receptor antagonist) prevented the antidepressant-like effect of YH-200 (60 mg·kg^-1, ig) in FST. Besides that, the pretreatment of mice with yohimbine (1 mg·kg^-1, ip, an α₂ adrenoceptor antagonist) augmented the antidepressant-like effect of YH-200 (30 mg·kg^-1, ig) in FST. After 14 d administration, YH-200 (30 and 60 mg·kg^-1, ig) did not develop drug resistance, but the potency was strengthened, meanwhile, it did not influence the changes in mice body weight. CONCLUSION YH-200 may possess the therapeutic potential for the treatment of depression via the multi-targets including the noradrenergic (α₁, α₂ and β-adrenoceptors), dopaminergic (D₁/D₅ and D₂/D₃ receptors) and AMPAergic systems.

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神经科学

Effects of Gastrodia elata and Uncaria rhynchophylla water extract on cerebral ischemic rat revealed by LC-FTICR-MS metabolomics approach

Jia Wen XIAN, Tao HUAN, Liang LI, Wing Nang LEUNG, Chun Wai CHAN

2015, 29(S1): 44-45.

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OBJECTIVE To study the treatment effect of Gastrodia elata and Uncaria rhynchophylla water extract (GUW) against cerebral ischemia, and the metabolomic profile of cerebrospinal fluid in GUW treated cerebral ischemia in rat model. METHODS Middle cerebral artery occlusion was performed in male SD rat. GUW or control (PBS) was administered orally for 7 d post-operationally. At day 7, the brain of the rat was harvested. The infarct volume of the brain was measured after TTC staining. The brain was subjected to H&E and cresyl violet staining in histological analysis. The cerebrospinal fluid (CSF) of another set of animals was aspirated. The CSF samples were labeled with ¹³C-dansyl and ¹²C-dansyl chloride. The reaction mixture was put into liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry for analysis of the CSF metabolome. Multivariate statistical analysis will be carried out to determine the differences of metabolomic profile in CSF of GUW versus control group. RESULTS After GUW treatment, the neurological deficit score was significantly improved from day 3 to 7 (P<0.001). GUW treated group significantly reduced 55.7% infarct volume on MCAO rats in TTC stained brain slices (P<0.001) and preserved brain histological structure observed in H&E and cresyl violet staining. On the other hand, according to various statistical analyses to compare CSF metabolite in the GUW and control group, volcano plot analysis showed 48 metabolites (P<0.001); OPLS-DA analysis showed 129 metabolites (VIP score >2.0) and 45 metabolites in ROC curve analysis. Among identified metabolites, L-aspartic acid (60%) and glycine (62%) decreased in GUW while threoninyl-alanine increased by 1.6 fold significantly. CONCLUSION GUW ameliorated cerebral ischemic injury, and the metabolomic approach may be a potential means to find biomarker of the Chinese medicine treatment on cerebral ischemia.

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神经科学

Effects of tetrahydroxystilbene glucoside in Parkinson's disease mice model induced by MPTP

Ru-yi ZHANG, Li ZHANG, Yan-chuan WU, Lin LI

2015, 29(S1): 45-45.

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OBJECTIVE To observe effects of tetrahydroxystilbene glucoside (TSG) on behavior, content of dopamine and its metabolites in striatum of Parkinson's disease (PD) model mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection. METHODS Mice were randomly divided into control group, model group, TSG low dose (60 mg·kg^-1) and high dose (120 mg·kg^-1) groups. The behavior changes of mice were observed by pole test, rotarod test and spontaneous movement test. The tyrosine hydroxylase (TH) positive cells were detected by immunohistochemical method. The content of dopamine (DA) and its metabolites in striatum were determined by HPLC-ECD. RESULTS MPTP model mice showed behavior deficit. The number of TH positive neurons in substantia nigra, the content of dopamine and its metabolites in striatum in model mice decreased significantly compared with control group. TSG ameliorated mice behavior, increased the number of TH positive neurons in the substantia nigra about 18.8%, and elevated the content of dopamine in striatum about 34.5% compared with model mice. CONCLUSION TSG protected dopaminergic neurons against MPTP-induced damage, and may become a candidate drug for prevention and treatment of Parkinson's disease.

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神经科学

Cornel iridoid glycoside inhibits tau hyper-phosphorylation by enhancing PP2A activity

Cui-cui YANG, Xue-xian KUAI, Ya-li LI, Li ZHANG, Lin LI, Lan ZHANG

2015, 29(S1): 46-46.

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OBJECTIVE The aim of the present study was to investigate the effect of cornel iridoid glycoside (CIG) on tau hyperphosphorylation induced by wortmannin (WT) and GF-109203X (GFX) and the underlying mechanisms. METHODS Human neuroblastoma SK-N-SH cells were pre-incubated with CIG (50, 100, and 200 μg·mL^-1, respectively) for 24 h, and then exposed to WT 10 μmol·L^-1 and GFX 10 μmol·L^-1 for 3 h after washing out CIG. Immuno-fluorescence was used to observe the microtubular cytoskeleton of the cultured cells. Western blotting was used to measure the phosphorylation level of tau protein, glycogen synthase kinase 3β (GSK-3β) and protein phosphatase 2A (PP2A). The activity of PP2A was detected by a biochemical assay. RESULTS Pre-incubation of CIG significantly attenuated the WT/GFX-induced tau hyperphosphorylation at the sites of Thr205, Thr212, Ser214, Thr217 Ser396 and PHF-1, and improved the damage of morphology and microtubular cytoskeleton of the cells. CIG did not prevent the decrease in p-AKT-ser473 and p-GSK3β-ser9 induced by WT/GFX. However, CIG significantly elevated the activity of PP2A by reducing the demethylation of PP2AC at Leu309 and the ratio of PME-1/LCMT in the WT/GFX-treated cells. CONCLUSION CIG obviously attenuated WT/GFX-induced tau hyper-phosphorylation at multiple AD-related sites by increasing the activity of PP2A. The mechanism may be involved in the reduced demethylation of PP2A via down regulating the ratio of PME/LCMT.

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神经科学

Analysis of potential amino acid biomarkers in brain tissue and the effect of galangin on cerebral ischemia

Jian GAO, Chang CHEN, Fei-peng DUAN, Jing FANG, Shao-jing LI

2015, 29(S1): 46-47.

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OBJECTIVE Galangin, a potent scavenger of free radicals, is used as herbal medicine for various ailments for centuries in Asia. With complex pathophysiology, ischemic stroke is one of the most frequent causes of death and disability worldwide. We have reported that galangin provides a direct protection against ischemic injury as a potential neuroprotective agent and has potential therapeutic effects on the changes of serum amino acids for ischemic stroke; however, its mechanism on changes of amino acids in the ischemic brain tissue has not yet been clarified. METHODS In this paper, we explored the amino acid biomarkers of brain tissue in the acute phase of cerebral ischemia and the effect of galangin on those potential biomarkers with a rapid, sensitive and accurate methodology of simultaneous quantification of 12 AAs in rat brain tissue by the RRLC/QQQ. RESULTS we identified that glutamic acid, alanine and aspartic acid all showed significant change in galangin-treated groups compared to vehicle-treated rats and four pathway-related enzymes were identified by multiplex interactions with the three amino acids. With metabolite-protein network analysis and molecule docking, six of 28 proteins were identified and may become the potential biomarkers of galangin for acute ischemic stroke. CONCLUSION All data in our study provide thought for exploring the mechanism of disease, discovering new targets for drug candidates and elucidating the related regulatory signal network.

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神经科学

Effect of Naojian Tablet on cerebral ischemic injury and its prevention mechanism

Hong-ping LONG, Pan MENG, Yuan-shan HAN, Yu-hong WANG

2015, 29(S1): 47-47.

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OBJECTIVE To investigate the prevention and its mechanism of Naojian Tablet, the adjusted prescription from Buyang Huanwu Decoction, during focal ischemic brain injury condition, and study on the main bioactive substance for this function. METHODS To apply the modified middle cerebral artery occlusion (MCAO) model in rats, immunohistochemistry, RT-PCR and Western blotting were applied to determine the expression of CDK5,CDK4/cyclinD1 in the hippocampus tissues in MCAO rat by treating with Naojian Tablet. On the other hand, the processes of metabolism and disposition of several active components in Naojian Tablet and Buyang Huanwu Decoction were analyzed and characterized by using HPLC-Q-TOF method. RESULTS Naojian Tablet (ig, 2.41 g·kg^-1) could significantly decrease the levels of CDK5, CDK4/cyclinD1 in hippocampus tissues in MACO rat after treated by 3 d, 7 d, 14 d, 28 d (P<0.05). The similar mechanism was observed in the Buyang Huanwu Decoction treated MACO rat. Calycosin, fermononetin, senkyunolide and ligustilide in Naojian Tablet was determined simultaneously as the main active ingredient of Naojian Tablet. CONCLUSION The above-mentioned in vivo studies suggested that Naojian Tablet maybe play its prevention effect on nerve cells via its acting on CDK5 signaling and cell cycle pathway, which verified the multi-target and multi active pathways efficiency of tradition Chinese medicine in treating diverse disease.

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神经科学

Effect of cornel iridoid glycoside on experimental autoimmune encephalomyelitis attenuation through JAK/STAT signaling pathway, at least partially

Lin-lin YIN, Yong-yan CHEN, Zhao QU, Li ZHANG, Qi WANG, Qi ZHANG, Lin LI

2015, 29(S1): 47-48.

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OBJECTIVE In order to investigate whether cornel iridoid glycoside (CIG), the main component extracted from Cornus officinalis, can treat demyelinating diseases of the central nervous system (CNS) such as multiple sclerosis (MS). METHODS CIG (30, 60 and 120 mg·kg^-1) or vehicle was intragastrically administered once daily to rats, starting immediately after purified myelin basic protein (MBP) 68-86 peptides immunization until day 20 post immunization (p.i.). Histopathological staining, enzyme-linked immunosorbent assay, biochemical methods and Western blotting approaches were used to evaluate the disease incidence and severity, neuroinflammatory and neurotrophic response in the CNS. RESULTS Neurological deficit and proportion of incidence seen in EAE rats were significantly reduced by CIG treatment in a dose-dependent manner. Histopathological staining showed that CIG treatment alleviated demyelination and inflammatory infiltration, increased the number of oligodendrocytes, enhanced the expression of brain-derived neurotrophic factor (BDNF). Production of proinflammatory molecules such as interleukin-1β(IL-1β), tumour necrosis factor-α and interferon-γ were also inhibited by CIG administration. CIG could ameliorate phosphorylation of STAT1, STAT3 and JAK1 as well as IL-6/IL-6R expression, which involved in immune response and inflammation. CONCLUSION Our results demonstrated that CIG may ameliorate EAE rats through down-regulation of JAK/STAT signaling pathway. This study gave new insight into the novel regulatory mechanism of CIG and highlight novel therapeutic targets and a potential therapeutic agent for the treatment of MS.

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神经科学

Inhibition of NMDA receptor and GSK3β mediates the fast onset of antidepressant like natural product

Qi ZHANG, Fei GUO, Zh-Wen FU, Bing ZHANG, Cheng-Gang HUANG, Yang LI

2015, 29(S1): 48-48.

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OBJECTIVE To investigate the fast antidepressant effect and the underlying mechanism of it. METHODS C57BL/6 mice were exposed to a 6-week chronic unpredictable mild stress (CUMS) protocol, and treated with YY-23 or a positive drug fluoxetine in the last 3 weeks, behavioral assessments proceed every week. NMDA-induced current were recorded by whole-cell patch clamp on dissociated hippocampal CA1 neurons of Sprague-Dawley rats. Phosphorylation of the signaling proteins was evaluated by Western blotting. RESULTS The electrophysiological results showed that YY-23 is a non-competitive NMDA receptor antagonist, neither in 'use-dependent' nor 'voltage-dependent' way, with no prominent effect on AMPA induced current. This antagonism of YY-23 could be partially prevented by proven blockers MK801 and Mg²⁺, laterally indicating the nature of NMDA receptor antagonist both pharmacologically and physiologically. Behavioral and Biochemical study showed that YY-23 could act as antidepressant and exerted strong inhibitory effect on GSK3β in PFC of CMS depression mice model. CONCLUSION YY-23 showed promising antidepressant effect with rapid onset, which might be related to NMDA receptor antagonism and GSK3β inhibition.

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神经科学

Effect of Tong-Qiao-Huo-Xue decoction on the expressions of related proteins of the blood-brain barrier and analysis of constituents in cerebrospinal fluid of cerebral ischemic rats

Ning WANG, Li-li LI, Qi-zhong JIN, Ya-fang LIU, Guang-yun WANG, Yan WANG

2015, 29(S1): 49-49.

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OBJECTIVE To evaluate the effects of Tong-Qiao-Huo-Xue decoction (TQHXD) on the blood-brain barrier (BBB) permeability and the expressions of related proteins on the rats; and to analyse the constituents in the cerebrospinal fluid on the rats with cerebral ischemic injury. METHODS Cerebral ischemia rats were induced by middle cerebral artery occlusion (MCAO). Adult male sprague-dawley (SD) rats were randomly divided into seven groups: sham-group; model group; nimodipine(NMP)-treated group and nao mai tai (NMT)-treated group were set as positive drug control groups; TQHXD-treated group (3, 6 and 12 g·kg^-1 body weight); The neurological function of rats was estimated by neurological defect scoring after the 1, 7 and 15 d after administration. Histological structure of the brain in rats were observed by hematoxylin and eosin (H&E) staining. Ultramicrostructural features of hippocampus neurons and the opening of tight junction (TJ) of BBB in rats were observed by transmission electron microscope (TEM). Western blotting was performed to detect the expression of ZO-1, occludin, claudin-5, AQP-4 and MMP-9 in BBB after cerebral ischemia injury. Component analysis experiments: adult male SD rats were randomly divided into four groups: Distilled water was administered intragastrically sham-operated rats; Distilled water was administered intragastrically model rats by MCAO; TQHXD was administered intragatrically to rats in sham-operated group; TQHXD was administered intragestrically to rats in model group by MCAO. GC and HPLC was used to detect three compounds, namely, muscone, ligustilide and hydroxysafflor yellow A, in rats cerebrospinal fluid (CSF) after oral administration of TQHXD. Finally, samples of cerebrospinal fluid of rats in each group were compared with single medicine so as to explicit the three compounds come from which herb. RESULTS TQHXD significantly reduced the neurological defect scores. Histological examination indicated that dense neuropil and largely surviving neurons had been seen in TQHXD-treated rats. TEM observation revealed that TQHXD could significantly inhibit the damage of hippocampal neurons and reduce the opening of TJ. The decreased protein expression levels of claudin-5, occludin, ZO-1 and the increased protein expression levels of AQP-4 and MMP-9 in cerebral ischemia tissue were significantly prevented by treatment of TQHXD. Analysis of experimental results showed that muscone, ligustilide and hydroxysafflor yellow A could penetrate the BBB into the CSF, and the content of the model group was lower than that of sham group after intragastric administration of TQHXD. CONCLUSION These results demonstrated that TQHXD may act as a potential neuroprotective agent against BBB damage for cerebral ischemia through protecting of hippocampus neurons, reducing the opening of TJ and decreasing the permeability of BBB by up-regulating ZO-1, occludin, claudin-5 expressions, down-regulating AQP-4 and MMP-9 expressions. The effect of TQHXD on the decrease of the opening of TJ also reduced the content of muscone, ligustilide and hydroxysafflor yellow A in cerebrospinal fluid.

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神经科学

Development of an ERK1/2 activation assay to determine relaxin-3/RXFP3 activation

Jayakody JPT, Dawe GS

2015, 29(S1): 50-50.

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OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists. METHODS Plated HEK-RXFP3, CHO-RXFP3, HEK293T and CHO-K1 cells in poly-L-lysine coated well plates. The cells were serum starved and treated with either human relaxin-3 (H3 relaxin) (10 nmol·L^-1), R3 B1-22R (10 μmol·L^-1) and pertussis toxin (PTX, 100 ng·mL^-1). The cells were lysed and the ERK1/2 activation was determined by SDS-PAGE followed by immunoblotting for phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) for the lysates. RESULTS The quantification of the data revealed that the peak of ERK1/2 activation can be detected precisely at 10 min post stimulation with 10 nmol·L^-1 H3 relaxin in both HEK-RXFP3 and CHO-RXFP3 cell lines in all three trials compared to the cells treated with vehicle (P<0.05). However, HEK-RXFP3 cells demonstrated a transient activation of ERK1/2 and CHO-RXFP3 cells demonstrated a continuous activation of ERK1/2 which was inhibited by the Gi inhibitor, PTX. Activation of ERK1/2 was significantly inhibited by pre-treating the cells with RXFP3 antagonist R3 B1-22R in HEK-RXFP3 cells. ERK1/2 activation was observed neither in wild type HEK293T nor in CHO-K1 cells. CONCLUSION The developed assay can detect RXFP3 activation or inhibition by agonists and antagonists via the detection of pERK1/2 in multiple cell lines. This assay will also be useful to detect signaling pathways upstream to ERK1/2 activation mediated by RXFP3. Activation of ERK1/2 in CHO-RXFP3 cells was mediated by Gi proteins at 10 min as well as at 25-35 min time points.

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炎症与免疫

Andrographolide restores steroid sensitivity to block LPS/IFNγ-induced IL-27 and airway hyper-responsiveness in mice

Wu-peng LIAO, Wan Shun Daniel TAN, Shiu Fred WONG

2015, 29(S1): 50-51.

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OBJECTIVE Steroid-resistant airway hyper-responsiveness (AHR) has been proposed to be related to the activation of innate host defense pathways such as those induced by LPS, IFN-γ, and LPS/IFN-γ-stimulated essential mediator IL-27. We investigated whether andrographolide, a previously demonstrated anti-inflammatory bioactive molecule extracted from the plant Andrographis paniculata, could restore steroid sensitivity to block LPS/IFN-γ-induced IL-27 production and AHR via its anti-oxidative property. METHODS Mouse macrophage cell line Raw264.7, mouse primary pulmonary monocyte/macrophage, and BALB/c mouse were treated with LPS/IFN-γ, in the presence and absence of increasing doses of dexamethasone and/or andrographolide. mRNA and protein levels of IL-27 in vitro and in vivo were examined, and mouse AHR was assessed. RESULTS Dexamethasone alone failed to inhibit LPS/IFN-γ-induced IL-27 and AHR in mice. Andrographolide significantly facilitated the suppressive effect of dexamethasone on LPS/IFN-γ-induced IL-27 level in macrophage cell line and primary monocyte/macrophage, mouse bronchoalveolar lavage fluid and lung tissue, and furthermore on the incurring AHR. LPS/IFN-γ did not impede nuclear translocation of glucocorticoid receptors but diminishthe protein level of histone deacetylase-2 (HDAC2), an essential epigenetic enzymeresponsible for steroid anti-inflammatory action. Andrographolide at low doses (5 μmol·L^-1 in vitro; 1 mg·kg^-1, ip in vivo) restored nuclear HDAC2 protein levels both in cells and in mouse lungs,possibly via suppression of PI₃K/Akt signaling pathway and up-regulation of the anti-oxidative transcription factor Nrf-2 level. CONCLUSION Our data suggest that andrographolide may resensitize steroid action on blocking LPS/IFN-γ-induced IL-27 and resultant AHR by restoring HDAC2 level.

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炎症与免疫

Vitamin E isoform γ-tocotrienol alleviates cigarette smoke-induced chronic obstructive pulmonary disease

Hong Yong PEH, Wan Shun Daniel TAN, Tze Khee CHAN, Chen Wei POW, Choon Nam ONG, Wai-Shiu Fred WONG

2015, 29(S1): 51-51.

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OBJECTIVE Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a leading cause of death, where inflammation and oxidative stress are involved in the pathogenesis. Vitamin E isoform γ-tocotrienol possesses both anti-oxidative and anti-inflammatory properties. We hypothesized that γ-tocotrienol may have protective effects against COPD. METHODS BALB/c mice were exposedto cigarette smoke daily for 2 weeks with oral γ-tocotrienol treatment in the second week. Bronchoalveolar lavage (BAL) fluid was assessed for total and differential cell counts, oxidative damage biomarkers, and cytokine levels. Lung tissues were examined for the expression of antioxidants and pro-inflammatory biomarkers. In order to measure changes in lung functions in COPD, another set of mice was exposed to cigarette smoke for 2 months with oral γ-tocotrienol treatment in the last 2 weeks. RESULTSγ-Tocotrienol dose-dependently abated cigarette smoke-induced elevation of BAL fluid total and neutrophil cell counts, cytokine and chemokine (IL-1β, IL-6, IL-17, LIX, G-CSF, KC, RANTES and VEGF) levels, as well as oxidative/nitrosative damage biomarker (advanced oxidation of protein products, 8-isoprostane, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine) levels. γ-Tocotrienol promoted total lung antioxidant capacity and endogenous antioxidant activities of superoxide dismutase, catalase and glutathione peroxidase. More importantly, γ-tocotrienol markedly restored work of breathing and lung functions (total lung capacity, static compliance and FEV100/FVC) in chronic experimental COPD. Furthermore, γ-tocotrienol demonstrated better anti-oxidative, anti-inflammatory, and restoration of lung functions in COPD than prednisolone. CONCLUSION We have shown for the first time the efficacy of vitamin E isomer γ-tocotrienol in protection against cigarette smoke-induced COPD by direct neutralization of free radicals, abating oxidative damage, and restoring antioxidants activities, coupled with anti-inflammatory actions in the inflamed airways.

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炎症与免疫

In vitro and in vivo pharmacological investigations of Pinus roxburghii for prevention of oxidative, inflammatory responses against osteoporosis

Sharma ADITI, Goyal ROHIT, Bhattacharya S

2015, 29(S1): 52-52.

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OBJECTIVE To evaluate antioxidant, anti-inflammatory, antimicrobial activities of Pinus Roxburghii stem bark in vitro and evaluation of in vivo anti-osteoporotic activity in female rats. METHODS The antioxidant activity of plant extract was assessed using DPPH, nitric oxide and H₂O₂ radical scavenging methods. In vitro anti-inflammatory activity was evaluated using albumin denaturation, membrane stabilization methods at various concentrations. The antibacterial and anti-fungal activity of extracts was also done against micro-organisms: Pseudomonas aurignosa, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans. Quantitative analysis of total phenolics, flavonoids and tannins were also estimated. Female Wistar albino rats (220-260 g) were employed and ovaries were surgically removed bilaterally in anaesthesia which causes post-menopausal syndrome in all the groups except sham operated rats. Extracts of Pinus roxburghii stem at 100 and 200 mg·kg^-1, po. and tamoxifen as standard 1 mg·kg^-1, po. were given. Acute toxicity study was also done. RESULTS Treatment with Pinus roxburghii extract in two doses significantly (P<0.05) ameliorated surgical ovariectomy induced biochemical and biomechanical changes like decrease in bone density, bone strength, alkaline phosphatase, serum calcium, serum phosphorous and increase in urine hydroxylproline levels. Plant extract also has significant (P<0.05) antioxidant, antimicrobial and anti-inflammatory activities. CONCLUSION The assessments done in present investigation may conclude that the constituents of Pinus roxburghii has significant potential in ameliorating oxidative, inflammatory reactions and possesses anti-microbial; osteobalstic and osteoclastic activities used for calcium homeostatis and improved bone strength in female rats.

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炎症与免疫

Isofuranodiene protect D-galactosamin/lipopolysacchride induced liver injury in rats

Wen-ping LI, Jing-shan SHI, Filippo MAGGI, Xiu-ping CHEN

2015, 29(S1): 52-53.

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OBJECTIVE Isofuranodiene (ISO) is a natural product isolated in Chinese herb. Our recent results showed anticancer effect in vitro. In this study, we investigated its live protective effect with a D-galactosamine/lipopolysacchride (GalN/LPS) induced rat model. METHODS SD rats were treated orally with or without ISO (20 and 50 mg·kg^-1, ig) for 3 d and then treated with GalN/LPS for 8 h. The serum were collected and the concentration of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) were determined. The liver injury was examined by H&E staining. The mRNA expression of IL-1β, IL-6 and inducible nitric oxide synthase (iNOS) in liver tissues were determined by real-time PCR. RESULTS Oral administration of ISO (20 and 50 mg·kg^-1) dramatically inhibited GalN/LPS-induced serum elevation of AST, ALT, and MDA levels. The liver injury was also significantly ameliorated as evidenced by the histological improvement in H&E staining. Furthermore, ISO treatment significantly inhibited GalN/LPS-induced mRNA expression of IL-1β, IL-6, and inducible nitric oxide synthase (iNOS) in liver tissues. CONCLUSION This data showed that ISO has hepatoprotective effect in rats.

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SRS27, a semisynthetic diterpenoid lactone, inhibits airway inflammation and hyper-responsiveness via NF-κB pathway in an allergic mouse asthma model

Jonathan Chee Woei LIM, Fera Yiqian GOH, Sreenivasa Rao SAGINEEDU, Audrey Chee Hui YONG, Shiran Mohd SIDIK, Nordin Haji LAJIS, Wai-Shiu Fred WONG, Johnson STANSLAS

2015, 29(S1): 53-54.

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Andrographis paniculata contains two main diterpenoid constituents, andrographolide (AGP) and 14-deoxy-11,12-didehydroandrographolide (DDAG), which were found to exhibit antiasthma effects in a mouse asthma model. However, due to inadequacies of both compounds in terms of drug-likeness, DDAG analogues were semisynthesised to tackle these shortcomings. The objective of the study was to investigate the potential of DDAG analogues as new antiasthma agents. Among the analogues, (SRS27) was proven to inhibit cysteinyl leukotrienes (CysLT) and nitric oxide (NO) synthesis in mouse macrophages, like AGP. DDAG, on the other hand, failed to exhibit such activities. SRS27 is less cytotoxic than AGP, suggests that a simple chemical modification of DDAG produces a compound with CysLT and NO inhibitory activites similar to AGP while maintaining toxicity profile similar to DDAG. It is interesting to note that other analogues such as SRS28, SRS49, SRS76 and SRS83 with chemical modifications on the same carbon numbers 3 and 19 of DDAG were unable to show inhibition of CysLT and NO synthesis. Consequently, the potential anti-inflammatory effect of SRS27 was investigated in ovalbumin (OVA)-induced mouse asthma model. The compound was administered in a prophylactic manner and showed a substantial decrease in mouse asthma model parameters. SRS27 at 3 mg·kg^-1 significantly reduced OVA-induced total cell such as macrophages, eosinophils, lymphocytes and neutrophils, as well as inflammatory cytokines such as IL-4, IL-5, IL-13 and eotaxin in bronchoalveolar lavage BAL fluid. The compound also suppressed serum IgE production. In addition, SRS27 suppressed mucus hyper-secretion and expression of inflammatory mediators such as TNF-α, MCP-1, Muc5ac, RANTES, IL-33 and iNOS. SRS27 is the first known DDAG derivative tested positive in mouse asthma model and as such SRS27 could serve as a prototype prophylactic agent.

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Non-typeable haemophilus influenzae-induced exacerbation on a cigarette smoke lung injury model: Protective effect of andrographolide

Wan-shun Daniel TAN, Hong-yong PEH, Chu-hui PANG, Wu-peng LIAO, Tze-khee CHAN, Suk-hiang LAU, Vincent T. K. CHOW, Wai-shiu Fred WONG

2015, 29(S1): 54-54.

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OBJECTIVE To develop a 2-week cigarette smoke (CS) acute lung injury model exacerbated by haemophilus influenzae (NTHi) and study the protective effect of andrographolide in this COPD model. METHODS Female BALB/c mice, 6-8-week-old, were exposed to 4% 3R4F CS delivered using a peristaltic pump daily for 2 weeks to induce an acute lung injury model. After 2 weeks of smoking, mice were inoculated intratracheally with NTHi to induce exacerbation on the model. Mice were sacrificed 48 h after last bacteria challenge and lung samples were collected for various analyses. RESULTS After developing a 2-week CS acute lung injury model exacerbated by NTHi, the CS+NTHi group was shown to have a higher inflammatory response, higher bacterial clearance, an upregulation of MMP12 mRNA levels and decrease in TIMP1 mRNA levels in the lungs. Administration of Andrographolide suppressed BALF lung cellular infiltrates, TNF-α, CXCL1/KC, IL-1β and 8-OHdG protein levels, together with increased HO-1 and GR mRNA levels and decreased MMP-8 and MMP-9 mRNA levels. Andrographolide was able to ameliorate lung histopathology as observed with H&E staining and inflammation scoring. Andrographolide was also shown to reduce Keap-1 level in lungs without affecting DJ-1 level. CONCLUSION This study demonstrates the protective effect of andrographolide in a novel 2-week CS acute lung injury model exacerbated by NTHi and presents it as a potential therapeutic for COPD.

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Anti-malarial drug, artesunate, as a novel therapeutic drug target for airway wall remodeling in asthma

Sheryl TAN, Benjamin ONG, Chang CHENG, Wanxing Eugene HO, John K.C TAM, Alastair G. STEWART, Trudi HARRIS, Wai-Shiu Fred WONG, Thai TRAN

2015, 29(S1): 54-55.

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OBJECTIVE Airway wall remodeling (AWR), which refers to structural changes in the airway, is a key characteristic of asthma. Airway smooth muscle (ASM) cell hypertrophy and hyperplasia contributes to AWR. Glucocorticoids, which are used as first line therapy for the treatment of asthma, reduce ASM proliferation but the magnitude of their anti-proliferative actions is dependent on the mitogen used. Moreover, glucocorticoid therapy is accompanied by many side effects. Artesunate, a semi-synthetic artemisinin derivative, has been widely used to treat malaria with minimal toxicity. Artesunate has been shown to attenuate allergic airway inflammation in mice. However, its role in treating AWR in asthma is not known. In this study, we hypothesize that artesunate has anti-proliferative actions on ASM cells, potentially reversing AWR. METHODS and RESULTS Quiescent primary human ASM cells were pre-treated (1 h) with artesunate (3, 10, 30 μmol·L^-1) before being stimulated with either FBS (10%) or thrombin (0.3 U·mL^-1). Following 48 h stimulation with mitogen, cells were counted using a haemocytometer. Dexamethasone (Dex, 100 nmol·L^-1) was used as a positive control. Artesunate concentration-dependently reduced cell number and the magnitude of inhibition appeared to be non-mitogen dependent. Moreover, we examined the effect of artesunate on two important signalling proteins involved in cell proliferation, ERK1/2 phosphorylation and cyclin D1 protein levels. Artesunate reduced cyclin D1 protein levels significantly following 20 h stimulation with either thrombin or FBS but had no effect on ERK1/2 phosphorylation following 6 h stimulation. Importantly, artesunate (30 μmol·L^-1), but not Dex, inhibited the phosphorylation of Akt, which is upstream of cyclin D1. Next, we show that the inhibitory effect of artesunate, but not Dex, on ASM cell number is retained at least 24 h post-treatment following stimulation with FBS. In an acute murine model of allergic asthma, artesunate treatment decreased sm-α-actin positive cells and cyclin D1 protein abundance in the ovalbumin sensitized and challenged mice. CONCLUSION We have shown that artesunate regulates the PI3K/Akt pathway to inhibit the proliferation of primary human cultured ASM cells. This is an alternative mode of action, in comparison to glucocorticoids such as Dex. The anti-proliferative effect of artesunate was further validated in vivo. Thus, our study provides a basis for the future development of artesunate as a novel anti-AWR agent that targets ASM hyperplasia via the PI3K/Akt pathway. Moreover, artesunate may be used as an add-on therapy for asthmatic patients.

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Houttuyniacordata extracts and constituents inhibit the infectivity of dengue virus type 2 in vitro

Meng Chee PHOON, Mei-lan XIE, Sheng-xiong DONG, Benny KH Tan, Vincent TK CHOW

2015, 29(S1): 55-56.

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OBJECTIVE Dengue virus belongs to the Flaviviridae family, and causes dengue fever and its related complications. Currently, there are no antiviral agents or licensed vaccines against dengue in humans. Houttuyniacordata Thunb. (Saururaceae) is documented to possess antiviral activity against several medically important viruses. METHODS The in vitro activities of the total ethyl acetate (EA) extracts of two batches of H. cordata, five EA fractions, and four of its constituent polyphenols (chlorogenic acid, hyperoside, quercetin, and quercitrin) against the new guinea C strain of dengue-2 virus were investigated. RESULTS Using the plaque reduction test, the total EA extracts of H. cordata inhibited viral infectivity when pre-incubated with virus before the viral adsorption stage, but exhibited no dose-dependent response when added to the cells at 6 h post-infection. The 50% inhibitory concentration (IC₅₀) of total EA extracts from two batches of H. cordata added before the viral adsorption stage were 0.24±3.1 μg·mL^-1 and 0.04±4.6 μg·mL^-1. However, only one out of five tested EA fractions showed considerably weaker IC₅₀ of 333 μg·mL^-1. The pure polyphenol compounds displayed some anti-dengue activity, with their combinations yielding greater antiviral effects, especially the combination of chlorogenic acid and hyperoside with a high selectivity index. However, the enhanced efficacy of the polyphenol combinations was still less than that of the total EA extracts which revealed absence of cytotoxicity. Therefore, there may be other compounds in H. cordata that contribute to the superior efficacy of the EA extracts. CONCLUSION We conclude that H. cordata possesses anti-dengue-2 virus activity, and harbors potential for the development of antiviral agents against dengue.

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Development of an in vitro cell model for LPS- and TNFα-induced airway inflammation

Jia Ying Joey LEE, Wai-Shui Fred WONG, Lit-Hsin LOO

2015, 29(S1): 56-56.

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OBJECTIVE To develop an in vitro airway epithelial cell model suitable for the large-scale studies of compounds that can suppress lipopolysaccharide (LPS)- and tumor necrosis factor alpha (TNFα)-induced airway inflammation. METHODS We have optimized the protocols to culture BEAS-2B, a normal human bronchial epithelial cell line, in glass-bottom 384-well microtiter plates. The cells were stimulated with TNFα and LPS from Pseudomonas aeruginosa, a common lunginfection pathogen in cystic fibrosis and chronic obstructive pulmonary disease. We used ELISA to measure the secretion levels of two pro-inflammatory cytokines,interleukin (IL)-6 and -8, after 0, 4, 8, 16, 24 h of stimulation; and immunofluorescence microscopy to measure the nuclear translocation of RelA, a subunit of the NF-κB complex, after 0, 15, 30, 60, 120 min of stimulation. To suppress the inflammatory response, we pre-treated the cells with a specific IκB kinase-2 inhibitor, TPCA-1; the main bioactive component of Andrographis paniculata, andrographolide; and DMSO control for 1 h. RESULTS We found that individual stimulant (either TNFα 10 ng·mL^-1 or LPS 10 μg·mL^-1) increased the IL-6 and IL-8 secretion levels by ~12-17 foldsas compared to DMSO controls after 8 h of stimulation. The combined stimulation (10 ng·mL^-1 TNFα and 10 μg·mL^-1 LPS)induced even higher IL-6 and -8 levels (~18-21 folds) at the same time points. Importantly, our imaging study shows that the NF-κB activation is early but transient under TNFα stimulation, late but sustained under LPS stimulation, and early and sustained under the combined stimulation. Finally, we also found that TPCA-1 10 μmol·L^-1 or andrographolide 30 μmol·L^-1 drastically reduced the IL-6 and -8 levels down to 4.5-9 folds as compared to the controls. CONCLUSIONThe combined TNFα and LPS stimulations induce faster and more sustained inflammatory responses, which can still be suppressed by anti-inflammatory compounds in our cell model. These more comprehensive activations of inflammatory signaling pathways will enable us to study and distinguish the mechanisms of different anti-inflammatory compounds or natural products.

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Therapeutic effects of velvet antler polypeptides on hepatic fibrosis in rats

Leng-xin DUAN, Cai-e WANG, Ji-le XIN, Yi-meng DUAN, Jian-gang WANG

2015, 29(S1): 56-57.

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OBJECTIVE To explore the therapeutic effects and underlying mechanisms of velvet antler polypeptides (VVAPs) in CCl₄-induced experimental hepatic fibrosis in rats. METHODS Anti-hepatic fibrosis properties of VAPs were tested by Subcutaneous injection (SC) into male Wistar rats of CCl₄-induced experimental hepatic fibrosis. After SC injections for 45 consecutive days at doses of 5 mg·kg^-1 (low dose, VAPsL), 10 mg·kg^-1 (mid-dose, VAPsM) and 20 mg·kg^-1 (high-dose, VAPsH), the rats were sacrificed and the various indicators were evaluated and tested. Observed hepatic cells degeneration and necrosis, inflammatory infiltration and levels of serum enzymes to assess treatment of VAPs; The expression levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), MDA, and hydroxyproline (HYP) in liver tissue were analyzed; RT-PCR analysis was carried out to detect the expression levels of matrix metalloproteinases 2 (MMP-2) and tissue inhibitor of metalloproteinases 1 (TIMP-1) in liver tissue. RESULTS VAPs has obvious anti-hepatic fibrosis effects. Hepatocyte swelling, fatty degeneration was significantly reduced, reducing infiltration of inflammatory cells. Release of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) decreased significantly, reduction of hyaluronic acid (HA) and laminin (LN) obviously, at the same time, the content of total protein and albumin increased significantly in serum. Activity of SOD and GSH-Px was significantly raised and the content of MDA and HYP was reduced significantly in liver tissue. Expression levels of MMP-2 and TIMP-1 mRNA in liver were decreased significantly. These improvements were more significant in high-dos and mid-dose groups (P<0.05 or P<0.01 vs model group). CONCLUSION These findings suggest VAPs can significant treat the hepatic fibrosis, which may be due to protect liver cells and improve liver functions by hydroxyl radical scavenging activity and great effect of antioxidation, and decrease the gene expression of MMP-2, improving exist-environment of liver cells and decreasing the gene expression of TIMP-1, prompting degradation of extracellular matrix.

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Pharmacokinetic of nasal curcumin: An approach for asthma medication

Subhashini SINI, Preeti Singh CHAUHAN, Ruchi CHAWLA, D DASH, Rashmi SINGH

2015, 29(S1): 57-58.

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OBJECTIVE The study aims to evaluate intranasal (i.n.) curcumin at 5 mg·kg^-1, its absorption through nasal mucosa reaching blood and lungs and investigate its anti-allergic and anti-asthmatic potentiality in ameliorating ovalbumin induced asthma in mouse model. METHODS A simple and sensitive high performance liquid chromatography method using UV detection (HPLC-UV) was developed and validated for the determination of nasal curcumin 5 mg·kg^-1 in nasal mucosa, plasma and lungs from 15 min-6 h of post dosing and further applied to determine the pharmacokinetics parameter. Further, for the anti-asthmatic study, BALB/c mice were sensitized (day 1, 7 and 14) and challenged with ovalbumin (day 19-22) and treated with intranasal curcumin 5 mg·kg^-1 (in the form of nasal drops) before an hour of challenge (day 19-22) to investigate its therapeutic effect on various parameters of airway inflammation as detected in the bronchoalveolar lavage fluid, serum and lung tissue. Serum was also used to study the liver kidney function test for the toxicity. RESULTS The validated method of HPLC was sensitive with a lower limit of quantitation of 5 μg·mL^-1 and the calibration curve represented good linearity (r²≥0.999) over the linear range of 5-50 μg·mL^-1. HPLC study reveals, absorption and quantification of curcumin as 1.9 μg·mL^-1 in the nasal mucosal scrapping at 15 min elevating to 4.9 μg·mL^-1 till 1 h and declining to 3.2 μg·mL^-1 till 3 h after intranasal administration of curcumin (5 mg·kg^-1). The plasma showed 0.9 μg·mL^-1 after 15 min spiking upto 1.5 μg·mL^-1 till 3 h while lung homogenate retained up to 3.6 μg·mL^-1 of curcumin till 3 h, which was detectable from 15 min (0.27 μg·mL^-1) and was on higher side as compared to earlier studies. The same pharmacological dose (5 mg·kg^-1, i.n.) has shown anti-asthmatic potential by inhibiting airway inflammation (eosinophilic inflammation), bronchoconstriction and modulation in cytokines level of Th2 (IL-4, IL-5), Th1 (IFN-γ) and proinflammatory (TNF-α) cytokines in ovalbumin induced asthma without having any side effect as detected by liver kidney function test. CONCLUSION The study reveals nasal mucosa as a viable route for the absorption of intranasal curcumin and accommodating increased transportation to the blood and lungs. Also, the nasal route is effective in retaining the level of curcumin till 6 h without any degradation and hence could be a promising route to improve its biological activities. Present study may prove the possibility of curcumin as complementary medication in the development of nasal drops or through nebulizer in human subjects. Further, pharmacodynamic study is in progress to prove its effectiveness not only in pulmonary disorders but also for systemic disorders.

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Synergistic effect of rosmarinic acid with antibiotics against Staphylococcus aureus and MRSA

Sanmuga Priya EKAMBARAM, Nathiya MARAPPAN, Vinodhini VISWANATHAN, Sabari Srinivasan G, Ajay B

2015, 29(S1): 58-58.

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OBJECTIVE To evaluate the synergistic effect of rosmarinic acid (RA) with antibiotics against Staphylococcus aureus(S. aureus) and MRSA and to identify the possible mechanism of action responsible for synergism if any. METHODS The antibacterial activity of Rosmarinic acid was studied for its zone of inhibition by agar well diffusion and MIC determination by liquid broth dilution technique against MRSA and VRSA. The synergistic effect of RA with antibiotics like amoxicillin, ofloxacin and vancomycin was evaluated by Broth checker board method and further confirmed with time kill kinetic studies. Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) were isolated by protein precipitation technique and its expression was studied by SDS PAGE. Further RA was evaluated for its beta lactamase inhibition property. RESULTS Rosmarinic acid exhibited antibacterial activity against S. aureus and MRSA by showing a MIC value of 0.8 mg·mL^-1 against S. aureus and 10 mg·mL^-1 against MRSA. Rosmarinic acid at 1/4 X MIC value reduced the MIC of vancomycin, amoxicillin and ofloxacin by 1/4 times against S. aureus. But against MRSA, vancomycin was found to be synergistic. All the synergistic combinations have shown FIC value of 0.5. In order to measure the kinetics of the anti-bacterial activity, the bacterial growth rate with RA, antibiotics and synergistic combinations against S. aureus and MRSA was studied. It is observed that the synergistic combinations showed better time kill kinetics as compared to RA and antibiotics. Further RA could able to destruct the cell surface proteins MSCRAMMs which was studied by SDS PAGE. RA was also found to show β lactamase inhibiting property. CONCLUSION It is concluded that RA possess antibacterial activity but at a very higher concentration (in mg/mL) against S. aureus and MRSA. However it shows synergism with antibiotics and could able to reduce the MIC of antibiotics. Thus RA could be developed as an adjuvant for antibiotics against S. aureus and MRSA caused infections. Further studies are needed to identify the mechanism for its synergism with antibiotics.

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Protective effects of NYG-1 on CCl₄-induced acute liver injury in rats

Hong-liang LI, Xuan-bin WANG, Ming LIU, Yi-bin FENG, Qiu-fang ZHANG

2015, 29(S1): 59-59.

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OBJECTIVE To study the protection effects and mechanisms of NYG-1 on CCl₄-induced acute liver injury. METHODS Acute liver injury model of rats was established by using CCl₄. 48 male SPF SD rats were weighed and randomly divided into six groups with 8 in each group, normal group, model group, positive control group (silibinin), low-, medium- and high-dose NYG-1 group. Silibinin was given orally to rats in the positive control group, NYG-1 (high-, medium- and low-dose) was given orally in the high-, medium- and low-dose NYG-1 group, respectively. Those rats were administered appropriately according to the group once daily for seven consecutive days. On the seventh day, rats were treated with 10% CCl₄ (10 mL·kg^-1 of 0.1% CCl₄ solution in olive oil) intraperitoneally injecting (ip) to induce acute liver injury, except the normal group. At 16 h after CCl₄ treatment, rats were weighed, then anaesthed with ether, the blood and liver were collected. Serum ALT, AST, LDH and ALP were measured. MDA content and SOD activity in liver homogenate were detected. The histopathological changes of liver were observed by H&E staining. RESULTS Acute liver injury model was established successfully in rats by intraperitoneally injecting CCl₄. Pretreatment with medium and high dose NYG-1 decreased the increase of ALT, AST and MDA induced by CCl₄, but it had no influence on serum LDH, ALP level and SOD activity in the liver homogenate. CONCLUSION The obtained results suggest that oral administration of NYG-1 hasve the protective effects against CCl₄-induced acute hepatic injury in rats, Its mechanism may be related to antioxidant-like action.

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In vivo and in vitro anti-arthritic efficacy of a herbal formula consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos

Brian Chi Yan CHENG, Hui GUO, Tao SU, Xiu-qiong FU, Ting LI, Zhi-ling YU

2015, 29(S1): 59-60.

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OBJECTIVE Rheumatoid arthritis (RA) is the most common inflammatory autoimmune disease, affecting around 1% of the world population. Toll-like receptor 4 (TLR4) signalling has been found to be involved in the pathogenesis of RA. It is a potential therapeutic target for RA treatment. A herbal formula (RL) consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos has traditionally been used in treating various inflammatory disorders. In this study, we would evaluate the anti-arthritic effect of RL on collagen-induced arthritis (CIA) in rats and investigate the involvement of TLR4 signaling in the mode of action of RL in vivo and in vitro. METHODS In vivo anti-arthritic efficacy was evaluated using CIA rats induced by bovine typeⅡ collagen. The treatment groups were treated with various concentrations of RL or positive control indomethacin for 35 d. Clinical signs (hind paw volume and arthritis severity scores), changes in serum inflammatory mediators, histological and radiographic changes of joints were investigated. Spleens and peritoneal macrophages were used to determine the effects of RL on innate and adaptive immune responses in CIA rats. The involvement of TLR4 signalling pathways in the anti-arthritic effect of RL was examined in cartilage tissue of CIA rats, murine RAW264.7 macrophages and human THP-1 monocytic cells. RESULTS The severity of arthritis in the CIA rats was significantly attenuated by RL. Histological score and radiographic score were efficiently improved by RL. RL could also dose-dependently inhibit pro-inflammatory cytokines in serum of CIA rats. RL significantly inhibited the production of various pro-inflammatory mediators, the expression and/or activity of the components of TLR4 signalling pathways in animal tissue and cell lines. CONCLUSION RL possesses anti-arthritic effect on collagen-induced arthritis in rats. The therapeutic effect of RL may be related to its inhibition on pro-inflammatory cytokines in serum. The inhibition of the TLR4/TAK1/NF-κB and TLR4/TAK1/MAPK pathways participate in the anti-arthritic effects of RL. This provides a pharmacological justification for the use of RL in the control of various arthritic diseases. Further investigation should be done to develop RL into a modern anti-arthritic agent.

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Protective effects of 3'-daidzein sulfonate sodium on chronic hepatic injury by regulating the T lymphocyte subsets in mice

Jing ZENG, Rui-zhen LIU, Gui-lin WEI, Liang-dong LI, Zhi-hua HUANG

2015, 29(S1): 60-60.

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OBJECTIVE To explore the protective effects of 3'-daidzein sulfonate sodium on chronic hepatic injury induced by carbon tetrachloride(CCl₄) in mice and investigate the mechanism about regulating the T lymphocyte subsets. METHODS Healthy Kunming male mice were randomly divided in 5 groups: control group, model group, bifendate positive control group(2.5 mg·kg^-1), low and high dose 3'-daidzein sulfonate sodium groups(0.1 and 0.3 mg·kg^-1). The chronic hepatic injury mice were made by intraperitoneal injection of 10% CCl₄ plant oil solution twice a week, and sustained for six weeks. At the same time, the mice were treated with normal saline, bifendate (2.5 mg·kg^-1) and 3'-daidzein sulfonate sodium (0.1 and 0.3 mg·kg^-1), respectively by ig administration once a day and continued for six weeks. After the last administration, the mice blood and liver were taken. Using automatic biochemical analyzer survey the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum. Flow cytometry was used to detect T lymphocyte subsets, enzymes analysis technique was used to observe the liver function(CD3⁺,CD4⁺ and CD8⁺), HemateinEosin stain was used to explore the changes in liver morphology. RESULTS The level of ALT and AST in the serum in model group mice increased significantly. While the level of ALT and AST in the serum in 3'-daidzein sulfonate sodium (0.1 or 0.3 mg·kg^-1, ig) groups mice decreased compared with the model group (P<0.05). And compared with the model group, the ratios of CD3⁺,CD4^-CD8^- and CD4⁺/CD8⁺ decreased,and the ratios of CD8⁺ increased in 3'-daidzein sulfonate sodium groups(0.1 and 0.3 mg·kg^-1, ig; P<0.05). CONCLUSION3'-daidzein sulfonate sodium have a protective effect on CCl₄-induced liver injury mice, and it can result in the changes of T lymphocyte subsets, which may be one of the factors leading to hepatic injury by CCl₄.

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Icaritin requires phosphatidylinositol 3 kinase/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

Zong-kang ZHANG, Jie LI, Wing-nang LEUNG, Ge ZHANG, Bao-ting ZHANG

2015, 29(S1): 61-61.

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OBJECTIVE Skeletal muscle undergoes rapid and profound atrophy in response to decreased mechanical loading, e.g., limb immobilization and bed rest. Phosphatidylinositol 3 kinase (PI₃K)/Akt signaling pathway is critical for regulating the balance between protein synthesis and degradation during disuse/inactivity-induced muscle atrophy. The present study aimed to investigate whether natural product Icaritin (ICT) required PI₃K/Akt signaling to exert counteractive effect on skeletal muscle atrophy following mechanical unloading. METHODS Two oral dosages of ICT (80 and 120 mg·kg^-1·d^-1) were administrated daily to adult male rats with or without daily injection of PI3K/Akt signaling inhibitor wortmannin (15 μg·kg^-1·d^-1) during 28-d hindlimb suspension (HS). Ex vivo muscle functional testing, histological and immunohistochemical analysis were performed to determine the changes of soleus muscle function, mean muscle fiber cross-sectional area (CSA) and fiber type distribution. Western blot and real-time PCR analysis were also performed to evaluate the protein or mRNA expression of the markers involved in PI₃K/Akt signaling pathway. RESULTS After 28-d HS, soleus muscle underwent profound muscle atrophy (-52.7% muscle mass vs. pre-HS baseline). The high dose ICT treatment significantly attenuated the decreases in soleus muscle mass (+22.6% vs. HS), muscle fiber CSA (+52.8% vs. HS), as well as the muscle functional testing parameters during the unloading. Molecularly, the high dose ICT treatment significantly attenuated the decreases in protein synthesis markers at protein levels (phosphorylation of Akt and its downstream proteins) during the unloading, whereas the increases in protein degradation markers at mRNA (atrogin-1 and MuRF-1) and protein (nuclear FOXO1 and FOXO3a) levels during the unloading were significantly attenuated by the high dose ICT treatment. The low dose ICT treatment moderately attenuated the above changes induced by the unloading. Mechanistically, Wortmannin could abolish the above effects of ICT. CONCLUSION ICT requires participation of PI3K/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading in a dose-dependent manner.

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Anti-inflammation and anti-fibrosis actions of resveratrol on experimental pneumoconiosis

Jie LI, Zong-kang ZHANG, Yue QU, Wing-Nang LEUNG, Bao-ting ZHANG

2015, 29(S1): 62-62.

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OBJECTIVE To investigate the therapeutic effects and related signaling pathways involved in the actions of resveratrol on experimental pneumoconiosis in vivo and in vitro. METHODS The pneumoconiosis animal model was induced by exposing male SD rats to 15 mg·m^-3 silica aerosol in an inhalation chamber system for 6 h·d^-1, 5 d·week^-1 for up to 8 weeks. The vehicle or resveratrol (10 or 20 mg·kg^-1) was preventively or remedially administered to the rats during or after the 4- or 8-week silica exposure (SE) period, respectively. After 4-, 8-, and up to 14-week treatment, in vivo near-infrared fluorescence imaging analysis and histological analysis were performed to evaluate the pathological changes in rat lung. Inflammatory cytokines level in bronchoalveolar lavage fluid (BALF) was measured by ELISA testing, and the deposition of fibrotic collagen proteins in lung parenchyma was determined by western blotting and immunohistochemistry analysis. Microarray analysis was performed to screen the signaling pathways involved in the actions of resveratrol on pneumoconiosis in vitro models. Anti-inflammation action and signaling of resveratrol was evaluated on silica-stimulated rat alveolar macrophage, which is one of the crucial effector cells for silica-induced inflammatory response; anti-fibrosis action and signaling of resveratrol was evaluated on TGF-β-induced human lung fibroblast, which acts as a promoter in the later fibrotic process of pneumoconiosis. RESULTS Silica aerosol exposure significantly increased macrophage infiltration and matrix metalloproteinases activity in lung tissue concomitant with the increased levels of inflammatory mediators in BALF. Preventive treatment with resveratrol (20 mg·kg^-1·d^-1) reversed all these biochemical indices as well as histopathological alterations induced by silica exposure. Post-SE resveratrol treatment mildly reduced silica-induced inflammatory response in rat lung with no statistical significance. In vitro study revealed that resveratrol could inhibit alveolar macrophage cell death and decrease the levels of IL-1β and TNF-α induced by silica particle exposure to cultured alveolar macrophages. Resveratrol was further shown to inhibit the nuclear transition of NF-κB and formation of cleaved caspase-1. Encouragingly, resveratrol preventively attenuated the lung fibrosis, evidenced by less fibrotic nodules formation and collagen proteins expression. No significant improvement on lung fibrosis was observed with post-SE resveratrol treatment. In vitro study further demonstrated that resveratrol suppressed TGF-β-induced lung fibroblast proliferation and collagen deposition, concomitant with the depressed activity of TGF-β/Smad signaling in lung fibroblast. CONCLUSION Resveratrol shows the anti-inflammation and anti-fibrosis actions on experimental pneumoconiosis in vivo and in vitro models. The depression of NF-κB, NALP3-inflammasome, and TGF-β/Smad signaling pathways may be involved in the anti-inflammation and anti-fibrosis actions of resveratrol, respectively. Resveratrol could be a potential therapeutic agent for the intervention of pneumoconiosis.

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Enhanced scratching elicited by a pruritogen and an algogen in a mouse model of contact hypersensitivity

Kai FU, Lin-tao QU, Steven G. SHIMADA, Hong NIE, Robert H LaMOTTE

2015, 29(S1): 62-63.

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OBJECTIVE To investigate whether normally pruritic and algesic chemicals evoked abnormal itch- or pain-like behaviors in the mouse after the development of contact hypersensitivity (CHS), an animal model of allergic contact dermatitis. METHODS Mice previously sensitized to a hapten (squaric acid dibutylester) applied to the abdomen, exhibited spontaneous itch-like scratching and pain-like wiping directed to the site on the cheek of the CHS elicited by a subsequent challenge with the same hapten. At 24 h after the second challenge, each mouse was placed in the test container and its pontaneous behavior was recorded for 30 min. Then each mouse received an intradermal injection into the previously challenged cheek of 5 μL of a chemical solution(a normal saline vehicle alone or the vehicle containing either histamine(5 μg), BAM8-22 (1 μg) or bradykinin (2.65 μg) and returned to the container to have its behavior recorded for another 30 min. RESULTS In comparison with the mean numbers of site-directed behaviors evoked by the injection of normal saline vehicle,bradykinin evoked significantly more wipes but not more bouts of scratching whereas histamine and BAM8-22 each elicited significantly more bouts of scratching but not more wipes. In comparison with acetone treated controls, the CHS mice displayed a significantly greater number of bouts of spontaneous site-directed scratching and wiping to the SADBE challenged skin after the second challenge (scratching: 1.2±0.67 vs 16.4± 3.40, wiping: 1.0±0.75 vs 27.8±5.96, respectively, P<0.05). BAM8-22 and bradykinin, but not histamine, each evoked a significantly greater number of scratching bouts in CHS mice than that in control mice. There were no significant differences in the mean number of wipes evoked by these chemicals except for BAM8-22 which elicited a greater number in CHS mice, when the mean number of bouts of scratching (or wipes) in response to saline injection was subtracted from the respective number of responses to each chemical. CONCLUSION CHS enhanced the itch-like scratching behavior evoked by a histamine-independent pruritic chemical, BAM8-22, but not the itch of histamine. Bradykinin, which evoked only pain-like behavior of wiping in normal skin, evoked scratching in addition to wiping in an area of CHS. It is speculated that these alterations in sensory behaviors may result from the selective sensitization of peripheral and/or central pruriceptive neurons.

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Protective effect of Nigella sativa L. to gastric mucous of indomethacin-induced gastric ulcer rats

Umi KALSUM, Renata PRIMASARI, Dewa AYU, Mudjiwijono HANDARU

2015, 29(S1): 63-64.

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OBJECTIVE To investigate the effect of Nigella sativa L. as gastric protective on indomethacin-induced rats. METHODS the design of this research is randomized post test control group design. The rats were randomly divided into 5 groups which 5 rats in each. Rats were fasted for 8 h before treatment. The first group was a control group (only gave aquadest as vehicle orally). The second group was subjected to induced with indomethacin 30 mg·kg^-1. The rest groups were subjected to induced by indomethacin and methanolic extract of Nigella sativa L. 200, 300 and 400 mg·kg^-1 every 8 h for 24 h, respectively, for third, fourth and fifth group. Rats were sacrificed after anesthetized with ketamine and gastric were washed before observed. Macroscopic observation based on a score of lesion and microscopic observation on gastric based by histological HE staining. Whole data were analysis of an ANOVA statistical program. RESULTS The administration of Nigella sativa L. significantly decreased gastric ulcer macroscopically starting at dose 100, 200 and 300 mg·kg^-1(P<0.05). Microscopic observation showed significant decreasing at dose 200 and 300 mg·kg^-1(P<0.05). Interestingly, there was no significant different between control and dose 300 mg·kg^-1. Negative correlation between lesion and doses were -0.919, -0.953 for macroscopic and microscopic lesion respectively. It means there was strong correlation between dose and lesion, higher dose lesser lesion. The mechanism of gastric protective of Nigella sativa L. may caused by the bioactive compound such as thymoquinone which known as antiinflammation and antioxidant. CONCLUSION Methanolic extract of Nigella sativa L. decreased peptic ulcer both macroscopic and microscopic conditions on indomethacin-induced rats.

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炎症与免疫

In vitro and in vivo immunomodulatory activities of polysaccharides purified from four species of Dendrobium

Lei SHI, Hui CAI, Seng Poon ONG, Feng TIAN

2015, 29(S1): 64-64.

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OBJECTIVE To investigate the isolation, extraction, purification, and immunomodulatory activities in vitro and in vivo of polysaccharides from four different species of Dendrobium herbs widely used in Singapore. METHODS To apply the classic method of water extraction and ethanol precipitation and the gel column chromatography technique, the polysaccharides from four species of Dendrobium herbs (D. moniliforme, D. crystallinum, D. primulinum, D. chrysanthum) were obtained. The polysaccharides were tested first on RAW 264.7 cell line with MTT method to test the effect of polysaccharides on macrophages. The two polysaccharides from D. moniliforme were found to promote proliferation of the cells, and were then tested on mice splenocyte primary cell culture. The same polysaccharides were also injected to female BALB/c mice at various doses for 21 d to see whether they would have affected the organs, especially the livers, spleens and thymuses. RESULTS Out of the 4 Dendrobium species tested, it was found that polysaccharides from D. moniliforme to be most supportive of the proliferation of macrophages in a dose-dependent manner, with increasing effect with the increasing doses used. Polysaccharides from D. chrysanthum and D. crystallinum were also found to act similarly albeit to a lesser extent. The two D. moniliforme polysaccharides were also found to greatly enhance the proliferation of mouse splenocytes in vitro and growth of the spleen in vivo. CONCLUSION The immunomodulatory activities in vitro and in vivo of polysaccharides from four species of Dendrobium herbs were different. Their diverse chemical characters might contribute to their varied bioactivity, which should be further investigated. In addition, it was suggested that the polysaccharides from D. moniliforme could be potential immunostimulants used in the healthcare and food industry.

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The compounds from Derris laxiflora Benth suppresses lipopolysaccharide- induced inflammatory response in murine Raw264.7 cells

Ai-jen YANG, Hsi-lin CHIU, Ming-yu CHAO, Jui-ching CHEN

2015, 29(S1): 64-65.

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OBJECTIVE The emerging role of chronic inflammation is the major degenerative diseases of modern society such as periodontitis, atherosclerosis, rheumatoid arthritis, Parkinson's disease and even cancer. Eight components were isolated from Derris laxiflora Benth., In this study, we found these compounds from Derris laxiflora Benth suppress lipopolysaccharide-induced inflammatory response in murine macrophage (RAW 264.7) cells. METHODS RAW 264.7 cells were cultured in DMEM media supplemented with 10% (V/V) heated-inactivated FBS, penicillin 100 U·mL^-1 and streptomycin 100 μg·mL^-1. The cells were incubated at 37℃ in a humidified atmosphere of 5%CO₂ in air. RAW264.7 cells were seeded in a 24-well plate at a density of 2×10⁵ mL^-1 and then incubated with or without LPS (100 ng·mL^-1) in the absence or presence of compounds for 24 h. Effects of these isolates on NO production were measured indirectly by analysis of nitrite levels using the Griess reaction. Quercetin was used as a positive control. RESULTS ight components were isolated from Derris laxiflora Benth., including three new pterocarpans 7,6'-dihydroxy-3'-methoxypterocarpan (1), derrispisatin (2), derriscoumaronochromone (3) and three new flavonoids cis-3,4'-dihydroxy-5,7-dimethoxyflavan (4), derriflavanone B (5), iso-lupinenol (6) as well as two known ones, lonchocarpol A (7) and lonchocarpol D (8). The structures of these new compounds were determined by analysis of their spectroscopic data. Raw264.7 cells were treated with the compounds from Derris laxiflora Benth for 24 h. Among them, compounds 5, 7 and 8 significantly suppressed the NO production in LPS-treated RAW264.7 cells with IC₅₀ values <10 μg·mL^-1. CONCLUSION In this study, we found that compounds from Derris laxiflora Benth suppresses lipopolysaccharide-induced inflammatory response in murine Raw264.7 cells.

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炎症与免疫

Effect of mangosteen (Garcinia mangostana Linn) peel extract on migration of endothelial cells treated with lipopolysaccharide

Dian NUGRAHENNY, Nur PERMATASARI, Adianto JAYANAGARA, Titin Andri WIHASTUTI, Djanggan SARGOWO

2015, 29(S1): 65-65.

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OBJECTIVE This study was aimed to evaluate the effect of mangosteen peel extract (MPE) on migration of human umbilical vein endothelial cells (HUVECs) treated with LPS. METHODS Cultured HUVECs were divided into five groups: normal (untreated) group, positive control group treated with LPS at dose of 20 ng·mL^-1 for 24 h, and groups pre-treated with LPS at dose of 20 ng·mL^-1 for 24 h followed by incubation with MPE at doses of 1, 2, and 4 μg·mL^-1, respectively. HUVECs migration was evaluated using wound-healing migration assay after 24 h of treatment. RESULTS LPS significantly (P<0.05) impaired HUVECs migration compared to the normal. MPE at dose of 1 μg·mL^-1 increased migration of LPS-treated HUVECs significantly (P<0.05) compared to the positive control and near to the normal level. CONCLUSION The mangosteen peel extract is able to dose-dependently preserve the endothelial cells function.

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炎症与免疫

Methanolic extract of ceplukan leaf (Physalis minima L.) attenuates ventricular fibrosis through inhibition of TNF-α in ovariectomized rats

Lestari BAYU, Permatasari NUR, SaifurRohman MOHAMMAD

2015, 29(S1): 65-66.

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OBJECTIVE To investigate the effects of ceplukan leaf (Physalis minima L), which contain phytoestrogen physalin and withanolides, toward ventricular TNF-α level and fibrosis in ovariectomized rats. METHODS Wistar rats divided into six groups (K1: normal; K2: 5 weeks ovariectomy (OVX); K3: 9 weeks ovariectomy (OVX), K4, K5, and K5: 9 weeks OVX+4 weeks ceplukan leaf's methanolic extract dose 500, 1500, and 2500 mg·kg^-1, respectively. TNF-α level measured with ELISA method. Fibrosis measured as blue color percentage in Masson's Trichrome staining. RESULTS This study showed that prolonged hypoestrogen increase ventricular fibrosis (P<0.05). Ceplukan leaf treatment also resulted in a decreased ventricular fibrosis and TNF-α level in dose dependent manner compared with those of without treatment group (P<0.05). Furthermore, the TNF-α level normalized in rat treated with 2500 mg·kg^-1 Physalis minima L (P<0.05). Reduction of fibrosis positively correlated with TNF-α level (P<0.05, r= 0.873). CONCLUSION Methanolic extract of ceplukan leaf decrease ventricular fibrosis through inhibition of ventricular TNF-α in ovariectomized rats. Duration of hypoestrogen increase ventricular fibrosis.

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炎症与免疫

Cucurbitacin E induces apoptosis and attenuates activation of hepatic stellate cells via PI₃K/Akt-AMPK-mTOR signaling pathway

You-li YAO, Li-hua LIAN, Shuang JIANG, Ji-xing NAN, Yan-ling WU

2015, 29(S1): 66-66.

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OBJECTIVE Hepatic fibrosis is a wound-healing response for injury. Activated hepatic stellate cells (HSCs) are the preferred targets of anti-hepatic fibrotic therapies. cucurbitacin E (CuE) is, one well-known natural compound derived from traditional Chinese medicine, used in Asian countries for the prevention and treatment of hepatic disease. Therefore, the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs. METHODS The murine HSC (t-HSC/Cl-6) cell line were incubated in 96-well plates and treated with TNF-α and CuE at various concentrations and indicated times. Cell viability was assessed with MTT assay. Another, t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α, CuE, AICAR or metformin for the indicated time and concentration. Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR. RESULTS CuE inhibited cell proliferation of activated HSC/T-6 cells in concentration- and time-dependent manner. CuE triggered the activation of caspase-3, cleaved the PARP, ration of bcl-2/bax, and cytochrom c protein in a time- and concentration-dependent manner. CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK. CuE inhibited the protein and mRNA expressions of α-SMA, TIMP-1 and collagenⅠ in activated HSC-T6. CuE broadly blocked p-PI3K, p-Akt, p-mTOR and p-p70S6K expressions. CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin. And metformin showed significantly higher activation of AMPK than AICAR. Ability of CuE on activation of AMPK was between AICAR and metformin. It's also found that CuE significantly decreased p-mTOR as well as AICAR and metformin. CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment. The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI₃K/Akt-AMPK-mTOR signaling.

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炎症与免疫

Inhibitory activity of Cinnamomum Cassia extract on hypersensitivity in mice

Seunggyeong LEE, Jungbin SONG, Donghun LEE, Young-Sik KIM, Hyun Soo KIM, Hailing GUO, Juyeon PARK, Hocheol KIM

2015, 29(S1): 67-67.

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OBJECTIVE Cinnamomum cassia has been used in traditional medicine in East Asian countries to treatallergic disease. Previous studies have demonstrated that C. cassiainhibits the development of mite antigen-induced skin lesions in NC/Nga mice by suppressing the Thelper 2 cell response. The objective of this study is to investigate whether C. cassia has an efficient inhibition on systemic anaphylaxis and contact hypersensitivity in mice. METHODS To induce systemic anaphylaxis,mice were injected intraperitoneally with compound 48/80. Mortality was monitored for 1 h after the injection. Contact hypersensitivity reaction was induced by topical application of 2,4-dinitrofluorobenzene (DNFB) to the ears of mice. After DNFB-challenge,ear thickness was measured 0, 24 and 48 h. RESULTS We discovered that C. cassia significantly inhibited compound 48/80-induced anaphylaxis and DNFB-induced ear swelling. Compound 48/80-induced anaphylaxiswas completely inhibited at the dose of 60 mg·kg^-1 body weight. Ear swelling at 24 h after DNFB-challenge was significantly less than that in the control group (0.16±0.03 vs. 0.32±0.03 mm, P<0.01), while C. cassia was administered at the dose of 200 mg·kg^-1. CONCLUSION These findings indicate that C. cassia suppresses allergic reactions, thus has potential value for mitigation of atopic dermatitis.

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炎症与免疫

P2X7 receptor inhibitor suppressed extracellular ATP/LPS-primed human hepatic stellate cells activation via downregulating NLRP3 inflammasome

Shuang JIANG, Quan JIN, Yan-ling WU, You-li YAO, Ji-xing NAN, Li-hua LIAN

2015, 29(S1): 67-68.

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OBJECTIVE To investigate the effect of P2X7 receptor (P2X7r) inhibition, using a specific inhibitor (A438079) to prevent the development of liver fibrosis on human hepatic stellate cells, LX-2. METHODS The supernatant from lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophages was supplemented to LX-2 cells for 24 h. LX-2 cells were primed with LPS for 4 h and subsequently stimulated for 30 min with 3 mmol·L^-1 of adenosine 5'-triphosphate (ATP). A438079 (10 μmol·L^-1) was supplemented to LX-2 cells 10 min prior to ATP. RESULTS Directly treated with LPS on LX-2 cells, mRNA expressions of IL-1β, IL-18 and IL-6 were increased, as well as P2X7r. And caspase-1, ASC and NLRP3 mRNA expressions were increased with LPS stimulation. LPS stimulation also increased α-SMA and collagenⅠ mRNA expressions. Interestingly treatment of LX-2 cells with mediums from LPS-primed RAW264.7 mouse macrophages exhibited greater increase of mRNA expressions of above genes than those in LX-2 directly treated with LPS. Pretreatment of directly or indirectly LPS-stimulated LX-2 cells with A438079 both suppressed IL-1β mRNA expression. In addition treatment of LPS-primed LX-2 cells with 3 mmol·L^-1 ATP induced the significant increase of IL-1β, IL-6, caspase-1, pannexin-1, α-SMA and collagenⅠ mRNA expression, the increasing of α-SMA protein expression and cleavage of IL-1β. These events were significantly suppressed by pretreatment with P2X7r antagonist A438079. P2X7r blockade also significantly reduced the protein expression of α-SMA. CONCLUSION Our results suggest that the involvement of the P2X7r-NLRP3 inflammasome pathway in the secretion of IL-1β from extracellular ATP/LPS-stimulated human hepatic stellate cells. This study demonstrated that repression of the P2X7r represents a novel potential therapeutic approach to control liver fibrosis.

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炎症与免疫

Possible role of mitochondrial permeability transition pore in hepatoprotective potentials of epigallocatachin gallate in paracetamol induced hepatotoxicity in rats

Gurfateh SINGH, Deepika BHATIA, SLHARIKUMAR

2015, 29(S1): 68-68.

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OBJECTIVE To present study was designed to investigate the hepatoprotective effect of epigallocatechin gallate (EGCG) in experimentally induced liver dysfunction in rats. METHODS Rats were administered paracetamol (3 g·kg^-1, po) on 3rd and 5th day to 7 d of experiment to produce hepatotoxicity in Wistar rats. The atractyloside (MPTP opener) was administered at the dose (5 mg·kg^-1, po) for 7 d. RESULTS Paracetamol caused increase in SGPT, SGOT, bilirubin, TBARS and decrease in SOD level in rats. However, pretreatment with EGCG (40 mg·kg^-1 po) in paracetamol group significantly reversed the effect of this hepatotoxicant and further resulted in decrease inflammatory cell infiltration, vacuolization and centrilobular necrosis as revealed by histological findings of rat liver in present study. Atractyloside (a selective MPTP opner) at the dose of 5 mg·kg^-1, po was given with EGCG for 7 d in paracetamol treated rats. Treatment with EGCG and atractyloside significantly a bolished the hepatoprotective effect of EGCG in paracetamol treated rats when compared with EGCG pretreated paracetamol group. CONCLUSION The finding of present investigation may conclude that paracetamol causes severe liver damage which is characterized by increased oxidative stress and mitochondrial dysfunction due to opening of MPTP. This study demonstrates the heptoprotective potentiation of EGCG may be due to cellular protective mechanisms like preventing the opening of MPTP, anti-oxidative, anti-inflammatory, membrane stabilization effects in paracetamol induced hepatotoxicity in rats.

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Active component of Isatidis Radix on insulin resistance in diabetes mellitus rat

Ji-ping LI, Tian-jiao HU, Zhen JIANG, Mo-xiang LIU

2015, 29(S1): 68-69.

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OBJECTIVE To investigate the role of active component of Isatidis Radix on insulin resistance in the diabetes mellitus rat. METHODS To induce diabetic rat model with long-term high sugar and high fat plus low-dose streptozotocin(25 mg·kg^-1). Then rats were randomly divided into 6 groups: control group, model group, rosiglitazone maleate group (0.3 mg·kg^-1), high(100 mg·kg^-1), middle(50 mg·kg^-1) and low(25 mg·kg^-1) active component of Isatidis Radix group. Drugs were adiministered orally once a day. After four weeks, following substances were measured: serum fasting blood glucose, total cholesterol, triglycerides, high density lipoprotein, low density lipoprotein, free fatty acids, fasting insulin, insulin index, pathological observation and immunohistochemistry technology of pancreas islet. RESULTS High and middle active component of Isatidis Radix group could decrease serum FBG, TC, TG, LDL, FFA, FINS and increase serum HDL, ISI; the damage of the pancreas islet has been restoration partly. CONCLUSION Active component of Isatidis Radix could improve insulin resistance in diabetic rats, which might be related to improvement of the function of pancreas islet.

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代谢性疾病

Investigating the prescription compatibility of Rhodiolarosea, Cordycepsmilitaris, and Rhubarb root for the treatment of metabolic syndrome

Jin-ying TIAN, Ling CHEN, Xiao-lin ZHANG, Jing HAN, Pei-cheng ZHANG, Fei YE

2015, 29(S1): 69-70.

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OBJECTIVE To investigate the compatibility of the prescription Rhodiolarosea, Cordycepsmilitaris, and Rhubarb root. METHODS The DIO mice accompanying with insulin resistance, hypercholesterolemia and obesity were induced by high fat diet (HFD) in C57BL/6 mice. For evaluating the insulin sensitivity in vivo, the values of serum insulin, HOMA-IR, and the glucose infuse rate (GIR) in hyperinsulinemic-euglycemic clamp test were determined; the time-dependent blood glucose changes were measured after insulin loading in insulin tolerance test (ITT), or after glucose loading in glucose tolerance test (GTT), respectively. Recombinant human protein tyrosine phosphatase 1B (PTP1B) was overexpressed by hGST-PTP1B-BL21 E.coli and purified by GST affinity chromatography. The activities of PTP1B and glutamine: fructose 6-phosphate amidotransferase (GFAT) were measured by the pNPP and GDH method, respectively. The activity of α-glucosidase was determined with the substrates of p-nitrophenyl-alpha-D-glucopyranoside (pNPG). The IC₅₀ value was calculated using the software GraphPad Prism 5. RESULTS 1st, the standard for the raw materials and for the extraction process of Rhodiolarosea, Cordycepsmilitaris, and Rhubarb root are formulated according to the inhibition on PTP1B, α-glucosidase, and GFAT in vitro, and the improvement on impaired glucose tolerance (IGT), hypercholesterolemia, and obesity in vivo, respectively. 2nd, the composition was determined by the synergistic effects of Rhodiolarosea, Cordycepsmilitaris, and Rhubarb root on insulin resistance in DIO mice. 3rd, in accordance with the IC₅₀ values from the L₉(3⁴) and L₈(2⁷) orthogonal experiments targeting on PTP1B and α-glucosidase, respectively, the best rate of Rhodiolarosea:Cordycepsmilitaris:Rhubarb root was selected. 4th, the effective dose for the treatment of metabolic syndrome was assessed based on the effects on IGT, hypercholesterolemia, obesity, insulin resistance, respectively, in DIO mice. CONCLUSION The prescription compatibility of Rhodiolarosea, Cordycepsmilitaris, and Rhubarb root with the rate of 20:1:1 was formed to treat the metabolic syndrome, such as IGT, hypercholesterolemia, and obesity, with the effective dose of 200 mg·kg^-1 by the major mechanism of the improvement on insulin sensitivity. Monarch drug, Rhodiola, can clear away the lung-heat, tonify Qi, resolve stasis and nourish the heart. Adjuvant drug, Cordycepsmilitaris, can tonify the lung Qi and the kidney essence, strong waist and knee, compatibility with Rhodiola to enhance the function of invigorating lung and kidney. Assistant drug, Rhubarb, can clear heat, detoxify, remove blood stasis. These three herbal are compatible to show the effects of tonifying Qi, nourishing essence, cleaning heat, reducing phlegm and resolving masses for the treatment of metabolic syndrome.

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Pandanus amaryllifolius leaf extract improves insulin sensitivity in high-fat diet-induced obese mice

Suphaket SAENTAWEESUK, Jarinyaporn NAOWABOOT, Nuntiya SOMPARN

2015, 29(S1): 70-70.

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OBJECTIVE To investigate the effect of Pandanus amaryllifolius leaf in high-fat diet-induced insulin resistance in mice model. METHODS To induce obesity, male ICR mice were fed with a high-fat diet (45% fat) for six weeks. The mice were divided into four groups (n=8): non-obese control mice were treated with 5% gum arabic and obese mice were treated with Pandanus amaryllifolius (125 and 250 mg·kg^-1 ·d^-1), or 5% gum arabic. After six weeks of treatments, the fasting blood glucose, serum insulin, OGTT and fat cell protein expression of glucose transporter 4 (GLUT4) were determined. RESULTS Administration of Pandanus amaryllifolius showed significantly (P<0.05) reduced the high blood glucose, inhibited the abnormal increase in blood glucose level during OGTT, and decreased the high level of serum insulin. Moreover, it is interesting that the protein expression of GLUT4 was effectively increased by Pandanus amaryllifolius. CONCLUSION These findings demonstrate that the extract from Pandanus amaryllifolius leaf possesses antihyperglycemic action in obese mice by improving insulin sensitivity and stimulating GLUT4 expression in adipose tissue.

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Potential role of Tinosporacordifolia extract in prevention of diabetic complications

Pratibha NADIG, Roshni REVANKAR, Vikram SARANGAM, Shekhar DETHE

2015, 29(S1): 70-71.

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OBJECTIVE To evaluate the effect of aqueousstem extract of Tinsporacordifolia (TCSE) on 1. experimentally induced diabetic peripheral neuropathy (DPN) 2. Serum triglycerides (TG), high density lipoproteins (HDL) and atherogenic index (AI) in type 2 diabetic patients. METHODS Experimental study: TCSE was evaluated on streptozotocin induced diabetic Wistar albino rats on the reaction time(RT) to radiant heat in tail flick test. The diabetic animals were grouped into diabetic control group (DC), standard control group(SC) that received glibenclamide+metformin; TCSE 1, TCSE2, and TCSE 3 that received 100, 200 and 400 mg·kg^-1 of TCSE respectively. Another group served as the normal control group (NC). The medications were administered once daily orally for 2 weeks after the induction of diabetes. RT was compared among these groups. Clinical study: 20 Type 2 diabetes mellitus patients with TG >200 mg·dL^-1 and HDL<50 mg·dL^-1 were randomised to receive either Rosuvastatin 10 mg or TCSE 300 mg per day once daily for 12 weeks. After 12 weeks, TG, HDL and AI (calculated as the log 10 of the ratio of TG with HDL) were analysed within the group and compared between the groups. Mann Whitney U test was applied for analysis for both. RESULTS Experimental study: RT was 10.0±0.7 s in the NC, 7.83±1.19 s in DC (P<0.05 compared to NC) indicating hyperalgesia. SC, TC1, TC2, and TC3 showed 12.83±0.31, 13.33±0.88; 13.67±1.09 and 15.25±0.23 s, respectively (P<0.05 compared to DC). Clinical study: In the clinical study, in theTCSE group, TG reduced from 251.62±15.22 to 212.2±11.83 mg·dL^-1; HDL increased from 34.53±0.74 to 40.81±0.71 mg·dL^-1 and mean AI reduced from 0.83±0.028 to 0.70±0.027. In the rosuvastatin group TG levels decreased from 255.8±13.03 to 196.3±12.84 mg·dL^-1; HDL increased from 37.38±0.77 to 43.3±1.21 mg·dL^-1 and mean AI reduced from 0.83±0.02 to 0.65±0.03. The change was statistically significant within each groups but not between the groups. CONCLUSION The aqueous extract of Tinosporacordifolia appears to be effective in reducing the diabetic peripheral neuropathy and atherogenic index.

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microRNA-32 promotes white fat browning through augmenting FGF21 expression in brown adipose tissue

Raymond NG, Attiqah Nurul HUSSIAN, Feng XU

2015, 29(S1): 71-71.

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OBJECTIVE To investigate the effect of microRNA-32 on cold-induced thermogenesis and brown adipocyte energy metabolism. METHODS To apply the cold-induced thermogenesis model in mice, 8-10 week old male C57Bl6 mice were placed within a 6℃ fridge for 7 d. Control microRNA inhibitor or miR-32 inhibitor (10 mg·kg^-1) was administered via intraperitoneal injection 16 h before the mice were placed in the fridge. Daily core body temperatures were taken using a rectal temperature probe. Mice were euthanized after 7 d and brown adipose tissue (BAT), inguinal and epididymal white adipose tissue (WAT), skeletal muscle and liver tissue analysed for changes in morphology and gene expression. RESULTS miR-32 inhibition in vivo inhibits the emergence of beige cells, which function like BAT cells, within WAT. In silico prediction and gene ontology analysis identified Tob1 as a likely target gene of miR-32. miR-32 inhibition led to increased expression of Tob1 whilst mutation of target sequence abolished this effect. Expression of brown adipose markers such as Ucp1, Pgc1α, Pparα and Prdm16 were significantly reduced in inguinal white adipose tissue (P<0.05). There was also a significant decrease in serum fgf21 levels due to the inhibition of Fgf21 expression in BAT (P<0.05). p38/MAPK signalling in brown adipose tissue was also significantly inhibited within brown adipose tissue leading to decreased fgf21 expression and secretion. CONCLUSION Our study shows that miR-32 plays a crucial role in stimulating beige cell emergence by activating p38/MAPK signalling during cold thermogenesis. miR-32 may prove effective as a treatment for obesity by activating cold-induced thermogenesis leading to increased energy metabolism.

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Effect of low molecular weight fucoidan on diabetic cardiovascular complications in Goto-Kakizaki type 2 diabetic rats

Wen-tong CUI, Wen-zhe YANG, Zhi-qiang WANG, Bo-lu ZHOU, Da-li LUO

2015, 29(S1): 72-72.

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OBJECTIVE To investigate the protective effect against diabetes cardiovascular complications of low molecular weight fucoidan (LMWF) from L. japonica in Qindao, China. METHODS LMWF (50, 100 and 200 mg·kg^-1·d^-1) or probucol (100 mg·kg^-1·d^-1) were given orally to Goto-Kakizaki type 2 diabetic rats for 12 weeks. Basal blood pressure, acetylcholine- or flow-mediated relaxation of mesenteric and paw arteries, endothelium-dependent dilation of aorta, eNOS phosphorylation and NO production were measured using laser Doppler flowmetry, force myograph, HE staining, Western blot and an NO assay, respectively. The establishment of diabetic cardiomyopathy (DCM) model and were evaluated by echocardiography and isolated heart perfusion. Ventricle staining with HE or Sirius red was performed to investigate the structural changes in myocardium. Oxidative stress and apoptosis were evaluated by enzyme activities, protein expressions and cell stainings in both myocardial tissues and cultured cardiomyocytes. RESULTS In aorta, LMWF robustly ameliorated the basal hypertension and impairment of endothelium-dependent relaxation in the aorta, as well as mesenteric and paw arteries in diabetic rats. In addition, the reduction in endothelial nitric oxide synthase (eNOS) phosphorylation at Ser1177, eNOS expression and NO production due to diabetes were partially reversed by LMWF treatment. However, probucol, a lipid-modifying drug with antioxidant properties, displayed only mild effects. Moreover, LMWF induced, in a dose-dependent manner, endothelium-dependent vasodilation and eNOS phosphorylation at Ser1177 in normal aorta, and also promoted Ser1177 phosphorylation and NO synthesis in primary cultured vasoendothelial cells. On DCM, LMWF has a beneficial effect by enhancing myocardial contractility and mitigating cardiac fibrosis as well as the production of reactive oxygen species (ROS) and myocyte apoptosis in diabetic hearts. CONCLUSION These data demonstrate for the first time that fucoidan protects vasoendothelial and cardiac function against diabetic injury in type 2 diabetes rats via, at least in part, preservation of eNOS function, amelioration of PKCβ-mediated oxidative stress and subsequent cardiomyocyte apoptosis. Fucoidan is therefore a potential candidate drug for protection of endothelium and heart in diabetic cardiovascular complications.

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Ganoderma Lucidum polysaccharide peptides protect kidney from renal ischemia reperfusion injury via counteracting oxidative stress

Dan-dan ZHONG, Hong-kai WANG, Ming LIU, Ying-li JIA, Ming HUANG, Hong ZHOU, Shu-qian LIN, Zhi-bin LIN, Bao-xue YANG

2015, 29(S1): 72-73.

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OBJECTIVE Ganoderma lucidum polysaccharide peptides (GLPP) have an anti-oxidant activity. The oxidative stress implicates in the pathogenesis of renal ischemia-reperfusion injury (RIRI). The objective of this study was to determine whether GLPP could attenuate RIRI via counteracting the oxidative stress. METHODS Mice subjected to uninephrectomy with the right kidney ischemia for 35 min and reperfusion for 24 h were used to explore the protective activity of GLPP against RIRI. In GLPP-treated group, 100 mg·kg^-1·d^-1 of GLPP were intraperitoneally injected for 7 d before the procedure. In vitro, NRK-52E cells subjected to hypoxia-reoxygenation (H/R) and tunicamycin were used to explore the protective effect of GLPP against oxidative stress. The mechanisms in which GLPP protected kidney from RIRI were studied using a series of physiological and molecular biological methods. RESULTS Kidneys undergone ischemia-reperfusion showed renal dysfunction and characteristic morphological changes including cellular necrosis, brush border loss, cast formation, vacuolization and tubular dilatation while these damages were significantly attenuated by GLPP treatment. The abnormal levels of MPO, MDA and SOD caused by renal ischemia-reperfusion were significantly reversed by GLPP treatment. More apoptotic cells were found in the renal ischemia-reperfusion group than the sham group whereas GLPP reduced apoptotic cells in the ischemia-reperfusion mice by 21.75% (P<0.01). The GLPPs (25 μg·mL^-1) alleviated H/R induced cell viability loss by 20.12% (P<0.01) and Δφm dissipation by 27.3% (P<0.01) in vitro as well and its pretreatment dramatically reduced H/R and tunicamycin induced cell injury. CONCLUSION Our study found that GLPP had a protective effect on RIRI via its anti-oxidative capacity, which suggests that GLPP may be developed as a candidate drug for preventing acute kidney injury.

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Role of metabolomics assisting the pharmacological activity and molecular mechanism in Panax notoginseng

Xue JIANG, So-Hyun KIM, Lin-fang HUANG, Hyung-Kyoon CHOI

2015, 29(S1): 73-74.

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OBJECTIVE The strategy and techniques of metabolomics was applied for the pharmacology and molecular mechanism research of Panax notoginseng (PN) in traditional Chinese medicine. METHODS The global metabolic profiles of PN were investigated by the NMR-based metabolomics. The different parts of PN were scanned into metabolic profiles by ¹H-NMR. The significant differences of these metabolic profiles were analyzed by PCA, PLS-DA, PLS-R, etc. The pharmacological effects including free radical scavenging activity (FRSA), anti-proliferation to human colorectal cancer cell line (HCT116), xanthine oxidase inhibition, were followed in vitro. Additionally, the molecular mechanism of xanthine degrading process by PN was attempted by ¹H-NMR. RESULTS The NMR-based metabolic profiles of different parts (upper part of root, middle part of root, lower part of root, hairy root, leaf and stem) of PN presented significant differences by multivariate statistical analysis. The hairy root and leaf revealed highest anti-proliferative effect to HCT116; the leaf and stem of PN showed highest level of FRSA; the leaf, stem, hairy root effected the xanthine degrading metabolic pathway. And the ¹H-NMR based molecular mechanism experiment showed that the xanthine metabolic pathway degraded by PN depended on the direct inhibition to xanthine. CONCLUSION The metabolomics strategy provided complementary chemical profiling to medicinal herbs, which accelerated the development of pharmacology and mechanism of action in traditional medicine. The subsidiary parts of PN, as leaf, stem and hairy root, have the potential to develop new drugs in curing cancer, inflammation and gout.

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Role of TRPM2 ion channel, an oxidative stress sensor, in hyperglycemia- induced pro-inflammaotry IL-1β in human monocytes

Hui-ling TSENG, Chi-teng VONG, Pui-man HOI

2015, 29(S1): 74-74.

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OBJECTIVE To investigate the role of transient receptor potential melastatin 2 (TRPM2), a calcium-permeable non-selective cation channel which acts as an oxidative stress sensor, in mediating the production of pro-inflammatory IL-1β in high glucose condition. METHODS Human pro-monocytic leukemia cell U937 was purchased from ATCC and cultured in RPMI 1640 (Life Technologies). Prior to high glucose (HG) stimulation, U937 cells were cultured in medium with glucose 5.5 mmol·L^-1 for 48 h. The cells were then incubated in high glucose concentration (30 mmol·L^-1) or mannitol (30 mmol·L^-1) for 48 h. The protein expression of TRPM2 and the production of human IL-1β were evaluated by ELISA. TRPM2 inhibitors (DPQ and AMP) and TRPM2 siRNAs were employed to further investigate the role of TRPM2 in HG-induced IL-1β production. RESULTS The TRPM2 protein expression was significantly up-regulated by 2-folds in U937 cells after the treatment of high glucose (30 mmol·L^-1 for 48 h) (P<0.01). The production of IL-1β in U937 was also significantly increased by HG treatment and was time- and dose-dependent (10, 20 or 30 mmol·L^-1 glucose for 24, 48 or 72 h) (P<0.01). The HG-induced IL-1β production in U937 could be abolished by using TRPM2 inhibitors DPQ (100 μmol·L^-1 for 45 min) and AMP (100 μmol·L^-1 for 45 min) as well as by the transfection of TRPM2 siRNAs (60 nmol·L^-1). CONCLUSION High glucose condition (such as in diabetes) might mediate pro-inflammatory environments via the modulation of TRPM2 channels on immune cells.

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Effects of Liuwei Dihuang decoction and its compatible prescriptions on the function of hypothalamus-pituitary-adrenal axis in senescence-accelerated mouse

Ning JIANG, Si-di LI, Wen-xia ZHOU, Yong-xiang ZHANG

2015, 29(S1): 74-75.

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OBJECTIVE To investigate age-related functional change of hypothalamus-pituitary-adrenal (HPA) axis in senescence accelerated mouse (SAM) and the effects of Liuwei Dihuang decoction (LW) and its San-bu (three tonics) and San-xie (three eliminators) components on the function of HPA axis. METHODS Male SAM-resistance/1 (SAMR1) and SAM-prone/8 (SAMP8) at the ages of 6 and 12 months old were used. SAMP8 were orally administered with LW, three tonics and three eliminators at the doses of 10, 6.4 and 3.6 g·kg^-1·d^-1, respectively, for consecutive 60 d. Serum level of CORT was assayed with ELISA method. The levels of hypothalamic CRH and pituitary ACTH was determined with radioimmunoassay. RESULTS The levels of hypothalamic CRH, pituitary ACTH and serous CORT were much higher in 6 and 12 months old SAMP8 than those in age-matched SAMR1, which suggested the abnormal function of HPA axis in SAMP8. Oral administration of LW and three tonics significantly decreased the level of hypothalamic CRH of 6 and 12 months old SAMP8, and reduced the levels of pituitary ACTH and serous CORT of 6 month old SAMP8. Three eliminators significantly decreased the level of hypothalamic CRH of 6 months old SAMP8. The results indicated that oral administration of LW, three tonics and three eliminators improved the function of HPA axis of SAMP8. CONCLUSION The results showed the hyperactivity of HPA axis of SAMP8, and LW improved the hyperactivity of 6 month old SAMP8. Three tonics and three eliminators had similar effects as LW, which had better effect after compatibility.

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Behavior and metabolomics study of the anti-aging effects of Scutellaria baicalensis Georgi extract on D-galactose-induced rats

Yu-shi ZHOU, Yan-fen CHANG, Guan-hua DU, Xue-mei QIN

2015, 29(S1): 75-76.

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OBJECTIVE Behavior research and urinary metabolomics method were applied to evaluate the anti-aging effects of Scutellaria baicalensis Georgi extract (SBG) in D-galactose-induced rats. METHODS Fifty rats were randomly divided into five groups (n=10 in each group). Group 1 served as vehicle control with injection of saline (vehicle control group), and the other groups of rats received daily subcutaneously injected with D-galactose (aged model group) at dose of 100 mg·kg^-1 for ten weeks, respectively. At the same time, rats in groups 3-5 were intragastrically administered SBG (extracted twice with 60% (V/V) ethanol) at doses of 50, 100 and 200 mg·kg^-1 for ten weeks, and the rats of groups 1 and 2 were administrated an equal volume of the vehicle. At the tenth week, the learning and memory abilities were examined by Morris water maze. The urine was collected using metabolic cages and analyzed by high-resolution ¹H-NMR spectroscopy combined with multivariate statistical analyses. Principal component analysis (PCA) was utilized to classify and reveal the differences between the model group and control group. Then, the concentration of these differences was analyzed with t-test to determine whether SBG was possible to influence the metabolic pattern induced by D-galactose. RESULTS Compared with the vehicle control group, the D-galactose-treated aged model group markedly spent longer time (P<0.05) in finding the platform on days 3-5 in the spatial learning acquisition training of Morris water maze test. However, the escape latency was significantly reduced (P<0.05) by long-term administration of SBG (50, 100 and 200 mg·kg^-1) compared with the D-galactose-treated aged model group on days 3-5. In the probe test, the D-galactose-treated aged model group made fewer (P<0.05) platform crossings and distance travelled in target quadrant (P<0.05) than the vehicle control group, and the SBG at doses of 50, 100 and 200 mg·kg^-1 treatments groups could significantly increase (P<0.05) the number of times of crossing over the platform site. The SBG at doses of 100 and 200 mg·kg^-1 treatments groups could significantly increase (P<0.05) the distance travelled in target quadrant compared with the D-galactose-treated aged model group. In addition, the significant difference in metabolic profiling was observed from model group compared with drug-dose group by using PCA, indicating the recovery effect of SBG on D-galactose induced aging rats. Some significantly changed metabolites like glycine, glucose and hexadecanoic acid have been identified. These biochemical changes are related to the the disturbance in aimno acid metabolism, energy metabolism and glycometabolism, which are helpful to further understanding the D-galactose induced aging rats and the therapeutic mechanism of SBG. CONCLUSION These results demonstrate that SBG extract has protective effect on the D-galactose-induced aging in rats.

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Effect of Urena lobata leaves extract on glucagon like peptide-1 serum level by inhibition of dipeptidyl peptidase-Ⅳ on diabetic rats

Yudi PURNOMO, Djoko Wahono S, Sutiman B SUMITRO, M Aris WIDODO

2015, 29(S1): 76-76.

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OBJECTIVE To investigate effect of Urena lobata leaves extract on glucagon like peptide-1 (GLP-1) serum level by inhibition of dipeptidyl peptidase-Ⅳ (DPP-Ⅳ) on diabetic rat. METHODS This study uses control group post test only with male spraque dawley rats. Diabetic rats was induced by High Fructose Diet (HFD) and single dose streptozotocin 25 mg·kg^-1 bw intra peritoneal. The rat was administrated orally with ethanolic extract of U.lobata leaves in dose of 250, 500 and 1000 mg·kg^-1 for 4 weeks. Blood sample were collected from the tail vein at 15 min after oral glucose administration and then DPP-Ⅳ serum level and GLP-1 were examined using a rat elisa kits of DPP-Ⅳ and GLP-1. The data was analyzed using ANOVA test continued with LSD test (P<0.05). RESULTS The oral administration of U.lobata leaves extract at dose of 250, 500 and 1000 mg·kg^-1 bw were able to prolong GLP-1 bioavaibility approximately 5, 2 and 2.5-fold respectively compared to diabetic group (P<0.05), while the DPP-Ⅳ serum level was decreased by 60%, 50% and 40% (P<0.05), respectively. In diabetic groups, DPP-Ⅳ serum level was increased more and less 4-fold compared to normal group (P<0.05) while the GLP level were decreased by 8-fold (P<0.05). CONCLUSION U.lobata leaves extract could prolong GLP-1 bioavaibility by reducing of DPP-Ⅳ serum level. This effect may be related to active compounds that act as an DPP-Ⅳ inhibitor in U.lobata extract.

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Anti-hyperglycemic effect of ethanolic leaves extract, hexane and water of Urena lobata on diabetic rats induced by high fructose diets and streptozotocin

Yudi PURNOMO, Doti WAHYUNINGTYAS, M Aris WIDODO

2015, 29(S1): 76-77.

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OBJECTIVE To investigate anti-hyperglycemic effect of ethanolic leaves extract, hexane and water of Urena lobata on diabetic rat induced by high fructose diets and streptozotocin. METHODS This study uses control group post test only with male spraque dawley rats. Diabetic rats was induced by high fructose diet (HFD) and single dose streptozotocin 25 mg·kg^-1 bw intra peritoneal. The rat was administrated orally with ethanolic leaves extract, hexane and water of U.lobata in concentrations of 500 mg·kg^-1 bw for 4 weeks. Blood sample were collected from the tail vein at 0, 15, 30, 60 and 120 min after oral glucose administration and then blood glucose level were measured using a commercially available glucometer. The integrated area under the postprandial glucose curves (AUCs) was calculated by the trapezoidal method. Insulin serum level examined by rat insulin elisa kits. The data was analyzed using ANOVA test continued with LSD test (P<0.05). RESULTS The oral administration of ethanolic leaves extract, hexane and water of U.lobata were able to decrease glucose AUC about 40%, 20% and 60% respectively compared to diabetic group (P<0.05), whereas the insulin level was increased by 2, 2 and 4-fold (P<0.05), respectively. In diabetic groups, glucose AUC was increased approximately 70% compared to normal group (P<0.05) while the insulin level were decreased by 14-fold (P<0.05). CONCLUSION U.lobata leaves extract could control the increase of blood glucose level after glucose administration by improve of insulin secretion. This effect may be related to active compounds that act as an anti-hyperglycemic and antioxidant in U.lobata leaves extract.

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Combined anti-hyperuricemia effect of Di.E and Gpm.E on hyperuricemia rats induced by high-purine diet

Min-xia PANG, Ying-ying MA, Hui-ming HU, Jie SU, Su-hong CHEN, Gui-yuan LYU

2015, 29(S1): 77-77.

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OBJECTIVE Di.E and Gpm.E were separately extracted from two kinds of traditional Chinese medicine. The aim of this research is to investigate the combined anti-hyperuricemia effect of Di.E and Gpm.E on hyperuricemia rats induced by high-purine diet, and to study the underlying mechanism. METHODS Sprague Dawley (SD) rats were divided into some groups according to their serum uric acid (UA), the normal control group received standard diet, while others were fed with high-purine diet. After the success of modeling, SD rats were divided into 5 groups according to UA: normal control group, model control group, Di.E and Gpm.E(high, middle, low dosages). The treatment groups were simultaneously orally administered. Two days before the last administration, the urine N- acetyl beta-D Glucosaminidase (NAG), Lysozyme(LYS) activity were separately detected using the urine collected from metabolic cage for 24 h. After the last administration, the blood was taken from postcava to detect the level: ① The related indexes of liver and kidney function: the serum UA, blood urea nitrogen (BUN), creatinine(CR), alanine aminotransferase (ALT); ② The uric acid metabolism related enzymes: the serum and liver xanthine oxidase (XOD), adenosine deaminase (ADA), guanine deaminase (GD), xanthine dehydrogenase (XDH) activity; ③ Fibrosis related indexes of renal tissue: connective tissue growth factor (CTGF), monocyte chemotactic protein-1 (MCP-1), TGF-β1, TNF-α, NF-κB levels. RESULTS Di.E and Gpm.E (high, middle dosages) could markedly reduce the serum UA level significantly, also can decrease XOD, ADA, TNF-α, MCP-1, NF-κB activity significantly. CONCLUSION The results show that Di.E and Gpm.E can decrease the serum UA level significantly, improve the liver and kidney function and slow down the process of renal fibrosis; the therapeutic effects may be related to their inhibition of XOD, ADA activity.

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Exploration on the establishment of animal models for hyperuricemia and the application of traditional Chinese medicine

Su-hong CHEN, Min-xia PANG, Ying-ying MA, Hui-ming HU, Rui-li YU, Gui-yuan LYU

2015, 29(S1): 78-78.

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OBJECTIVE In recent years, the frequency of hyperuricemia has gradually risen along with the improvement of living standards, the irregular of diet and overfeeding greasy and surfeit flavor. However, hyperuricemia is a disorder of purine metabolism, and is strongly associated with insulin resistance and abnormal glucose metabolism. It is important to obtain a more stable and sustained animal model for the efficacy evaluation of traditional Chinese medicine (TCM). METHODS To manufacture the rodent model of hyperuricemia, the theory of increasing the source of the uric acid, reducing uric acid excretion and inhibiting uricase were used. We observed the influence on the serum uric acid and other indicators of rats induced by some factors: the lipid emulsion, high purine diet, beer with sugar, beer with sugar and high purine, and so on. Then we choose one of the stable and sustained animal models, studying the effects of Plo.E(which extracted from a TCM) on modulating the level of serum uric acid and the preliminary mechanism in this abstract. RESULTS 1 At the 2nd week, the level of serum UA, BUN, Cr, TC, LDL-c of rats in the lipid emulsion group raised significantly. ② At the 6th weeks, the serum UA in both the high purine diet group and lipid emulsion group raised obviously. ③ The effects of Plo.E on hyperuricemia rats induced by high purine diet: After 8 d administration, the Plo.E(three dosages) can reduce the UA level, and the middle and low dosages can reduce the TG level. After 25 d administration, Plo.E can reduce the plasma viscosity, UA, TG, the whole blood viscosity level, the high dosage also can reduce the TC level. CONCLUSION The rats induced by the high purine diet and lipid emulsion can raised the serum UA obviously, while the way of lipid emulsion is earlier and more stable. Plo.E has a therapeutic effect on hyperuricemia, with an activity of reducing plasma viscosity and blood lipid. The above models conform to the pathogenesis of humans, can be used to study the causes and pathogenesis of hyperuricemia complicating metabolic disorder and the related treatment drug screening.

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Potential role of nimbolide in chemoprevention and therapy of prostate cancer

Jing-wen ZHANG, Muthu K. SHANMUGAM, Amudha DEIVASIGAMANI, Alan Prem KUMAR, Kam Man HUI, Gautam SETHI

2015, 29(S1): 78-79.

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OBJECTIVE Prostate cancer is one of the most commonly diagnosed cancers worldwide. Current therapies for metastatic prostate cancer are only marginally effective, and hence novel treatment modalities are urgently needed. Considerable evidence suggests that chronic inflammation plays a pivotal role in the development and progression of prostate cancer. Thus agents that can suppress these inflammatory mediators could form the basis of novel therapy for prostate cancer patients. In our study, we focused on analyzing the potential anticancer effects of nimbolide, a terpenoid lactone derived from Azadirachta indica (Neem tree) against prostate cancer. METHODS Molecular biology techniques such as western blot analysis, DNA binding, luciferase assays, and immunohistochemistry were used for both in vitro and in vivo experiments. RESULTS Data from the in vitro studies indicated that nimbolide could inhibit cell proliferation, induce apoptosis and suppress cellular invasion and migration. Interestingly, nimbolide also abrogated the activation of pro-inflammatory STAT3 transcription factor, and this effect was found to be mediated via an increased production of reactive oxygen species (ROS), whereas depletion of ROS attenuated p-STAT3 inhibitory effects of the drug. The in vivo efficacy of nimbolide was also noted in transgenic adenocarcinoma of mouse prostate (TRAMP) model, in which this triterpenoid significantly suppressed the tumor progression and growth without exhibiting any substantial adverse effects. CONCLUSION Overall our findings indicate that nimbolide exhibits significant anticancer effects in prostate cancer, and these effects may be mediated at least in part through the modulation of STAT3 signaling pathway.

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Ascochlorin overcomes chemoresistance and regulates the plasticity of doxorubicin induced EMT via modulation of the NF-кB pathway in hepatocellular carcinoma

Xiao-yun DAI, Kwang Seok AHN, Chulwon KIM, Muthu K SHANMUGAM, Feng LI, Ji-zhong SHI, Alan Prem KUMAR, Ling-zhi WANG, Boon Cher GOH, Junji MAGAE, Tina H ONG, Kam M HUI, Gautam SETHI

2015, 29(S1): 79-80.

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OBJECTIVE Doxorubicin-based therapy has been found to be not significantly effective for the treatment of advanced stage hepatocellular carcinomas (HCCs), which often undergo epithelial-mesenchymal transition (EMT) during tumor progression. Increasing evidence suggest(s) that epithelial cell transformation to mesenchymal state canenhance the ability to self-renew and confer greater resistance to the conventional chemotherapeutic drugs.The aim of this study was to examine the potential efficacyof ascochlorin, an isoprenoid antibiotic to overcome drug resistance induced by doxorubicin in HCC cell lines and to elucidate its underlying mechanism(s) of action. METHODS The effect of doxorubicin and ascochlorin on HCC cell lines was determined by MTT, Western blotting, immunofluorescence and NF-кB DNA binding assays. RESULTS Our results indicate that HCC cells that show a mesenchymal-like phenotype,are resistance to the doxorubicin therapy which directly correlated with an increased slug expression. We also observed that activation of NF-кB pathway plays an essential role in doxorubicin induced-chemoresistance and pharmacological inhibition of this pathway with ascochlorin can significantly reverse drug-induced invasion/migration and resistance in HCC cells. CONCLUSION Our results indicate that combination treatment of doxorubicin with ascochlorin has the potential to inhibit HCC growth and metastasis.

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Garcinol sensitizes human head and neck carcinoma to cisplatin in a xenograft mouse model despite downregulation of proliferative biomarkers

Feng LI, Muthu K. SHANMUGAM, Kodappully Sivaraman SIVEEN, Fan WANG, Tina H ONG, Ser Yue LOO, Mahadeva MM, Somnath MANDAL, Alan Prem KUMAR, Boon Cher GOH, Tapas KUNDU, Kwang Seok AHN, Ling-zhi WANG, Kam Man HUI, Gautam SETHI

2015, 29(S1): 80-80.

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OBJECTIVE Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated whether garcinol, a polyisoprenylated benzophenone can chemosensitize HNSCC to cisplatin. METHODS The effect of garcinol and cisplatin on HNSCC was assessed by MTT, Western blotting, real time PCR, FACS, immunohistochemistry, DNA binding assay and xenograft mouse model. RESULTS We found that garcinol inhibited the viability of a panel of diverse HNSCC cell lines, enhanced the apoptotic effect of cisplatin, suppressed constitutive as well as cisplatin-induced NF-κB activation, and downregulated the expression of various oncogenic gene products (cyclin D1, Bcl-2, survivin and VEGF). In vivo study showed that administration of garcinol alone (0.5 mg·kg^-1, ip five times/week) significantly suppressed the growth of the tumor, and this effect was further increased by cisplatin. Both the markers of proliferation index (Ki-67) and microvessel density (CD31) were downregulated in tumor tissues by the combination of cisplatin and garcinol. The pharmacokinetic results of garcinol indicated that good systemic exposure was achievable after ip administration of garcinol at 0.5 and 2 mg·kg^-1 with mean peak concentration (c_max) of 1825.4 and 6635.7 nmol·L^-1 in the mouse serum, respectively. CONCLUSION Overall, our results suggest that garcinol can indeed potentiate the effects of cisplatin by negative regulation of various inflammatory and proliferative biomarkers.

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Glycyrrhetinic acid-triggered cytoprotective autophagy via inositol- requiring enzyme 1α-c-Jun N-terminal kinase cascade in non-small cell lung cancer cells

Zheng-hai TANG, Le-le ZHANG, Ting LI, Jia-hong LU, Chung-hang LEUNG, Xiu-ping CHEN, Yi-tao WANG, Jin-jian LU

2015, 29(S1): 81-81.

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OBJECTIVE Glycyrrhetinic acid(GA), one of the main bioactive constituents of the famous Chinese medicinal herb Glycyrrhizauralensis Fisch, presents potent cytotoxicity to various cancer cells both in vitro and in vivo. Herein, we'd like to determine whether GA triggers autophagy in non-small cell lung cancer cells and the mechanisms involved. METHODS Cell proliferation was determined by MTT assay and colony formation. AnnexinⅤ/PI staining, Hoechst33342 staining, and Western blotting were used to detect GA-induced apoptosis. GA-induced autophagy was measured by expression of the lipid modification of light chain-3 (LC3) and transfected with GFP-LC3 or GFP-RFP-LC3 plasmid. Pharmacological regulators, siRNA, and plasmid transfection were used to study the mechanisms of GA-triggered autophagy. RESULTS GA inhibited cell proliferation and induced apoptosis in a concentration-dependent manner in non-small cell lung cancer A549 cells. GA induced autophagyas evidenced by up-regulation of LC3-Ⅱ expression when combined treatment with chloroquine and induction of the red punta after GFP-RFP-LC3 plasmid transfection. Knockdown of autophagy related proteins (ATG) 7, ATG 5, or beclin 1 by siRNA, the expression of LC3-Ⅱ and GFP-LC3 punt atriggered by GA were decreased. Furthermore, the c-Jun N-terminal kinase (JNK) pathways were activated after treatment with GA, and pretreatment with JNK inhibitor SP600125 or silence of JNK pathway by siRNA of JNK or c-jun obviously reduced GA-induced LC3-Ⅱ expression and GFP-LC3 punta formation. GA also stimulate dendoplasmic reticulum stress response by triggering inositol-requiring enzyme 1α (IRE1α) pathway, and knockdown of IRE1α inhibited the activation of JNK pathway and autophagy induced by GA. In addition, GA-induced cell proliferative inhibition and apoptosis were both enhanced when silence of autophagy as well as JNK pathway. CONCLUSION Our study demonstrated, for the first time,that GA induced a cytoprotective autophagy in non-small cell lung cancer cells by activating the IRE1 α-JNK pathway, which might decreased the anti-cancer effects of GA.

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In vitro and in vivo effect of artesunate against prostate cancer: Targetting STAT3 pathway to combat disease progression

Nachiyappan ALAMELU, Kwang Seok AHN, Kodappully Sivaraman SIVEEN, AlanPrem KUMAR, Gautam SETHI

2015, 29(S1): 81-82.

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OBJECTIVE In prostate cancer (PCa), signal transducer and activator of transcription factor 3(STAT3) has been strongly associated with tumor progression, through numerous means.Hence this allows STAT3 to be an important target for therapeutic action. Artesunate (ART), a well know antimalarial agentis making its way as an anticancer drug. In the present study, we investigated whether ART can control aberrant STAT3 signaling, and thereby take a toll on PCa development. METHODS Various PCa cell lines (DU145, PC3, LNCaP) and in vivo xenograft mouse model are used. Cytotoxic effects of ART against various PCa cell lines were evaluated by MTT assay. Flow cytometry cell cycle analysis and DNA fragmentation assay was done to detect the apoptotic effect of ART. Expression of STAT3 and its regulated gene in the presence and absence of ART were measured by WB, IHC and RT PCR. STAT3 DNA binding activities was analyzed by ELISA. RESULTS ART was found to dephosphorylate STAT3 at Tyr 405, thereby reducing its nuclear translocation and DNA binding efficiency in DU145 PCa cells. We proclaim that ART can prevent the PCa development, as it can inhibit proliferation, bring about cell cycle arrest at G₀/G₁ phase, AnnexinⅤ positive staining, DNA fragmentation, caspase 3 activation and PARP cleavage in PCa cell lines. Furthermore, inhibition of constitutive STAT3 expression was associated with the ability of ART to suppress its upstream kinases such as Janus kinase 1 and 2 (JAK1 and JAK2). SHP-1, protein tyrosine phosphatases which are considered to be one of the major regulators of STAT3 phosphorylation was upregulated in the presence of ART. We observed reversal in ART mediated inhibition of STAT3 in the presence of pervanadate, tyrosine phosphates inhibitor and during SHP-1 knock down. ART was able to inactivate STAT3 in DU145 cells exposed to conditioned media (CM) rich in cytokines. In the presence of ART we observed the down regulation of various STAT3 regulated gene products which are involved in proliferation, survival, and angiogenesis. ART even blocked the motility and invasion of PCa cells. ART substantially decreased the tumor volume in xenograft mouse which is implanted with DU145 cells. Also ability of ART to control aberrant STAT3 signaling was in accordance with its in vitro studies. CONCLUSION Over all through our findings, we have disclosed for the first time that ART could possibly exerts it antitumor effect by interrupting deregulated expression of STAT3 in PCa, both in vitro and in vivo.

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Garcinol suppresses the growth of human hepatocellular carcinoma by inducing abrogation of STAT3 phosphorylation, acetylation and dimerization

Muthu K SHANMUGAM, Snehajyoti CHATTERJEE, Peramaiyan RAJENDRAN, Feng LI, Parijat SENAPATI, KwongFai WONG, Alan Prem KUMAR, John M LUK, Kam Man HUI, Gautam SETHI, Tapas K KUNDU

2015, 29(S1): 82-83.

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OBJECTIVE Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third cause of global cancer mortality. Activation of signal transducer and activator of transcription 3 (STAT3) is commonly observed in tumor cells and is a critical mediator of on cogenic signaling in HCC and controls the expression of several genes involved in proliferation, survival, metastasis and angiogenesis. Current drug-targeted therapies, besides being expensive, are associated with serious side effects and morbidity. Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. In the present report, we investigated whether the potent HAT/KAT inhibitor, garcinol, (apolyisoprenylatedbenzophenone), could suppress STAT3 activation in HCC cells and in nude mice model. METHODS The effect of garcinol on HCC cell lines wasdetermined by MTT assay, immunoblotting, DNA binding assays, immuno-fluorescenceand immune-histochemical analysis. The effect of garcinolon the inhibition of tumor growth in vivo was also investigated using HCCxenograft tumor modelin athymic nu/nu mice. RESULTS We found that garcinol could inhibit constitutive STAT3 activation in a dose- and time-dependent manner both by inhibiting STAT3 phosphorylation and acetylation in HCC cells. When investigated for molecular mechanism(s), we found that garcinol interferes with the dimer formation of STAT3 thereby inhibits its nuclear localization. Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppresses its dimerization in vitro. To understand the cellular mechanism(s) of inhibition of STAT3 function by garcinol, we observed that upon inhibition of STAT3 dimerization bygarcinol, STAT3 DNA binding ability gets repressed. The inhibition of STAT3 activation by garcinol led to the suppression of various gene products involved in proliferation, survival, and angiogenesis. Finally, when administered i.p., garcinol inhibited the growth of human HCC xenograft tumors in athymic nu/nu mice. CONCLUSION Results from in vitro and in vivo studies suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling cascade in HCC by inhibiting its phosphorylation, acetylation and ultimately dimerization.

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Liguzinediol protects against apoptosis induced by doxorubicin in H₉C₂ cells

Yu LI, Chao LIN, Ya-yun ZHANG, Yuan YAO, Xiang WU, Wei LI, Hui-min BIAN

2015, 29(S1): 83-83.

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OBJECTIVE Liguzinediol is a derivative of the natural active ingredient ligustrazine, and we found that liguzinediol has significant positive inotropic effects, which are stronger than that of TMP. Besides, it does not lead to arrhythmia, hypotension and other side effects. This study aims to investigate the anti-apoptotic effects of liguzinediolon H₉C₂ cells. METHODS Apoptotic H₉C₂ cells induced by DOX were observed by electron microscope and FCM analysis. The protein expressions of Bax, Bcl-2, caspases 3 and NF-κB were detected by Western blotting. RESULTS Apoptotic H₉C₂ cells induced by DOX were observed, but without apoptotic bodies in liguzinediol group. Declined peak of H₉C₂ cell apoptosis was seen in liguzinediol group by FCM analysis. And downregulation of Bax, caspases 3, NF-κB and upregulation of Bcl-2 were found by Western blotting. CONCLUSION Liguzinediol protected cardiomyocytes against apoptosis through downregulation of Bax and caspases 3 and upregulation of Bcl-2. Liguzinediol can inhibited cardiomyocyte apoptosis through the NF-κB signal pathway.

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RNA helicase DDX20 as a surrogate marker of statin activity in invasive breast cancer

Chao WANG, Beiying QIU, Jean Paul THIERY, Gautam SETHI, Patrick J. CASEY, Celestial T. YAP, Soo Chin LEE, Vinay TERGAONKAR, Alan Prem KUMAR

2015, 29(S1): 84-84.

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OBJECTIVE To evaluate if RNA helicase DDX20, highly expressed in triple negative breast cancer (TNBC) cells, could serve as a surrogate marker for simvastatin treatment response. METHODS We first assessed correlation between 17 mevalonate pathway-related genes and expression of DDX20 in a cohort of 1325 breast cancer tumors. TNBC cells, MDA-MB-231, were then treated with simvastatin and mevalonate pathway intermediates to assess the alteration in DDX20 expression. In the mouse model, MDA-MB-231 cells were injected to tail veins of mice, groups of 8 mice each were injected intraperioneally with vehicle or simvastatin 25 mg·kg^-1 3 times a week for 6 weeks. The number of metastatic colonies formed was quantified and immunohistochemical (IHC) staining of DDX20 was carried out in the lung tissues. RESULTS Among the 17 genes evaluated, positive correlation with DDX20 expression was observed in eight of them, with HMGCR having the highest correlation. Our in vitro experiments show exposure of breast cancer cells to simvastatin lead to a Rho-dependent decrease in gene expression of DDX20, leading to decreased tumor proliferation in a mevalonate pathway-dependent manner. Conversely, ectopic overexpression of DDX20 significantly abrogated the anti-metastatic activity of simvastatin. A similar observation is seen in the mouse model, where simvastatin-injected mice show significantly fewer visible lung metastases compared to placebo-fed mice. IHC staining on these lung tissues showed decreased DDX20 expression in simvastatin-injected group, corroborating our observations in vitro. CONCLUSION DDX20 is a potential surrogate marker for simvastatin treatment response in breast cancer and a long term implication of our findings is the possibility of an effective combinatorial therapeutic intervention using statins (to suppress DDX20 gene expression) and a suitable first-line agent "for the kill" of invasive breast cancer.

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Anti-emetic mechanisms of Xiaobanxia Tang decoction on the chemotherapy-induced pica model in rats

Gong-chang YU, Yong ZHANG, Ke NIE

2015, 29(S1): 84-85.

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OBJECTIVE To investigate the mechanisms of Xiaobanxia Tang (XBXT) in the prevention and treatment of chemotherapy-induced nausea and vomiting. METHODS The chemotherapy-induced rat pica model was established by intraperitoneal injection of cisplatin 6 mg·kg^-1. Kaolin consumption was used as an indicator of nausea and vomiting. Wistar male rats were randomly divided into normal control, XBXT normal control, model, ondansetron treating, XBXT decoction high and low dose groups. The rats in ondansetron group, XBXT normal control group, XBXT high and low dose groups were gavaged ondansetron 2.6 mg·kg^-1·d^-1, XBXT 1.6, 3.2 and 1.6 g·kg^-1·d^-1, respectively 1 h before cisplatin injection, and the administration were given every 12 h. Kaolin consumptions were weighed every 12 h. After 24 h and 72 h of cisplatin injection, animals were sacrificed respectively. The contents of 5-HT, 5-HIAA, dopamine(DA), DOPAC, substance P (SP), TPH, MAO and TH were measured by ELISA. The mRNA expression of 5-HT transporter (SERT), 5-HT3A receptor, SP precursor (PPTA), NK1 and D2 receptors in rat ileum and medulla oblongata were measured by RT-PCR, the protein expression were measured by Western blotting. RESULTS The high and low dosages of XBXT could significantly inhibit kaolin consumptions in cisplatin-treated rats, and reduce 5-HT, increase 5-HIAA contents and reduce 5-HT3A receptor mRNA and protein expression, above effects are related to the reduction of TPH and the enhancement of MAOA levels. The two dosages of XBXT could significantly reduce SP and NK1 mRNA and protein expression, which was related to the reduction of PPTA mRNA expression. XBXT could also significantly reduce DA contents and D2 receptor mRNA and protein expression, which was related to the reduction of TH. CONCLUSION XBXT has significant antiemetic effect in chemotherapy-induced nausea and vomiting, the underlying mechanisms are related to the inhibition of 5-HT and 5-HT3A receptor, SP and NK1 receptor, DA and D2 receptor.

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Specific growth inhibition by alteration of metabolic pathway using Chinese herbal medicine in tyrosine kinase inhibitor resistant non-small cell lung cancer

Xing-xing FAN, Maria Pik WONG, Zhi-wei CAO, Jian-lin WU, Hua ZHOU, Zhi-hong JIANG, Liang LIU, Elaine Lai-han LEUN

2015, 29(S1): 85-86.

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OBJECTIVE Lung cancer is the leading cause of cancer death worldwide. Epidermal growth factor receptor (EGFR) mutation(s) is/are common in non-small cell lung cancer (NSCLC) in Asian population, resulting in lung tumor formation. L858R substitution mutation on exon 21 and in-flame deletion mutation on exon 19 are the two most common forms of EGFR mutation. Molecular targeted therapy using tyrosine kinase inhibitor (TKI) targeting EGFR shows promising initial response, however drug resistance is common. Therefore, it is needed to identify new inhibitors to tackle TKI-resistance. In this study, we aim to investigate the effect of multiple single purified compounds derived from Chinese herbal medicines (CMHs) on a panel of NSCLC cell lines with different EGFR mutational statuses and TKI sensitivity. We also examine the biological functional effect and drug action mechanism of these cell lines after drug treatment. METHODS We have reviewed the literature and selected ten single purified compounds derived from CMHs which exhibited the highest potential of cancer suppression effect in NSCLC. We have recruited three EGFR-dependent NSCLC cell lines for drug screening using cytotoxicity assay. A549 is used as EGFR wild-type control. Two TKI-resistant NSCLC cell lines were used, H1975 harbors double mutation (EGFR^L858R+T790M) and H1650 harbors EGFR^{exon 19 deletion}. H2228 is a NSCLC cell line which harbours EML4-ALK fusion gene and was used as EGFR-independent cell line control. MTT assay was used to determine the drug efficacy and IC₅₀ value. Then functional assays including cell cycle arrest analysis and apoptosis assay was used to determine the biological effect after drug treatment. RESULTS MTT assay revealed that six out of ten candidate agents showed significant cancer-inhibiting effects in H1650 and H1975 cells. Three compounds exhibited IC₅₀ value at micro-molar levels while another three compounds exhibited IC₅₀ at as low as nano-molar levels. One compound exhibited specificity on EGFR-dependent NSCLC cell lines, which showed 10-fold more selective than the EGFR-independent H2228 cells. Cell cycle analysis and immunoblotting assay showed that one effective compound, designated was MUST-1, altered the metabolic pathway of glucose metabolism and lipid metabolism, and induced cell cycle arrest at G₁ phase in NSCLC with EGFR mutation. However, the anti-proliferative effects were distinct in NSCLC cell lines with different EGFR mutation patterns. CONCLUSION MUST-1 significantly induced cell cycle arrest in four NSCLC in EGFR mutant cell lines but the inhibiting effect was not significant in EGFR wild-type cell line. Immunobloting assay revealed that overall intracellular lipid content, glycolytic enzymes PKM2 and cell cycle regulatory gene expression were altered after 72 h compound treatment in the responsive cells. Further investigation is required to elucidate the underlying reason of drug selectivity and the role of glucose and lipid metabolism in regulating drug sensitivity.

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Stellate cells regulate Nrf2 signaling to orchestrate metabolic changes and ROS detoxification in pancreatic cancer cells

Yuan-seng WU, Chung-yeng LOOI, Masamune ATSUSHI, Shin-yee FUNG, Chung IVY

2015, 29(S1): 86-87.

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OBJECTIVE We previously showed that human pancreatic stellate cells (HPSCs) promote pancreatic ductal adenocarcinoma (PDAC) cell growth by activating nuclear factor erythroid2-related factor 2 (Nrf2), a key transcriptional regulator of cytoprotective genes. We aim to investigate whether Nrf2-mediated metabolic reprogramming and reactive oxygen species (ROS) detoxification are involved in HPSCs-mediated cell growth. METHODS Nrf2-mediated metabolic genes expression of pentose phosphate pathway (PPP) for purine nucleotide synthesis; glutamine metabolism for nicotinamide adenine dinucleotide phosphate (NADPH)-equivalent producers and also glutathione biosynthesis both for intracellular ROS inactivation were examined using quantitative real-time PCR (qRT-PCR) after treated with conditioned media derived from HPSCs (HPSC-CM) in human PDAC cells (BxPC-3 and AsPC-1) with or without Nrf2 gene silencing using siRNA-mediated technique. Metabolites involved in PPP for purine nucleotide and NADPH generation were selected and their concentration was measured using UHPLC-MS/MS. Antioxidants, tiron and N-acetylcysteine (NAC) were used to attenuate the intracellular ROS rendered by Nrf2 before measuring PDAC cell growth and also phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B (AKT) using MTT and Western blotting, respectively. RESULTS Metabolically, HPSC-CMupregulated Nrf2-mediated genes involved in three metabolic pathways (G6PD, PGD, TKT, PPAT, MTHFD2, ME1, IDH1, GCLC and GCLM) in BxPC-3 and AsPC-1 cells. HPSC-CM was able to upregulate all the metabolic genes after Nrf2 gene silencing, and also significantly increased the metabolite concentration of ribose 5-phosphate and inosine 5'-monophosphate, which are involved in nucleotide synthesis for cell growth. Decreasing the intracellular ROS rendered by Nrf2 suppressed PDAC cell growth and also phosphorylation of ERK 1/2 and AKT protein. CONCLUSION Our findings reveal that HPSC-CM activates Nrf2-mediated metabolic reprogramming, which leads to purine nucleotide synthesis and ROS detoxification to promote PDAC cell growth.

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Targeting the undruggable KRAS: In vitro anticancer property of andrographolide and its semisynthetic analogues in pancreatic adenocarcinoma cell lines harbouring oncogenic mutant K-ras

Michelle Siying TAN, Yuan Han TEH, Kok Lian HO, Sreenivasa Rao SAGINEEDU, Johnson STANSLAS

2015, 29(S1): 87-87.

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OBJECTIVE To evaluate the anticancer activity of andrographolide (AGP) and its semisynthetic analogues (SRJ09 and SRJ23) in pancreatic adenocarcinoma (PDAC) cell lines harbouring therapeutically highly relevant oncogenic K-ras glycine-12 (KRAS-G12) mutant proteins. In a landmark publication, we revealed that AGP and its derivatives bind KRAS protein to inhibit RAS signaling PNAS, 110:10201-06). This discovery prompted the initiation of this investigation. METHODS The cell growth inhibitory effect of the compounds on PDAC cell lines (PANC-1 (KRAS-G12D), Capan-2 (KRAS-G12V), and MIA PaCa-2 (KRAS-G12C)), was assessed by MTT assay. RESULTS In comparison with AGP and SRJ09, SRJ23 showed the greatest growth inhibition in all PDAC cell lines with mutant KRAS proteins. The inhibitory effect of SRJ23 on the cell growth was similar for all PDAC cell lines. AGP exerted selective growth inhibition against PANC-1 (KRAS-G12D) cells, while the growth inhibition of SRJ09 was selective towards Capan-2 (KRAS-G12V) cells. CONCLUSION AGP and SRJ09 showed selectivity for PDAC cell lines with specific KRAS mutations. This suggests the mutational status of KRAS protein and the structural features of these two compounds orchestrally determined the magnitude of cell growth inhibition in PDAC cell lines. The higher potency of SRJ23 implies it could be developed into an anticancer agent for the treatment of mutant KRAS-driven malignancies. To this end, efforts are in progress to derive new molecules from this compound for further improvement of potency.

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Combination of kaempferol and chloroquine induces glioma cell death via expansion and subsequent rupture of lysosomes

In Young KIM, Mi Jin YOON, Min Jun SHIM, Jun Hee LIM, Kyeong Sook CHOI

2015, 29(S1): 87-88.

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OBJECTIVE Chloroquine is considered as a potential chemotherapy and radiotherapy sensitizer, but the anticancer effect of chloroquine alone is limited. Since we found that the flavonoid kaempferol effectively sensitizes glioma cells to chloroquine-mediated cell death, we investigated the underlying mechanisms of glioma cell death induced by the combination of kaempferol and chloroquine. METHODS To examine the effect of kaempferol and/or chloroquine on various glioma cells, cell viability assay using calcein-AM and EthD-1 was performed. The changes in the lysosomal structures following treatment with kaempferol and/or chloroquine were observed by electron microscopy and fluorescence microscopy using acridine orange or Lyso-tracker Red. The changes in cathepsin D proteins were analyzed by Western blotting, immunocytochemistry, and fluorescence microscopy using BODIPY FL-pepstatin. RESULTS Treatment with subtoxic doses of chloroquine, when combined with kaempferol, effectively induced cell death in various glioma cells, but not in normal astrocytes. While kaempferol treatment increased the numbers of lysosome, chloroquine treatment increased lysosomal masses. Combined treatment with kaempferol and chloroquine induced the expansion and subsequent rupture of lysosomes, leading to the spillage of the lysosomal contents into the cytosol. We found that while kaemfperol treatment increased the active mature forms of cathepsin D, chloroquine treatment completely blocked the processing of cathepsin D. The processing of cathepsin D was also blocked by the combined treatment, but the activity of cathepsin D, which was released from the lysosomes, was restored. The cell death induced by kaempferol and chloroquine in U251MG cells was accompanied by mitochondrial dysfunction, ER stress, and DNA damage. CONCLUSION Disruption of lysosomal membrane integrity and a resultant release of lysosomal proteases may critically contribute to the irreparable damage of various organelles and glioma cell death by chloroquine plus kaempferol.

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In vivo and in vitro structure and relationship analysis of andrographolide derivatives: Identifying AGL-2 as a novel anti-angiogenesis agent

Jing-jing LI, Yu-ran PENG, Shang LI, Judy Yuet-Wa CHAN, Guo-zhen CUI, Guo-chun ZHOU, Simon Ming-Yuen LEE

2015, 29(S1): 88-89.

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OBJECTIVE To analyse structure and relationship of several andrographolide derivatives in multiple in vivo and in vitro angiogenesis assays, and to demonstrate a novel compound named AGL-2 as a potential anti-angiogenesis agent. METHODS Human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish (Danio rerio) in vivo models were used to screen and identify the anti-angiogenesis activities of six andrographolide derivatives; namely, AGL-1, AGL-2, AGS-72, AGS-72a, AGS-79 and AGP-151. RESULTS AGL-2 exhibited the strongest anti-angiogenic activity among all the derivatives in zebrafish model. Interestingly, another compound named AGS-72 showed stronger anti-angiogenic activity than AGL-2 in VEGF-induced HUVECs proliferation, migration, invasion and tube formation assays. In addition, AGL-2 was found to suppress the VEGF-induced VEGFR-2 auto-phosphorylation and inhibit the activity of VEGFR-2 mediated signaling cascades in a dose-dependent manner. CONCLUSION AGL-2 was demonstrated to be a promising anti-angiogenic agent among all the tested derivatives. The mechanism underlying the anti-angiogenic activity of AGL-2 probably involve VEGFR-2 signaling pathway. Even though, how some of chemical structure alterations result in discrepancy between in vivo and in vitro activities still remains to be resolved, this study shall provide new insight into how modification of the chemical structure of andrographolide affects this newly identified anti-angiogenesis activity. Meanwhile, AGL-2 can be exploited as a potential therapeutic agent for the treatment of angiogenesis-related diseases.

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Cytotoxic effect some fractions of parasitic tea tree (Scurrulla atropurpurea Danser) and its potential for DNA demethylation in silico approach

Ni Luh Putu Eka SUDIWATI, Mulyohadi ALI, Edi Priyo UTOMO, AULANI'AM, Tatit NURSETA

2015, 29(S1): 89-89.

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OBJECTIVE To investigate the secondary metabolite flavonoids containing beside epigallo catechin 3-gallate (EGCG) from n-hexane, chloroform and ethanolic fraction, cytotoxic potential from some fractions of Scurrula artropurpurea (Blume) Danser (SAD), and its potential as DNA methyl transferase (DNMT) inhibitor. METHODS To identify 3 isolate from different regions, Lawang and Lembang (plateau region), Wlingi (lowland region) by 3 fractions (n-hexane, chloroform and ethanol) using thin layer chromatography (TLC) and a spectrometer. EGCG level measured by Liquid-Chromatography mass spectrophotometer (LC-MS). The viability of HeLa cells measured by MTT assay. Acridine ethidium-bromide staining was applied to evaluate cell death. Molecular docking conducted for DNMT inhibition by Autodock Vina using Pyrex 0.8 programs and molecular interaction by ligand scout V 2.0 program. RESULTS The different region showed different secondary metabolite. SAD from Lawang and Lembang (both are polite) have similar active compound in n-hexan fraction (didhydroflavonol, flavonone, flavanone), chloroform fraction (dihydroflavonol, flavanone, catechin) and ethanolic fraction (flavonone, flavonol and EGCG). While Wlingi isolate have no dihydroflavonol in chloroform fraction, but flavone. We used Lawang isolate for next research to evaluate cytotoxicity potential for HeLa cells for 24 h. IC₅₀ chloroform fraction was the highest, followed by ethanol and n-hexane fraction (96.16, 298.82 and 489.66 μg·mL^-1, respectively). Index apoptosis were observed at half IC₅₀ concentration of chloroform (99.97%), ethanol (66.79%) and n-hexane fractions (14.92%). In silico analysis showed some active compounds have specific binding site to the DNMT with low energy binding affinity such as -10.4 for EGCG, -8.4 for catechin, -8.2 for dihydroflavonol, -8.0 for flavone, -7.9 for flavonol and -7.8 kcal·mol^-1 for flavonone. The impact of of DNMT and bioactive compounds complex showed its potential for epigenetic manipulation such as demethylation. Inhibition of DNMT on ABCG2 gene could combat drug efflux which main problem of multidrug resistance in cervical cancer chemotherapy. CONCLUSION Chloroform fraction (without EGCG containing) have the highest potential for cytotoxic agent for Hela cells rather than ethanol and n-hexane fraction and could develop as chemotherapeutic agent due to its property against multi drugs resistance as well.

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Mechanisms of eriocalyxin B on antileukemia activity by human cDNA microarray

Zhi-ying WENG, Hui-hui XIANG, Ying-li WU, Ma-ling LI, Han-dong SUN

2015, 29(S1): 90-90.

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OBJECTIVE Eriocalyxin B (EriB) is a natural diterpenoid purified from Isodon eriocalyx var. laxiflora, traditional Chinese herbal medicines, possesses strong antileukemic activity with low toxicity. In murine t(8;21) leukemia models, EriB remarkably prolong the survival time and decreased the xenograft tumor size by targeting AML1-ETO oncoprotein.In angiogenesis research by the highly vascularized chorioallantoic membrane (CAM) of the chicken embryo further confirm its antiangiogenic activity. Microarray offers a high efficient approach to study the gene expression profile treated by EriB, so as to provide systematic information about potential mechanisms of EriB curing AML. METHODS The t(8;21)AML cell line Kasumi-1 is most sensitive to EriB. Cells are treated with Eri-B (0.5 μmol·L^-1) and collected in 2, 6, 12 and 24 h, respectively. Using human cDNA microarray, we investigate the changes of differential gene expression of Kasumi-1 cells by time before and after Eri-B treatment. Meanwhile, the mRNA expression of TRAF2, DEDD2, BAG3, SAT1, IQGAP1, C-myc and GRB2 is detected by semi-quantitative RT-PCR and real-time quantitative PCR. RESULTS The genes regulated significantly are correlated with cell proliferation,apoptosis, cell circle, regulation of transcription, response to stimulies and metabolism. Regulation of TNFR mediated apoptotic signaling by EriB plays a key role to induce apoptosis and cell circle, involved NF-κB, Ras-MAPK, cAMP/PKA, PI₃K/Akt, Cyt c/caspase and p53-Rb signal pathways. After detected by SqRT-PCR and real-time quantitative PCR, the mRNA expressions of BAG3, DEDD2, SAT1 and C-myc are significantly changed. CONCLUSION These findings describes the probable mechanisms involved and the value of EriB as a promising candidate targeting apoptosis cascade and cell circle in treatment of AML.

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A plant sesquiterpene lactone and its derivative reduce cutaneous side effect of vemurafenib, a BRAF inhibitor drug to treat late stage melanoma

Meng-ting CHANG, Jia-hua FENG, Kyoko NAKAGAWA-GOTO, Kuo-Hsiung LEE, Lie-Fen SHYUR

2015, 29(S1): 90-91.

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OBJECTIVE To investigate the pharmacological effect of a plant sesquiterpene lactone (designated D) and its semi-organically synthesized novel derivative (designated S) and the role of lipid mediators, viz., oxylipins in attenuating vemurafenib-induced cutaneous side effects. METHODS A DMBA/TPA-induced skin carcinogenesis mouse model mimicking cutaneous side effect caused by vemurafenib was established to evaluate the efficacy of compound D and S in reversal of vemurafenib side effect. Comparative oxylipin metabolomics platform using UPLC-TQD mass spectrometry coupled with partial least squares-discriminant analysis (PLS-DA) analysis, cell-based assays, and immunochemistry analysis were performed to elucidate the mechanism insights of DET and S compounds and the role of specific oxylipins in skin cancer carcinogenesis. RESULTS Vemurafenib treatment expedited the skin papillomas formation in DMBA-TPA treated mouse from week 6 to week 3. Both D and S compounds could suppress the vemurafenib side effect and also decrease total papillomas numbers (55% to 72%) and average sizes (66% to 89%). Oxylipins metabolome analysis shows that specific arachidonic acid metabolites may play a role in vemurafenib-induced squamous cell carcinoma or keratoacanthomas formation in mouse skin that can be deregulated by D or S compound treatment. Notably, S compound can inhibit vemurafenib-induced paradoxical activation of MAP kinases in mouse skin or in NRAS mutant melanoma cells. CONCLUSION Our results indicate that plant sesquiterpene lactone D and its novel analog can reduce cutaneous side effect of vemurafenib through novel modes of action by inhibiting paradoxical activation of MAP kinases and de-regulating pro-inflammation mediators COX-2 and specific ecosanoid-type of oxylipins. This study may suggest a novel adjuvant therapy approach in treatment of BRAFV600E mutant melanoma.

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Antiangiogenic effect of berberine α-hydroxy-δ-decanoylethyl sulfonate on Lewis lung carcinoma

Jing LIN, Hui QI

2015, 29(S1): 91-92.

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OBJECTIVE To investigate the antiangiogenic and antitumor effects of berberine α-hydroxy-δ-decanoylethyl sulfonate (HB) in vitro and in vivo. METHODS MTT assay was employed to determine the proliferation of tumor cells. Western blot analysis was used to detect the expression of VEGF and MMP-9. Transwell co-culture was used to determine the cell migration at the conditioned medium of Lewis lung carcinoma cells exposed to HB for 24 h. The C57BL/6 mice bearing Lewis lung carcinomas were treated with vehicle, different doses of HB (60, 90 and 120 mg·kg^-1, ig) for 20 d, or cyclophosphamide (50 mg·kg^-1, ig) at d 1, d 8 and d 15. Afterwards, the xenografted tumors were exercised and weighed, and the lung metastasis was determined by HE stainingof the lung tissues. Tumor angiogenesis was determined by CD34 staining and microvessel density (MVD) by immunohistochemistry. RESULTSHB inhibited the growth of Lewis lung carcinoma cells in a time-dependent manner with IC₅₀ value of 8.87, 2.26 and 0.98 mg·L^-1, respectively when the cells were treated with HB for 24, 48 and 72 h. Cell migration induced by the conditioned medium of Lewis lung carcinoma cells treated with 2.5, 5 and 10 mg·L^-1 HB was reduced by 37%, 54% and 63% respectively (P<0.01). HB suppressed the expression of angiogenesis factors in a concentration-dependent manner. HB was effective in inhibition of MMP-9 expression at 5, 10 and 20 mg·L^-1 (P<0.05), whereas HB 20 mg·L^-1 was minimal effective concentration in inhibition of VEGF expression (P<0.01). On C57BL/6 mice bearing Lewis lung carcinomas, HB significantly reduced the tumor burden and MVD in tumor mass as well as inhibited lung metastasis at doses of 90 and 120 mg·kg^-1. CONCLUSION HB produces reliable antiangiogenic effect, inhibits the growth and metastasis of Lewis lung carcinoma. These effects may be attributed to its ability of suppressing the expression of VEGF and MMP-9, and reducing MVD.

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天然药物研发与技术

Inorganic nanoparticles for hydrophobic anticancer drug delivery

Xiao-ming ZHU

2015, 29(S1): 92-92.

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OBJECTIVE Many drug candidates identified from natural products are poorly water-soluble. The surfactants used to disperse the hydrophobic anticancer drugs in water may cause a serious of acute hypersensitivity reactions. Nanotechnology provides an alternative strategy for delivery of anticancer drugs. In the present study, different inorganic nanoparticles are utilized as hydrophobic anticancer drug carriers. METHODS Different inorganic superparamagnetic iron oxide, platinum and gold nanoparticles were synthesized. The hydrophobic anticancer drugs such as curcumin, gambogic acid and doxorubicin (DOX) base were loaded into the porous area or onto the surface of the nanoparticles. Cellular uptake and biocompatibility of nanoparticles were studied in human glioblastoma U-87 MG cells. The anticancer effect of drug loaded nanoparticles was compared with that of free drugs. Photothermal conversion of platinum and gold nanoparticles was studied by irradiation of nanoparticles with a near-infrared laser. RESULTS The synthesized nanoparticles are readily internalized by U-87 MG cells, and the internalized nanoparticles are mainly localized in endosomes/lysosomes in cells. The nanoparticle-based drug carrier provides the aqueous dispersions of the hydrophobic drugs. In endosomes/lysosomes mimicking buffers with a pH of 4.5-5.5, pH-dependent drug release was observed from drug loaded nanoparticles. The intracellular drug content and cytotoxicity are significantly higher for drug loaded nanoparticles than free drug. Photothermal treatment has a synergistic effect on drug's anticancer activity. CONCLUSION These results suggested inorganic nanoparticles is a promising intracellular carrier for hydrophobic anticancer drugs.

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天然药物研发与技术

Killing effect of the combined treatment of ultrasound and hypocrellin B on Candida albicans

Chuan-shan XU, Albert Wing-Nang LEUNG, Margaret IP, Xin-na WANG, Pan WANG

2015, 29(S1): 92-93.

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OBJECTIVE To investigate the killing effect of the combined treatment of ultrasound and hypocrellin B, an active component isolated from a natural product Hypocrella bambuase, on Candida albicans. METHODS At first, Candida albicans are incubated with hypcrellin B and the uptake of hypocrellin B in Candida albicans was determined using fluorescence intensity analysis. And then the sensitized Candida albicans were exposed to low-intensity of ultrasound. After the combined treatment of ultrasound and hypocellin B, the growth of Candida albicans was evaluated using the colony counting method, and membrane integrity of Candida albicans was also analyzed by a flow cytometry with propidium iodide staining. RESULTS Hypocrellin B was fast absorbed by candida albicans. The growth of candida albicans was more significantly inhibited by the combined treatment of ultrasound and hypocrellin B than the controls (P<0.05). The more marked damages to membrane integrity were found in the combined treatment than the controls (P<0.05). CONCLUSION The combined treatment of ultrasound and hypocrellin B had significant killing effect on Candida albicans, indicating sonodynamic therapy with hypocrellin B from natural product might be a potential approach for combating fungal infections.

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天然药物研发与技术

HPTLC Analysis of multi-ingredient traditional Chinese medicine supplement

Martin CAI, Sheena GOH, Leng NEO, Feng TIAN

2015, 29(S1): 93-94.

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OBJECTIVE Analysis of traditional Chinese medicinal (TCM) supplements has always been a laborious task, particularly in the case of multi-ingredient formulations. Traditionally, herbal extracts are analyzed using one or few markers compounds. In the recent years, however, pharmaceutical companies are introducing health supplements of TCM ingredients to cater to the needs of consumers in the fast-paced society in this age. As such, new problems arise in the aspects of composition identification as well as quality analysis. In most cases of products or supplements formulated with multiple TCM herbs, the chemical composition and nature of each raw material differs greatly from the others in the formulation. This results in a requirement for individual analytical processes in order to identify the marker compounds in the various botanicals. The aim of this study is to establish a rapid and low cost analytical approach using high performance thin layer chromatography (HPTLC) for the analysis of 3 key components of the LAC Liver Protector, a supplement with TCM formulation aimed at improving liver health. METHODS The TCM herbs were extracted with methods derived from Chinese Pharmacopeia standards. The herbal extracts were then applied to HPTLC plates using CAMAG Linomat 5 and developed using fully saturated twin-trough chromatographic chambers. The developed plates were derivatized and documented with CAMAG TLC Visualizer. The images were processed with CAMAG winCATS software to calculate the Rf values and confirm the presence of key constituents of the herbs. RESULTS The 3 key components of LAC Liver Protector product, namely Radix notoginseng, Rhizoma alismatis and Radix paeoniae alba/rubra were identified in all 6 batches of products tested. CONCLUSION With the increasing trend of small and medium-sized enterprises (SMEs) bringing natural products and health supplements into the market, it is crucial that the qualities of both raw materials and end products be well-assured for the protection of consumers. With the technology of HPTLC, science can be incorporated to help SMEs with their quality control, thereby ensuring product quality.

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天然药物研发与技术

A fast screening method for multi-residue pesticide analysis of TCM herbs using liquid chromatography-quadrupole-time-of-flightmass spectrometry

Xue-jia XUE, Hong YANG, Feng TIAN

2015, 29(S1): 93-93.

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OBJECTIVE Identification of pesticide residues in TCM herbs is important for drug safety and public health. However, it is very challenging for the development of an instrument-based multi-residue pesticide analysis method for theherbalproducts due to their complex matrix. This study aims to develop a rapid method for the high-throughput detection of pesticide residues in TCM herbal products using liquid chromatography coupled to quadrupoletime of flight (LC-QTOF) mass spectrometry. METHODS In this study, we attempt to investigate possible pesticide residues in TCM herbal products which are commonly available in Singapore and to consolidate a comprehensive pesticide database for more than 250 regulated pesticides in FDA Regulations. We also attempt to perform non-targeted and targeted screenings of pesticide residues in herbal products by searching our database. RESULTS The developed LC-QTOF method demonstrates have high mass accuracy, resolution and selectivity, which makes it a very suitable tool for monitoring both target and non-target pesticides with a limit of detection at the low ppb level. Moreover, the LC-QTOF technology permits to retain all spectral data in a single run. It may avoid many difficulties related to matrix effects and will save much manpower. CONCLUSIONThis means that a variety of TCM herbs can be easily detected by this method for the presence or absence of pesticide residues that must conform to government regulations.

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天然药物研发与技术

Evaluation of photodynamic efficiency of an endogenous enzyme-NQO1 activated hypericin

Qi-cai XIAO, Wing-nang LEUNG, Chuan-shan XU

2015, 29(S1): 94-94.

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OBJECTIVE To investigate the therapeutic efficiency of an endogenous enzyme-NQO1 activated hypericin in photodynamic therapy. METHODS An endogenous enzyme-NQO1 responsive photo sensitizer was designed and synthesized by conjugating a quinonebased ligand to a natural photosensitizer derived from Chinese herb. The photophysical and photochemical properties were investigated through UV-visible and fluorescence spectrophotometer, and the photodynamic activity was evaluated with MTT assay. RESULTS An endogenous enzyme-NQO1 activated hypericin was prepared and fully characterized with various spectroscopic methods. The electronic absorption was almost the same with the free hypericin, indicating the introducing of the ligand to hypericin has little effect to its ground state, while there is almost no detected fluorescence and reactive oxygen species (ROS) generation in PBS solution indicating the introducing of the ligand can effectively quench the fluorescence emission and ROS generation. The in vitro study showed that both compounds have almost no dark toxicity, but they are highlyphotocytoxic with an IC₅₀ less than 1 μmol·L^-1 against A549 cell lines indicating the modified compound can be activated in the intracellular environment. CONCLUSION A simple and efficient hypericin-based activated photosensitizer was prepared. The ROS generation was quenched in PBS solution and it would be activated inside A549 cell lines. It may be served as a promising tumor selective fluorescent probe and photosensitizer for targeted photodynamic therapy.

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天然药物研发与技术

Identification of a natural product-like STAT3 dimerization inhibitor by structure-based virtual screening

Li-juan LIU, Ka-Ho LEUNG, Daniel Shiu-Hin CHAN, Dik-Lung MA, Chung-Hang LEUNG

2015, 29(S1): 95-95.

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OBJECTIVE To apply molecular docking techniques to identify STAT3 inhibitors from a database of over 90 000 natural product and natural product-like compounds. METHODS Molecular docking was used for the virtual screening campaign and hit validation of STAT3 inhibitor. To further evaluate the potency of candidates at inhibiting STAT3-DNA binding activity, a STAT3 and STAT1 transcription factor ELISA was performed. A dual-luciferase reporter assay, co-immunoprecipitation assay and Western blotting were carried out for the investigation of effect of compound 1 on STAT3-driven transcription, STAT3 dimerization and STAT3 phosphorylation. Finally, the cell toxicity of compound 1 was assessed by using MTT assay on different cell lines. RESULTS The virtual screening campaign furnished fourteen hit compounds, from which compound 1 emerged as a top candidate. Compound 1 inhibited STAT3 DNA-binding activity in vitro and attenuated STAT3-directed transcription in cellulo with selectivity over STAT1 and comparable potency to the well-known STAT3 inhibitor S3I-201. Furthermore, compound 1 inhibited STAT3 dimerization and decreased STAT3 phosphorylation in cells without affecting STAT1 dimerization and phosphorylation. Compound 1 also exhibited selective anti-proliferative activity against cancer cells over normal cells in vitro. CONCLUSION The benzofuran derivative 1 was identified as a potential inhibitor of STAT3 dimerization using in silico screening. Molecular docking analysis suggested that compound 1 might putatively function as an inhibitor of STAT3 dimerization by binding to the SH2 domain. To the best of our knowledge, compound 1 has not been reported as a STAT3 inhibitor and no biological activity of compound 1 has been presented in the literature.

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天然药物研发与技术

Pharmacophore modeling for the identification of small-molecule inhibitors of TACE

Li-juan LIU, Ka-Ho LEUNG, Sheng LIN, Daniel Shiu-Hin CHAN, Dewi SUSANTI, Wei-dong RAO, Philip Wai Hong CHAN, Dik-Lung MA, Chung-Hang LEUNG

2015, 29(S1): 95-96.

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OBJECTIVE To employ pharmacophore modeling to identify a TACE inhibitor from an in-house database of 66 organic compounds. METHODS To identify the common features required for TACE inhibition, we generated a pharmacophore model from a set of TACE-selective inhibitor using the Common Feature Pharmacophore Model protocol implemented in Discovery Studio 3.1.1. A fluorimetric assay was used to investigate the potential ability of compounds to inhibit TACE enzymatic activity. The ability of compound 1 to inhibit TACE activity in a human monocyte THP-1 cell line was evaluated by ELISA. RESULTS In this study, a pharmacophore model constructed from a training set of TACE inhibitors was used to screen an in-house database of organic compounds, from which compound 1 emerged as a top candidate. In a cell-free assay, compound 1 inhibited TACE enzymatic activity in a dose-dependent manner. Moreover, compound 1 inhibited the production of soluble TNF-α in human acute monocytic leukemia THP-1 cells without impacting nitric oxide production, and exhibited anti-proliferative activity against THP-1 cells. CONCLUSION Compound 1 was found to inhibit TACE enzymatic activity in a cell-free system and LPS-induced TNF-α secretion in cellulo. We envisage that compound 1 may be employed as a useful scaffold for the development of more potent TACE inhibitors.

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天然药物研发与技术

Effect of sonodynamic action of hypocrellin B on biofilm-producing Enterococcus faecalis

Xin PANG, Xin-na WANG, Margaret IP, Albert Wing-Nang LEUNG, Pan WANG, He-yu HUA, Chuans-han XU

2015, 29(S1): 96-96.

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OBJECTIVE To investigate the effect of sonodynamic action on biofilm-producing Enterococcus faecalis using hypocrellin B from a natural product Hypocrella bambuase as a sensitizer. METHODS The uptake of hypocrellin B in biofilm-producing Enterococcus faecalis was measured using the fluorescent analysis method. After the combined treatment of hypocrellin B and ultrasound, the growth of biofilm-producing Enterococcus faecalis was measured using the colony counting method, the membrane integrity was detected using a flow cytometric analysis with propidium iodide staining. Chromosomal DNA of bacteria was analysed using a pulsed-field gel electrophoresis (PFGE). RESULTS The uptake of hypocrellin B in biofilm-producing Enterocuccus faecalis reached a high peak at the 20 min incubation. After the combined treatment of hypocrellin B, the growth of biofilm-producing Enterocuccus faecalis significantly descreased and membrane integrity was remarkably damaged. However, no remarkable change in bacterial DNA was found. CONCLUSION Sonodynamic action of hypocrellin B had significant killing activity on Enterocuccus faecalis.

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天然药物研发与技术

In silico binding mode analysis of andrographolide and its derivatives to mutant and wild-type K-ras

Shun Ying QUAH, Pran KishoreDEB, Johnson STANSLAS

2015, 29(S1): 97-97.

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OBJECTIVE To investigate the structural requirements for effective binding of andrographolide (AGP) and its derivatives (SRJ09 and SRJ23) to mutant K-ras for inhibition of exchange factor binding viain silico docking simulations. METHODS The molecular docking studies were carried out by using SiteMap v3.4 andGlide v6.6modules (SchrÖdinger, Inc.). Surface mapping on the 3-D X-ray crystal structures of three mutant K-ras proteins-K-rasG12V (PDB ID: 4EPX), K-rasG12C (PDB ID: 4LDJ), and K-rasG12D (PDB ID: 4DSU), as well as wild-type K-ras protein (PDB ID: 4LPK), was performed to generate possible sites for ligand binding.Thirty conformers were generated for each of the studied compounds, and these conformers were docked into each possible binding site in both wild-type and mutant K-ras proteins. The free energy of binding of the compounds with the wild-type and mutant K-ras proteins was performed using prime molecular mechanics with generalized Born and solvent accessibility (MM-GBSA) approach. RESULTS The conformers of AGP, SRJ09 and SRJ23 that were found to form the most stable complex inside each possible binding siteas indicated by the highest binding free energy, both in wild-type and mutant proteins, were selected. A common binding site between switchⅠ and Ⅱ regions, where a pocket surrounded by amino acid residues Lys5, Leu6, Val7, Ser39, Asp54, Leu56,Tyr71, Thr74, and Gly75, was found in all K-rasG12 mutants.This site corresponds to the hydrophobic binding pockets having aliphatic side-chain portionsas found previously for other Ras binders, which are located between α-helix 2 and the core β-sheets (between switchⅠ and Ⅱ regions). This common binding pocket was not observed in the wild-type K-ras. A binding pocket adjacent to switchⅡ region (amino acid 60-72), where all ligands bind well, was found instead. All compoundsanchor well inside the common binding pocket in each of the K-fasG12 mutants and these compounds showed the strongest binding interactions to K-fasG12C. SRJ09 and SRJ23 showed stronger binding interactions to both wild-type and mutant K-ras proteins as compared with the parent compound. Overall, the compounds displayed higher binding energies toall three mutant proteins as compared to their wild-type counterpart. CONCLUSION AGP, SRJ09, and SRJ23 are potential K-ras-targeting anti-cancer agents. The compounds target both wild-type and mutant K-ras but they bind to a different binding pocket in the wild-type protein. Both binding pockets found in wild-type and mutant K-ras involve switchⅡ region that binds the guanine nucleotide exchange factor (GEF) such as Son of Sevenless. These suggest a possible inhibition of exchange factor binding to both wild-type and mutant K-ras proteins. Lower binding energies of the compounds to wild-type K-ras protein suggest a transient binding and inhibition. Stronger binding of all compounds to mutant K-ras proteins could lead to more targeted and prolonged inhibition.

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天然药物研发与技术

Integrative database for multi-compound drug discovery in complementary medicine

Sunyong YOO, Jaejoon CHOI, Moonshik SHIN, Suhyun HA, Kyungrin NOH, Hojung NAM, Doheon LEE

2015, 29(S1): 98-98.

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OBJECTIVE To construct an integrative database for multi-compound drug discovery. METHODS We designed and constructed a database system, which integrates traditional herbal medicine, functional food, and drug combination information. Our database consists of six entity tables, namely drug combinations, functional foods, prescriptions, herbs, compounds and phenotypes. We established strategies for data integration and entity resolution to facilitate heterogeneous information of multi-compound therapies. To standardize the data, instances of entity tables are mapped to international identifiers, and phenotype terms in narrative text are extracted by using the named entity recognition (NER) method. RESULTS The database integrates therapeutic information of traditional herbal medicine, functional foods and combination drugs which is acquired from Traditional Chinese Medicine Information Database (TCM-ID), Food and Drug Administration (FDA) and Drug Combination Database (DCDB). The herb information is mapped to NCBI taxonomy identifiers, and compound information is mapped to PubChem and ChEMBL identifiers for standardization. We also applied MetaMap, a tool for recognizing UMLS concepts from narrative text, to extract phenotype terms. The current version of the database contains 6 291 drug combinations, 1 615 functional foods, 20 091 prescriptions, 8889 herbs, 227 636 compounds and 11 744 phenotypes. CONCLUSION Our database provides various therapeutic information of multi-compound therapies which serve as a fundamental resource for the polypharmacology research.

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天然药物研发与技术

Virtual screening and optimization of typeⅡ inhibitors of JAK2 from a natural product library

Dik-Lung MA, Daniel Shiu-Hin CHAN, Guo WEI, Hai-jing ZHONG, Hui YANG, Lai To LEUNG, Elizabeth A. GULLEN, Pauline ChIU, Yung-Chi CHENG, Chung-hang LEUNG

2015, 29(S1): 98-99.

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OBJECTIVE To utilize a structure-based lead optimization approach to generate novel natural product-like typeⅡ inhibitors of JAK2 using the DOLPHIN protocol. METHODS Initially, the DOLHPIN computational protocol was employed to convert an active (DFG-in) conformation of JAK2 into a typeⅡ-compatible conformation, which was used as a model for the structure-based virtual screening of 150 000 natural product-like compounds in silico. The novel biflavonoid analogues were designed and synthesized based on the structure of lead compound and then tested for JAK2 and STAT3 inhibitory activity, cytotoxicity and HCV antiviral activity. RESULTS The top eleven highest-scoring compounds were generated from the initial high-throughput virtual screening. Amentoflavone 1a, a biflavonoid from the Chinese plant Gingko biloba, emerged as a promising candidate for further biological evaluation. In dose-response experiments, amentoflavone 1a inhibited JAK2 activity in a concentration dependent fashion with an IC₅₀ value of 5 μmol·L^-1. As a proof-of-concept, we designed nine analogues 1b-1j with the addition of one or more aliphatic side chains to the biflavonoid scaffold of 1a. The octyl (C₈) analogue 1b displayed superior potency against JAK2 activity and HCV activity compared to the parent compound 1a, validating the structure-based lead optimization approach used in this study. Moreover, kinetic analysis indicated that analogue 1b exhibited a non-competitive mode of inhibition, suggesting that this compound may be a putative typeⅡ inhibitor of JAK2. CONCLUSION Amentoflavone 1a has been identified as a JAK2 inhibitor by structure-based virtual screening of a natural product library. In silico optimization using the DOLPHIN model yielded analogues with enhanced potency against JAK2 activity and HCV activity in cellulo. Molecular modeling and kinetic experiments suggested that the analogues may function as typeⅡ inhibitors of JAK2.

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天然药物研发与技术

Identifying multi-component drug candidates in natural products via association rule mining

Suhyun HA, Kyungrin NOH, Moonshik SHIN, Sunyong YOO, Jaejoon CHOI, Hojung NAM, Doheon LEE

2015, 29(S1): 99-100.

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OBJECTIVE To identify compound combinations as candidate multi-component drugs for the type 2 diabetes from natural product information. METHODS Chemical composition information of herbs in natural medicine was acquired by integrating conventional databases; Traditional Chinese Medicine Information Database (TCM-ID) and Traditional Chinese Medicine Integrated Database (TCMID). Therapeutic effect of each herb on the type 2 diabetes was examined by analyzing annotated function information with a text-mining method. The Apriori algorithm, which is a classical method for extracting associations between object in large-scale databases, was employed to infer association rules between compound combinations and therapeutic effect on the target disease. The chemical composition and therapeutic information of each herb was used as a transaction, which consists of the chemical compound combination as an antecedent item set and the therapeutic effect as a consequent item. The association rules with high support and confidence value were suggested as candidate multi-component drugs for the type 2 diabetes. RESULTS Totally 40 941 association rules were inferred with support lower bound 0.05% and maximum rule length 4. With respect to support and confidence, the top-ranked compound combination was puerarin and daidzin (support=0.15%, confidence=100%). In addition, the top 16 compound combinations were composed of 11 individual chemical compounds; puerarin, daidzin, abscisic acid, batatisine, dopamine, cholesterol, daidzein, gamma-aminobutyric acid, stigmasterol, campesteryl ferulate, and campesterol. To validate therapeutic effect of the proposed compound combinations, literature evidences of each individual compound were investigated. Among the 11 individual compounds, six compounds were reported to be effective for the treatment of the diabetes mellitus. CONCLUSION By analyzing natural product in formation with association rule mining, 16 compound combinations are suggested as candidate multi-component drugs for the type 2 diabetes. These compound combinations are recommended for further investigation in the context of drug development.

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Cistanchetubulosa (Schenk) R. Wight amelioratesbisphenol A-induced testicular and sperm toxicity in rats through steroidogenesis and redox pathways

Zhi-hui JIANG, Wen-yan XIE, Meng WANG, Jian WANG, Xiao-ying ZHANG

2015, 29(S1): 100-100.

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OBJECTIVE Bisphenol A (BPA) is a commonly used phenolic environmental estrogen that impacts male reproductive system. Cistanchetubulosa (CT) is a traditional Chinese medicine used to boost male sexual activity.It has been proved for increasing the expression of steroidogenic enzyme and remarkable antioxidant activity. This study aimed to investigate the mechanisms behind the possible protective effects of CT against BPA-induced testicular and sperm toxicity in rats. METHODS CT was identified by 5.8s gene sequencing. The major compositions (echinacosideand acteoside) of CT were quantified by HPLC method. CT (200 or 400 mg·kg^-1) or VE (positive control, 300 mg ·kg^-1) and BPA (200 mg·kg^-1) were administered orally (one time per day) to adult male ratsfor 6 weeks. The sperm parameters were observed by dark-field microscope; serum hormone levels (FSH, LH and T) were tested by radio immunosorbent; LDH-x activity, redox balance status (content of MDA, GSH, H₂O₂ and OH^-, and activity of SOD) were evaluated using commercial kits; the expression of the key steroidogenic enzymes were evaluated by qRT-PCR, heat map, immunofluorescence and Western blotting. RESULTS The CT treatment reversed BPA-induced abnormal in sperm characteristics, testicular structure and LDH-x activity, as well as normalized serum testosterone. CT increased both mRNA and protein expressions of key steroidogenic enzymes including StAR, CYP11A1, 3β-HSD, 17β-HSD and CYP17A1, suggesting that CT enhanced sex hormone synthesis. Moreover, the remarkable antioxidant effect of CT contributed to its protection against BPA-induced testicular toxicity, which was evidenced by the normalization of testicular, redox markers (MDA, H₂O₂, OH^- and GSH contents, and SOD activity) after CT treatment. CONCLUSION CT effectively attenuated BPA-induced poor sperm quality and testicular toxicity in rats through steroidogenesis and redox pathways.

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Design, synthesis and bioevaluation of N-hydroxyquinolinones as potential anti-plasmodial, anti-bacterial and iron(Ⅱ)-chelating agents

Yan-bo TENG, Alice Soh-Meoy ONG, Laurent RÉNIA, Christina Li-Lin CHAI

2015, 29(S1): 101-101.

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OBJECTIVE To establish a small compound library via a versatile synthetic route for the investigation of natural-inspiring compounds containing N-hydroxypyridones motif as potential anti-plasmodial, anti-bacterial and iron(Ⅱ)-chelating agents. METHODS An amidation/cyclization approach was adopted to synthesize a library of N-hydroxyquinolinones. The anti-plasmodial susceptibility of lab clone 3D7 P. falciparum was measured using a protocol modified from the WHO microtest. The minimum bactericidal concentration (MBC) values were determined against Escherichia coli and Staphylococcus arueus. Nine compounds were selected to test their iron(Ⅱ)-chelating abilities. The iron(Ⅱ)-chelating ability was determined by measuring the absorbance of ferrozine-iron complex at 562 nm. RESULTS A new route for the facile synthesis of a library of N-hydroxyquinolinones based on one-pot palladium catalyzed C-N amidation/dehydrocyclizationsequence was implemented. Four compounds show anti-plasmodial activities with the range of 1.1-1.4 μmol·L^-1, 50% chelation abilities of the nine selected compounds were shown to be 0.24-0.29 mmol·L^-1. CONCLUSION Alibrary of N-hydroxyquinolinones was synthesized via a novel synthetic route. The anti-plasmodial and anti-bacterial activities of these compounds were evaluated. Four compounds show potent anti-plasmodial activities Nine compounds were examined for their propensities to undergo iron chelation and these compounds were shown to be promising iron(Ⅱ)chelators as compared to EDTA.

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Testa of Rhynchosia nulubilis down-regulates melanin synthesis in UVB-induced guinea pigs epidermal skin tissue

Hyun Jeong LEE, Donghun LEE, Jung-bin SONG, Young Sik KIM, Hai-ling GUO, Seunggyeong LEE, Juyeon PARK, Sun Yoeu KIM, Hocheol KIM

2015, 29(S1): 101-102.

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OBJECTIVE Synthetic compounds that are used in the clinic to regulate skin hyperpigmentation, such as arbutin, and kojic acid, are only moderately effective. But their use is limited by side effects. As part of an effort to overcome the limitations, there is a pressing need for developing effective natural product for whitening. Each of black soybean has been reported to produce anti-melanogenic effects. Especially, antioxidant and anti-aging compounds are richer in testa than the rest part of black soybean. Therefore, we hypothesized that testa of Rhynchosia nulubilis (TR) would show more anti-melanogenic effects than whole black soybean. METHODS Anti-melanogenic effect of TR was done by using mouse B16 F10 melanoma. The depigmenting efficacy was then observed following topical application of the TR to UVB-stimulated hyperpigmented dorsal skin of guinea pigs. RESULTS Treatment with TR extract resulted a 27.67% decrease in melanogenesis while whole black soybean showed 5.3% decrease at mouse melanoma cell line. Colorimetric analysis showed a significantly lower depigmenting value by day 14 following treatment with TR in UVB-irradiated guinea pigs the dorsal skin, indicating that TR produced a depigmentation effect. By staining with Hematoxylin-Eosin staing and Fontana-Masson stain, we found that the TR-treated group had more effect histopathologically in epidermal melanin production than resveratrol or rice alone-treated group. TR was associated with reduction in the levels of microphthalmia-associated transcription factor (MITF), and downregulation of tyrosinase and tyrosinase-related protein (TRP-1) expression, leading to inhibit epidermal melanin production by Western blotting analysis. CONCLUSION This study suggests that the testa of R.nulubilis may be a promising candidate in regulating skin pigmentation with UVB exposure.

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Comparative study of standardized Centella asiatica and combination of levodopa and benzerazid HCl on Parkinson's model zebrafish

Husnul KHOTIMAH, Wibi RIAWAN, LIZIYYANNIDA, Anggi NURKHAIRINA, Mulyohadi ALI, M Aris WIDODO, Sutiman B SUMITRO

2015, 29(S1): 102-103.

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OBJECTIVE To investigate the motility, dopamine, serotonin and pro-BDNF level of Centella asiatica (CA) comparable to the combination of levodopa and benzerazid HCL on zebrafish model Parkinson's. METHODS Rotenone 5 μg·L^-1 induced to adult zebrafish (8 months) for 28 d to made zebrafish model Parkinson's. CA concentration were used 5 and 10 μg·mL^-1. The combination of levodopa 100 g and 25 mg benzerazid (L-DOPA agonist) was given at 1 μg·mL^-1 at the same time to rotenone, and CA as well. Motility assessment conducted for every 7 d started on day 0 until day 28. Five fish were subjected in the 2 L tank (25 cm×16.5 cm×12 cm). Three vertical lines were drawn on the tank at equal distances, dividing the tank into four zones (the length of each zone was 6.25 cm). Locomotor activity was measured for 5 min by counting the number of lines that adult zebrafish crossed. Fish were sacrificed by decapitating on ice water. Dopamine level measured from whole brain by ELISA, serotonin and pro-BDNF by immunoreactivity at substantia nigra. RESULTS The locomotor activity of rotenone treated fish were significantly decrease starts at 7 d compared to control group. Interestingly on 10 μg·mL^-1 group there are increasing motility start at day 7 and slightly decrease until day 28, but on L-DOPA group increasing motility at day 7 followed by decreasing motility significantly (P<0.05) until day 28. Dopamine level of rotenone group decreased compared to control group, CA 10 μg·mL^-1 and L-DOPA group increased significantly to rotenone group which CA μg·mL^-1 higher than L-DOPA group. Serotonin and pro-BDNF level had the same profile. Its significantly increased on rotenone group compared to control group. Serotonin on group with CA administration significantly decreased compared to rotenone group, while rotenone plus CA 10 μg·mL^-1 had no significant differet to L-DOPA group (P>0.05). Pro-BDNF on rotenone with CA 5 μg·mL^-1 had no significant difference, while compare to rotenone with CA 10 μg·mL^-1 group and L-DOPA group were significantly different (P<0.05). CONCLUSION Rotenone produces reliable Parkinson's zebrafish model by decreasing motility and dopamine level. CA was more stable, increasing motility than L-DOPA administration. CA and L-DOPA increased dopamine level, but decreasing serotonin and pro-BDNF.

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Effects of Angelica sinensis on longitudinal bone growth rate in adolescent female rats

Hai-ling GUO, Donghun LEE, Young-Sik KIM, Jung-bin SONG, Hyun Soo KIM, Hyun Jung LEE, Sung Hyun LEE, Juyeon PARK, Hocheol KIM

2015, 29(S1): 103-103.

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OBJECTIVE To investigate the effect of A. sinensis on longitudinal bone growth rate in adolescent female rats. METHODS A. sinensis was extracted with 30% EtOH for 3 h at 90℃ in a reflux apparatus. Female Sprague-Dawley rats (33 d-old) were randomly divided into three groups: control (vehicle), recombinant human growth hormone (rhGH; 20 μg·kg^-1·d^-1), and A. sinensis (300 mg·kg^-1·d^-1). A. sinensis extracts or vehicle was administered orally twice daily for 4 d. Longitudinal bone growth rate in newly synthesized bone was observed using tetracycline labeling (20 mg·kg^-1, intraperitoneally). RESULTS A. sinensis significantly increased longitudinal bone growth rate in adolescent female rats at 300 mg·kg^-1 (6.1%, 357.34±32.67 μm·d^-1) compared to control group (336.80±14.47 μm·d^-1). CONCLUSION A. sinensis extract significantly increased longitudinal bone growth rate and growth plate height in adolescent female rats. These results suggest that A. sinensis could be helpful for increasing bone growth rate in children who have growth retardation.

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Effect of Dioscorea batatas on longitudinal bone growth rate in adolescent female rats

Hyun Soo KIM, Donghun LEE, Young-Sik KIM, Jung-bin SONG, Hyun Jung LEE, Seunggyeong LEE, Sung Hyun LEE, Juyeon PARK, Hocheol KIM

2015, 29(S1): 103-104.

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OBJECTIVE The root of Dioscorea batatas, rich in steroidal saponins, alkaloids, tannins, phytosterols, and starch, is an herbal medicine of tonifying qi and nourishing stomach after invigorating spleen with a long history of safe use for treatment of chronic dysentery and weakness of the spleen and stomach in Korea. This study was aimed to investigate the effect of D. batatas on longitudinal bone growth rate in adolescent female rats. METHODS D. batatas was extracted with 30% EtOH for 3 h at 90℃ in a reflux apparatus. In two groups, we administered a twice daily dosage of D. batatas extract (at 30 and 300 mg·kg^-1, respectively) per os for 4 d, and in a control group, we administered vehicle only under the same conditions. Recombinant human growth hormone (rhGH) was subcutaneously injected once daily. All rats were born at same day (33 d-old). On day 3, tetracycline was injected intraperitoneally to form a fluorescent band on the growth plates. RESULTS The bone growth rate in groups administered D. batatas 300 mg·kg^-1 and rhGH was significantly increased to 343.8±20.7, and 359.6±30.2 μm·d^-1 respectively from control group, 320.7±23.2 μm·d^-1. No difference was observed in the amount of food intake or mean body weight among all groups during the acclimation or administration period. CONCLUSION These results suggest that D. batatas extracts have the potential to induce height increase; however, further research, including clinical trials, is necessary.

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Basal repertoire of toxin-related transcripts in the zoanthid Protopalythoa variabilis (Cnidaria, Anthozoa)

Chen HUANG, Jean-Etienne RL MORLIGHEM, Hefeng ZHOU, Erica Patrícia LIMA, Paula Braga GOMES, Carlos Daniel PEREZ, Inchio LOU, Simon Ming Yuen LEE, G Radis-BAPTISTA

2015, 29(S1): 104-105.

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OBJECTIVE To analyze the whole transcriptome of zoanthid Protopalythoa variabilis (P. variabilis), a cnidarian, and discover the potential toxic substances in P. variabilis. METHODS The P. variabilis RNA deep sequencing was performed using the HiSeq 2500 automatic sequencing platform. All the unigenes generated from the assembly process were functionally annotated based on the similarity with databases. The multiple alignments of translated toxin-related sequences were performed with Clustalw2, and amino acid identity and similarity highlighted by using BoxShade tool. Three different methods including I-TASSER, PEP-FOLD and MODELLER were applied to predict tri-dimensional models of toxin-related polypeptides from translated transcript sequences of P. variabilis. The toxicity of one of the putative toxins, namely ShK/Aurelin-like peptide, was evaluated using zebrafish model. RESULTS A total of 67,549,914 pairs of quality-filtered, 90-base-pair Illumina reads from an mRNA sample were obtained. The de novo assemblies yielded 276,526 contigs. The sequence comparison of 130,121 unigenes with entries in Toxin database showed that 1542 unigenes were potential peptide toxins at which 11 unigenes were related to Stichodactyla toxin (ShK) domain (Pfam ID: PF01549). ShK is a 35 residues peptide sequence that was firstly discovered from the sea anemone Stichodactyla helianthus. Here, we found out one ShK-like peptide that processed a relatively higher sequence similarity with known ShK (Uniprot ID: P29186) of Bunodosoma granuliferum (red warty sea anemone). The Protopalythoa Shk-like peptide was submitted to Probis server to detect probable binding site and found to match with a protein AURELIN (PDB id: 2lg4, UniProt id: Q0MWV8) which possesses structural homology with previously identified antimicrobial peptides and K⁺-channel-blocking toxins. Our results showed that the ShK/Aurelin-like peptide was lethal to zebrafish embryos at concentrations above 30 μmol·L^-1, and could induce zebrafish locomotor deficit at 10 μmol·L^-1. CONCLUSION This study, for the first time, presented the whole transcriptome profile and a potential toxic peptide of P. variabilis.

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Transcriptome analysis of leaves, roots and flowers of Panax notoginseng on ginsenoside and alkaloid biosynthesis and amelioration of vascular insufficiency conditions by saponins from the flower buds

Bin-rui YANG, Kwok-Kuen CHEUNG, Ming-hua LIU, Wai-Fung CHEUNG, Xin ZHOU, Rui-fang XIE, You-hua WANG, Pui Man HOI, Stephen Kwok-Wing TSUI, Simon Ming-Yuen LEE

2015, 29(S1): 105-106.

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OBJECTIVE To investigate the transcriptomic details on the biosynthetic pathways in different parts of the Panax notoginseng and the pharmacological activity of the saponins extracted from the flower (FS) on vascular insufficiency conditions. METHODS RNA sequencing of three different Panax notoginseng tissues was performed using next generation DNA sequencing and differential gene expression was validated by real-time PCR. In order to determine pro-angiogenic and therapeutic effects of FS on myocardial infraction (MI), FS was examined on the endothelial cell migration assay, vascular insufficiency model in zebrafish and MI model in rats. RESULTS After assembling the high quality sequencing reads into 107 340 unigenes, biochemical pathways were predicted and 9 908 unigenes were assigned to 135 KEGG pathways. Among them, 270 unigenes were identified to be involved in triterpene saponin biosynthesis as well as 350 and 342 unigenes were predicted to encode cytochrome P450s and glycosyltransferases, respectively. One unigene was annotated as CYP716A53v2, probably participates in the formation of protopanaxatriol from protopanaxadiol and the differential expression of this gene was confirmed by real-time PCR. In addition, the pharmacological evaluation demonstrate that FS significantly promoted vascular endothelial growth factor (VEGF) induced the migration of human umbilical vein endothelial cells (HUVECs) and partially restored defective intersegmental vessels in a chemically induced vascular insufficiency model of zebrafish larva. Moreover, the two week post-treatment of the rat MI model with FS (25-50 mg·kg^-1·d^-1) induced approximately 3-fold upregulation of VEGF mRNA expression, with a concomitant increase in blood vessel density in the peri-infarct area of the heart by 50.7%, compared to 41.4% in the MI group. Furthermore, TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment. CONCLUSION We have established a global transcriptome dataset for Panax notoginseng and provided additional genetic information for further genome-wide research and analyses. Candidate genes involved in ginsenoside biosynthesis, including putative cytochrome P450s and glycosyltransferases were obtained. The transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of P. notoginseng. And the significant pro-angiogenic effect of FS in multiple experimental models renders the purified saponin preparation as potential preventive and therapeutic agent for cardiovascular diseases yet to be developed.

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Human urine metabolome in response to Fermentum rubrum (Hongqu Capsules) by HILIC-UHPLC-QTOF

Hong YANG, Edmund J. D. LEE, Seok Hwee KOO, Feng TIAN

2015, 29(S1): 106-106.

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OBJECTIVE "-omics" study represents an unbiased perspective to examine the bio-system to discover the novel biomarker(s) which might be overlooked when targeted analysis is performed instead. Urine, due to its ease of collection, minimal invasion involved and rich information of the downstream metabolites, has been extensively studied. Fermentum rubrum (Hongqu, HQ capsules) is a well-known traditional medicine with the claimed slimming effect which could be related to the reduction in the deposition of total cholesterol and glycerides. Lack in sufficient clinical evidence is always one of the leading reasons that hamper traditional medicines from gaining world-wide recognition. In our pursuit of scientific support for HQ capsules in managing obesity, we aim to examine the alteration of urinary metabolites in this small-scale human clinical research. METHODS 6 Chinese subjects were included and exposed to short-term administration of HQ capsules for 3 d, twice daily, two capsules each dosing. The urine samples were collected for three days prior to the dosing and on the day following the last dosing. In total, 96 urine samples were collected and then separated on hydrophilic interaction chromatography (HILIC) and analyzed by Agilent 6550 ESI-Quadrupole-time-of-flight (QTOF) mass spectrometer. RESULTS Under positive mode, two compounds were found to be present only in post-dosing urine, and one compound was significantly lowered in post-dosing samples. Those two compounds might be associated with the administered HQ capsules, which is formulated with multi-herbal constituents. CONCLUSION Further elucidation on the structures of these compounds is needed to enable better understanding of the mechanism of HQ capsules in managing obesity.

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Identification of genetic factors modulating doxorubicin-associated cytotoxicity by calcium

Ying Jun LIM, ThuyTrang NGUYEN, Melanie Hui Min FAN, Kim Kiat LIM, Wee Han ANG, Joo Chuan TONG, Kenneth Hon-Kim BAN, Ee Sin CHEN

2015, 29(S1): 106-107.

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OBJECTIVE To examine the effect of calcium on the response towards doxorubicin, a widely used chemotherapeutic agent in the clinic, using fission yeast (Schizosaccharomycespombe) and human cancer cells (MCF7)This project built on a previously performed genome-wide search of doxorubicin-resistance (DXR) genes in Schizosaccharomycespombe fission yeast, which identified a host of genes that counteracted doxorubicin cytotoxicity. METHODS Growth fitness of Schizosaccharomycespombe knock-out mutants of doxorubicin-resistance genes were tested on varying concentrations of calcium, doxorubicin or calcium+doxorubicin. Similar growth fitness experiments were also performed on MCF7 breast cancer cells. RESULTS We found that a subset of null mutants of DXR genes show concurrent hypersensitivity to calcium and doxorubicin. Interestingly, their hypersensitivity towards doxorubicinwas suppressed by calcium. This phenotype was dependent on the integrity of the proton pump vacuolar-ATPase (V-ATPase) as the disruption of the V-ATPase-assembly factors (Rav1 and Vph2) abolished the suppressive effect of calcium. CONCLUSION Our findings uncovered an unexpected negative regulation of chemotherapeutic drug efficacy by dietary micronutrient that may caution against the concurrent consumption of calcium-rich dietary products alongside doxorubicin treatment.

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Discovery of novel thieno[3,2-b]pyrrole 5-carboxamides as potent inhibitors of Chikungunya virus

K C CHING, Y W KAM, Lisa FP NG, Christina L.L CHAI

2015, 29(S1): 107-107.

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Chikungunya fever (CHIKF) is an arboviral disease that typically consists of an acute illness with fever, skin rash, and incapacitating arthralgia.The causative agent of CHIKF is Chikungunya virus (CHIKV), an alphavirus that is transmitted by the Aedes mosquitoes. Despite the re-emergence of CHIKV as an epidemic threat, there is no approved effective anti-viral treatment currently available for CHIKV. In our preliminary studies, selected small molecule inhibitors of arboviruses related to CHIKV were investigated and this led us to identify compounds with thieno[3,2-b]pyrroles scaffold as hits. Building on the discovery of our best hit compounds, 5-carboxylic acid thieno[3,2-b]pyrroles 1 and 5-carboxamide thieno[3,2-b]pyrrole 2, the main aim of this study is to optimize their anti-viral activities by synthesizing analogs of thieno[3,2-b]pyrroless 1 and 2 and examine their activities against CHIKV. In these two parallel optimization studies, we synthesized two series of thieno[3,2-b]pyrroles, namely the 5-carboxylic acids and 5-carboxamides that possessed a variety of substituents at N4, C2, C6 or C5 positions of the thieno[3,2-b]pyrroles scaffold. These compounds were then examined for their cytotoxicity effects and anti-viral activities using a luminescence-labelled CHIKV infectious clone. The most potent compound in our studies was found in the 5-carboxamide series. The synthesis, biological activity and structure-activity relationship (SAR) will be presented and discussed in detail.

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Substrate-dependent inhibition of human cytochrome P450 3A catalytic activity by specific isomers of vitamin E

Swee Fen CHAI, Sumit BANSAL, Aik Jiang LAU

2015, 29(S1): 107-108.

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OBJECTIVE Lithocholic acid, which is a secondary bile acid, has been reported to be hepatotoxic and carcinogenic. It is metabolized by human cytochrome P450 3A (CYP3A) to form 3-ketocholanoic acid. A previous study suggests that vitamin E isomers (tocotrienols and tocopherols) are metabolized by CYP3A. Given that substrates of an enzyme may competitively inhibit the enzyme, we determined whether alpha-tocotrienol, gamma-tocotrienol, delta-tocotrienol, tocotrienol-rich mixture (a mixture consisting of 25.7% d-α-tocotrienol, 2.6% d-β-tocotrienol, 28.6% d-γ-tocotrienol, 8.4% d-δ-tocotrienol, 25.6% d-α-tocopherol, and 4.3% d-α-tocomonoenol), and alpha-tocopherol inhibit human liver microsomal CYP3A activity, as assessed by the enzymatic conversion of lithocholic acid to 3-ketocholanoic acid and of testosterone to 6β-hydroxytestosterone. METHODS Enzymatic formation of 3-ketocholanoic acid via lithocholic acid 3-oxidation was determined in pooled human liver microsomes and recombinant CYP3A4 and CYP3A5. Enzyme inhibition assay was conducted in a mixture containing potassium phosphate buffer (pH 7.4), human liver microsomes, NADPH, lithocholic acid, and various concentrations of a test chemical. The amount of 3-ketocholanoic acid formed was quantified by a novel, validated ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method. RESULTS Lithocholic acid was metabolized to 3-ketocholanoic acid by human recombinant CYP3A4 and CYP3A5 enzymes and human liver microsomes. Alpha-tocotrienol, gamma-tocotrienol, delta-tocotrienol, and tocotriernol-rich mixture, but not alpha-tocopherol, inhibited 3-ketocholanoic acid formation in human liver microsomes. Concentration-response experiments indicated that tocotrienol-rich mixture and delta-tocotrienol inhibited 3-ketocholanoic acid formation with IC₅₀ values of 6.6±2.1 μg·mL^-1 and 19.0±1.0 μmol·L^-1, respectively. CONCLUSION Tocotrienols inhibited CYP3A-catalyzed lithocholic acid 3-oxidation but not CYP3A-catalyzed testosterone 6-beta-hydroxylation. This suggests that lithocholic acid and testosterone bind to different CYP3A binding sites and that tocotrienols preferentially inhibit the lithocholic acid binding site on CYP3A enzymes.

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Pharmacokinetic comparison among resveratrol and some of its naturally occurring analogs

Hai-shu LIN, Samuel Chao Ming YEO, Wan CHEN

2015, 29(S1): 108-108.

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OBJECTIVE To elucidate the structural-pharmacokinetic relationship and identify resveratrol analogs with favorable pharmacokinetic profiles for potential medicinal application. METHODSThe pharmacokinetic data of resveratrol (trans-3,5,4-trihydroxystilbene), pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene), resveratrol trimethyl ether (trans-3,5,4-trimethoxystilbene) and some other herbal resveratrol analogs were extracted from the authors' recent publications and compared. RESULTSAqueous solubility, to different extent, has been identified as a barrier to oral absorption of resveratrol and its analogs. In addition, the para hydroxyl group(s) on the aromatic ring was less liable to metabolism compared to the meta-hydroxyl group(s). Pterostilbene and resveratrol trimethyl ether displayed more superior pharmacokinetic properties than resveratrol, i.e. much slower clearance and abundant plasma exposure. CONCLUSION Pterostilbene appears to be a favorable candidate for further development. Resveratrol analogs with meta-hydroxyl group(s) might have poor metabolic stability and suffer from rapid clearance and low oral bioavailability.

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Effect of soybean milk for 12 weeks in six-weeks-old male rats' testis, prostate, epididymis, seminal vesicles and testosterone

Nurdiana NURDIANA, Imam SARWONO, R SETYOHADI, Stephen HARSONO, Priyo BUDIUTOMO, Feby ADIGUNA, Zaw M AUNG

2015, 29(S1): 109-110.

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OBJECTIVE To investigate whether soy milk may cause reproductive disorders and decrease testosterone. METHODS Thirty two six weeks old male rats were divided into 4 groups including control group (non treatment) and three other groups were treated with soy milk powder (7.1, 14.2 and 21.3 g·kg^-1) everyday for 90 d. Histopathological examination of testis, epididymis and seminal vesicles were done using HE staining. Blood testosterone levels were assayed by ELISA. RESULTS There were positive correlations between the doses of soy milk with spermatogenesis in the testes, prostate epithelial cell hyperplasia. There were also positively correlation between dose of soy milk with vacuoles forming on epididymal epithelial cells and apoptosis in epithelial cells of seminal vesicles. The blood testosterone levels were not significantly difference between groups. CONCLUSION Subchronically soy milk feeding in rats induce histopathology changes of reproductive organs that closely related to the process of endocrine disruptors.

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Pharmacological activities and herb-drug interactions of Panax ginseng and its chemical components: a review

Hai-Ning WEE, Hui-Chuing YEW, Hwee-Ling KOH, Chay-Hoon TAN

2015, 29(S1): 109-109.

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OBJECTIVE Panax ginseng C.A. Meyer (ginseng) is a well-known medicinal plant worldwide and a key ingredient in many commercially-available health products. It is used as a tonic for invigoration and for tification in times of fatigue and debility or declining capacity for work and concentration. Previous in-house study has surveyed over three hundred ginseng and ginseng products (including P. ginseng, P. quinquefolius, P. notoginseng, P. pseudoginseng) available in Singapore. This review presents an overview of the pharmacological activities and herb-drug interactions of P. ginseng and its ginsenosides. METHODS Literature searches of PubMed and ScienceDirect were done to identify pharmacological activities and herb-drug interactions of P. ginseng, its extracts and its chemical components, including ginsenosides. Studies of whole plant extracts include both White ginseng and Red ginseng. The studies for the pharmacological activities of whole plant extract were limited to those published from 2009 to 2015. There was no restriction on the time frame of other studies. Terms such as "P. ginseng", "Ginsenosides" were searched. Studies found included in vitro assays, in vivo animal studies, human clinical trials as well as individual case reports. RESULTS A total of 112 studies were found on whole plant extracts and 257 studies on its individual components. Whole plant extracts of ginseng were found to possess over fifty different pharmacological activities, while its individual components exhibit parts of this spectrum. P. ginseng was found to interact with drugs such as 5-fluorouracil, irinotecan, mitomycin C, docetaxel, cisplatin, alcohol, midazolam, warfarin, phenelzine, raltegravir and imatinib. CONCLUSIONP. ginseng and its components exhibit a wide range of pharmacological activities and interact with some drugs. There remain much opportunities for future research.

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Structure-activity relationships for anti-inflammatory effect of flavonoids

Peng ZHANG, Judith CW MAK, Ricky YKMAN, Susan WS LEUNG

2015, 29(S1): 110-110.

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OBJECTIVE The present study examined the potential of flavonoids in reducing airway inflammation and determined the structure activity relationships (SAR), if present, for their anti-inflammatory effects. METHODS Seventeen flavonoids with different chemical structures were selected for the study. Inflammation was induced in human bronchial epithelial BEAS-2B cells with lipopolysaccharide (LPS). BEAS-2B cells were incubated with or without different flavonoids (10 μmol·L^-1) 1 h before treatment with LPS (10 μg·mL^-1) for 24 h. The viability of the cells after exposure to LPS and/or flavonoids were determined by thiazolyl blue tetrazolium bromide (MTT) assay. The amount of the inflammatory mediators, interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1), were measured in the supernatants byenzyme-linked immunosorbent assay (ELISA). RESULTS Flavonoids (1 to 10 μmol·L^-1) and LPS (1 to 10 μg·mL^-1) did not affect the viability of BEAS-2B cells. LPS (10 μg·mL^-1) significantly stimulated the release of IL-6, IL-8 and MCP-1 in BEAS-2B cells. Among the flavonoids tested, only apigenin, luteolin and genistein (10 μmol·L^-1) significantly inhibited the release of the inflammatory mediators. CONCLUSION These findings suggested that a hydroxy group at C5 and C7 positions in the A ring, a double bond between C2 and C3 and acarbonyl group at the C4 position in the C ring of the flavonoid might play an important role for their anti-inflammatory effect. The presence of a hydroxy group at C3 position or glycosylation at C3 or C7 position reduces the effectiveness of a flavonoid as an anti-inflammatory agent.

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Effect of Weiliuan of different prescriptions on gastrointestinal regulation factor in spleen-deficiency rats

Xuan-xuan ZHU, Xue-fei ZHANG, Shen-lin LIU

2015, 29(S1): 110-111.

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OBJECTIVE To observe the regulating effect of Weiliuan on digestive enzyme and relevant gastrointestinal hormone in spleen-deficiency rats. METHODS The rats were given the decoction of Radix et Rhizoma combine with dietary disorder to induce spleen-deficiency syndrome. Spleen-deficiency rats were divided into 8 groups:normal control group, spleen deficiency model group,Mengtuoshi San group(0.15 g·kg^-1),Weiliuan 31.5 and 15.75 g·kg^-1 groups,Weiliuan splited prescriptionⅠ 31.5 and 15.75 g·kg^-1 groups, Weiliuan splited prescription Ⅱ 31.5 and 15.75 g·kg^-1 groups. At the end of experiment, the level of amylase(AMS), lactic dehydrogenase(LDH), succinate dehydrogenase(SDH), motilin(MTL), somatostatin(SS), gastrin(GAS) and organ coefficient were detected. RESULTS Weiliuan group,Weiliuan splited prescriptionⅠ group and Weiliuan splited prescriptionⅡ group can significantly reduce the spleen index,the level of SDH and LDH,it can also improve the level of the AMS(P<0.05, P<0.01)In the regulation of hormone levels,Weiliuan group,Weiliuan splited prescription Ⅰ group and Weiliuan splited prescription Ⅱ group can significantly improve the levels MTL and GAS(P<0.05), but had no obvious effect on SS. CONCLUSION Weiliuan group,Weiliuan splited prescription Ⅰ group and Weiliuan splited prescription Ⅱ group can improve the symptoms of spleen deficiency syndrome significantly. Its mechanism may be to regulate the levels of AMS, SDH, LDH, MTL and GAS.

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Regulative effect of aqueous extract of Eichhornia crassipes on the oxygen uptake capacity and motion responses of mice under wet/cool stress

Yu-xiang AN, Yuan YUAN, Man YAO, Xin ZHANG, Di ZHANG, Yue ZHAO

2015, 29(S1): 111-111.

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OBJECTIVE To investigate the regulative effect of aqueous extract of Eichhornia crassipes on the oxygen uptake capacity and motion responses of mice under wet/cool stress. METHODSKM mice were randomly divided into normal control group, model control group, Eichhornia crassipes low-dose group (0.5 g·kg^-1) and high dose group (1.5 g·kg^-1), then performed intragastric administration for a period of 7 d. Wet/cold stressed mice model was established using repeated intermittent (3 h·d^-1, consecutive 7 d) exposure method by placing mice into the exposure box which was covered by ice at the bottom (T<0℃; RH>85%). Oxygen consumption was observed during exposure. Inclined plane method (muscle coordination capacity test) was performed immediately after exposure. 60-second video of exhaustive swimming (to calculate swimming speed) was taken 1 h after the last exposure, the exhaustive swimming time was recorded. RESULTS (1) Compared with normal control group, model control group showed significant improvement in oxygen consumption (P<0.01), with an increase up to 77.33%, while inclined plate time, swimming speed and exhaustive swimming time showed significant reduction (P<0.01), at the rates 46.3%, 8.54% and 19.68% respectively. (2) Compared with normal control group, Eichhornia crassipes low dose group and high dose group both improved oxygen consumption significantly (P<0.01), at rates of 10.93% and 90.38% respectively. This indicated high-dose aqueous extract of Eichhornia crassipes could fulfil actual oxygen demand of wet/cold stressed mice effectively. Inclined plate and exhaustive swimming experiments indicated that low-dose aqueous extract of Eichhornia crassipes boosted physical power significantly, as its exhaustive swimming time was increased up to 56.61%. Meanwhile, high-dose aqueous extract of Eichhornia crassipes has significant regulative effect on muscle coordination capability and motion speed, which were increased by 17.38% and 11.17%, respectively. CONCLUSION Aqueous extract of Eichhornia crassipes has definite regulative effect on the oxygen uptake capacity and motion responses of wet/cold stressed mice. Aqueous extract of Eichhornia crassipes can achieve stress protective effect by improving oxygen uptake capacity, muscle coordination capacity and motion abilities.

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Antidiarrheal and phytochemical constituents of methanol extracts of some plants in Saudi Arabia

Khaled B. Al HARBi, Ibrahim M. El-ASHMAWY

2015, 29(S1): 112-112.

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OBJECTIVE The purpose of the present study was to evaluate the antidiarrheal effects and the phytochemical constituents of methanol extracts of some plants used traditionally in kingdom of Saudi Arabia using castor oil-induced diarrhea and gastrointestinal motility test using charcoal meal method were examined. METHODS The methanolic extracts were initially assayed for their effects in castor oil-induced diarrhea at different doses (250 and 500 mg·kg^-1 followed by their evaluation on the peristaltic movements in charcoal meal test. RESULTS The results of the present study indicates that, administration of the following methanol plant extracts; Rhazya stricta, Heliotropium bacciferum, Tribulus longipetalus, Achillea fragrantissima, Artemisia herba alba, Haloxylon salicornicum, Plantago coronopus, Cyperus conglomerates, Hordeum distichon, Eragrostis poaeoides, Astragalus spinosus, Lepidium sativum seeds induced a higher percentages of inhibition of diarrhea. While, the administration of Rhanterium epapposum, Moltkiopsis ciliate, Lasiurus hirsutus, Deverra triradiata, Lycium shawii. Echinops spinosus, Echinops hussoni, and Prosopis farcta were the least efficacy. The antidiarrheal effects of these plants might be due to their high contents of flavonoids and tannins. CONCLUSION It could be concluded that, the remarkable antidiarrheal effects of these plants attest to their utility in a wide range of stats of diarrhea.

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Anti-tick activity of some methanol plant extracts indigenous in Saudi Arabia

Khaled B. Al-HARBI, Ibrahim M. El-ASHMAWY, Hussein M. OMAR

2015, 29(S1): 112-113.

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OBJECTIVE Ticks are economically important parasites all over the world. They are vectors of different animal and human infectious diseases. Tick control is globally based on chemical acaricides, but the developing tick resistance to most of the currently used synthetic acaricides encouraged to look for alternative ways to control ticks,especially natural products that are relatively environment friendly. METHODS An in vitro screening of the effects of some indigenous plants was carried out. Twenty-one plant species were extracted and tried to have effects on the most prevalent tick species; Hyalomma drommederi of camels. Fully engorged female ticks of the same weight, were immersed in the different extracts for 5, 15, 30 and 60 min. RESULTS Treated ticks were revived and incubated with the control groups for daily observation of their activity and egg production. While some plant extracts have killing effects after 30-60 min exposure (Rhanterium epapposum, Achillea fragrantissima and Eragrostis poaeodes) others have stopped oviposition (Artemisia herba alba, Haloxylon salicornicum, Plantago coronopus, Moltkiopsis ciliate and Lasiurus hirsutus). Most of the extracts have reducing effect on the egg mass. Marked reduction of the egg masses associated with delayed oviposition was recorded on exposure to Lepidium sativum seeds extract. CONCLUSION The phytoconstituents were determined by a standard methods and further studies are on going to determine the bioactive components that may have the above mentioned acaricidal effects.

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Polyprenols isolated from Picea abies L. spruce needles influence atorvastatin-mediated muscle strength weakness in Wistar female rats

Baiba JANSONE, Zane DZIRKALE, Kaspars JEKABSONS, Vladimirs PILIPENKO, Ulrika BEITNERE, Raimonds SKUMBINS, Zita FREIBERGA, Elga POPPELA, Ugis KLETNIEKS, Ilona VANAGA, Karlis KLETNIEKS, Ruta MUCENIECE, Vija KLUSA

2015, 29(S1): 113-113.

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OBJECTIVE To assess the effects of polyprenols (isolated from Picea abies L. spruce needles) on muscle strength/tone and coordination, and to investigate whether polyprenols may protect atorvastatin-mediated muscle strength/tone weakness in female Wistar rats. METHODS Polyprenols at doses of 1, 10 and 20 mg·kg^-1, atorvastatin 80 mg·kg^-1 or their combination were administered once daily per os for 16 consecutive days in Wistar female rats (n=9-10 per group). Assessment of muscle strength was performed by grip strength test (on day 15) and wire hang test (on day 16). Rotarod test was used to measured locomotor coordination and muscle tone (on day 16). General locomotor activity was evaluated in open field test (on day 15). Assessment of plasma cholesterol level and creatine kinase activity was done on day 17. RESULTS Atorvastatin-treated rats exhibited a marked decrease in grasping strength and hanging time. PP (20 mg·kg^-1) significantly protected against atorvastatin-induced muscle weakness in grip strength test, and restored it to control values. At all doses, polyprenols prolonged hanging time which was decreased by atorvastatin in wire hang test. Polyprenols per se at 1 and 10 mg·kg^-1 did not show difference compared to control group animals, while only at 20 mg·kg^-1 hanging time was prolonged vs. control in wire hang test. No changes between control and tested groups were observed in rotarod and open field tests. Blood cholesterol level was not changed in any of tested groups in female Wistar rats. Polyprenols (20 mg·kg^-1) significantly (by 25%) increased plasma creatine kinase activity but it was not affected by the combined treatment. CONCLUSION Since polyprenols acted as protectors of atorvastatin-induced muscle weakness, the combination of polyprenols with atorvastatin may be helpful for reducing muscle-related side effects in patients receiving a long-term atorvastatin therapy.

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Solubility and pharmacokinetics determination of chlorogeninc acid

Li ZHANG, Wei-sheng XU, Su-bo WANG, Guan-hua DU, Yang LU

2015, 29(S1): 113-114.

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OBJECTIVE To enhance the quality and efficiency of chlorogeninc acid by investigating the differences among the chlorogeninc acid polymorphs in bioavailability and solubility. METHODS Determinative method was used to analyze the solubility of chlorogeninc acid polymorphs; solid chlorogeninc acid in different forms were orally administered to the rats, and a HPLC method was established to determinate plasma lever of metabolite-acyclovir and the bioavailability was analyzed. RESULTS The indirect pharmacokinetic parameters of Chlorogeninc acid,as the metabolites of the form Ⅰ, Ⅱ, Ⅲ, were as follows: c_max was 0.37, 0.34 and 0.44 mg·L^-1,respectively; AUC_0→t was 0.71, 0.76 and 0.79 mg·L^-1·h, respectively. CONCLUSION The solubility of form Ⅲ was larger than the other forms'. The solubility of Chlorogeninc acid polymorphs: form Ⅰ, form Ⅱ, form Ⅲ were merely the same, there was no statistically significant difference in pharmacokinetic parameters among these three forms.

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Uridine adenosine tetraphosphate acts as a pro-angiogenic factor in vitro through purinergic P2Y receptors

Zhi-chao ZHOU, Ihsan CHRIFI, Yan-juan XU, Dirk J DUNCKER, S Jamal MUSTAFA, Daphne MERKUS, Caroline CHENG

2015, 29(S1): 114-114.

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OBJECTIVE Uridine adenosine tetraphosphate (Up₄A), a dinucleotide, contains both purine and pyrimidine moieties, and exerts its vascular influence via activation of purinergic receptors. Here, we aimed to investigate the effects of Up₄A on angiogenesis and the putative purinergic receptors (PR) involved in this process. METHODS Tubule formation assay was performed in 3D matrix system. In this assay, human umbilical vein endothelial cells (HUVECs) were co-cultured with pericytes with various Up₄A doses (0, 1, 2.5, 5, 10 and 20 μmol·L^-1) in the absence and presence of P2Y₆R antagonist MRS2578 (10 μmol·L^-1) for 5 d. Expression profile of PR subtypes and angiogenic factors was assessed in HUVECs by q-PCR with and without P2Y₆R antagonist. RESULTS No difference in initial tubule formation was detected between Up₄A stimulation and control conditions at day 2. In contrast, a significant increase in vascular density in response to Up₄A was observed at day 5. Up₄A at a dose of 2.5 and 5 μmol·L^-1 promoted total tubule length (by -1.89 fold and -2.23 fold), number of tubules (by -1.71 fold and -1.89 fold) as well as number of junctions (by -2.24 fold and -2.80 fold), all of which were inhibited by MRS2578. Further increase in Up₄A dose to 10 and 20 μmol·L^-1 did not induce an increase in these vascular parameters as compared to non-treated controls. Moreover, Up₄A increased mRNA level of P2YRs (P2Y₂R, P2Y₄R and P2Y₆R) but not P2XR (P2X₄R and P2X₇R) or P1R (A_2AR and A_2BR), while Up₄A upregulated VEGFA and ANGPT1 but not VEGFR2, ANGPT2, Tie1 and Tie2 at mRNA level. Transcriptional upregulation of P2YRs and angiogenic factors by Up₄A was inhibited by MRS2578. CONCLUSION Up₄A is functionally capable of promoting tubule formation in vitro co-culture system. This process is likely mediated by activation of pyrimidine-favored P2YRs but not P2XR or P1Rs, and involves stimulation of well known angiogenic factors.

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In vitro and in vivo study of formulated valproic acid loaded nanoemulsion in rats for epilepsy treatment

SF TAN, M Basri, Kirby P Brian, J Stanslas, H Basri

2015, 29(S1): 115-115.

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OBJECTIVE To investigate the in vitro and in vivo potential of formulated valproic acid-encapsulated nanoemulsion in terms of drug penetrability across the blood-brain barrier. METHODSHuman cerebral microvascular endothelial cells (hCMEC/D3) and human cortical astrocytes (SC-1800) were cultured on the membrane of the transwell inserts as to build the co-culture model of in vitro BBB model. Drug penetration analysis was then conducted at various time intervals for 24 h. The in vivo study of this formulated nanoemulsion was also investigated using Sprague Dawley rats. They were treated with VANE 60 mg·kg^-1 or valproic acid 60 mg·kg^-1 via intraperitoneal route before being anesthetized and sacrificed by cardiac puncture at different time points after administration. The blood and brain were collected and processed. The samples were analyzed with HPLC to determine the pharmacokinetic profile and biodistribution of the drug in the brain. RESULTS The formulated VANE had apparent permeability coefficient of 0.205 cm·s^-1 in vitro studies. It was 1.06-fold better than the drug solution. It showed that the VANE can penetrate across the BBB faster than the drug solution. The cumulative amount was reported to be 1.12-fold higher that the drug solution. This briefly suggests the VANE can be good candidate to promote the uptake of drug into the brain. In in vivo study, the formulated nanoemulsion showed remarkably improved pharmacokinetic parameters with 3.85-fold higher of total area under the curve-time curve (AUC) and 2.81-fold higher in maximum concentration of drug in blood plasma (c_max). More interestingly, the half-life (T_1/2) is prolonged by 1.80-fold and its clearance (Cl) was reduced by 3.85-fold. CONCLUSION The formulated nanoemulsions were able to improve most of the pharmacokinetic parameter thus this formulation technique improves the bioavailability of the drug in the brain compared to the drug solution alone.

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Fitness profiling links topoisomeraseⅡ regulation of centromeric integrity to doxorubicin resistance in fission yeast

Thi Thuy Trang NGUYEN, Julia Sze Lynn LIM, Richard Ming Yi TANG, Lou-xin ZHANG, Ee Sin CHEN

2015, 29(S1): 115-116.

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OBJECTIVE To address the molecular implication of Top2 in the context of its interaction with doxorubicin resistance (DXR) genes. METHODS To perform epistasis analyses of top2 with 63 genes representing doxorubicin resistance (DXR) genes in fission yeast. Fission yeast cells with single and double mutants were serial diluted and spotted to plates containing 15-75 μg·mL^-1 doxorubicin. Plates were scanned after 3 and 7 d. Cell growth was measured and compared between single mutants and double mutants. Nucleus morphology was performed by staining the cells with 4',6-diamidino-2-phenylindole (DAPI) to observe chromosome segregation. Reverse transcriptase PCR (RT-PCR) was employed to visualize the changes in transcription level and evaluate the stability of chromatin structure. RESULTS Our findings revealed a subset that synergistically collaborate with Top2 to confer DXR and showed that the chromatin-regulating RSC and SAGA complexes act with Top2 in a cluster that is functionally distinct from the Ino80 complex. In various DXR mutants, doxorubicin hypersensitivity was unexpectedly suppressed by a concomitant top2 mutation. Several DXR proteins showed centromeric localization, and their disruption resulted in centromeric defects and chromosome missegregation. An additional top2 mutation could restore centromeric chromatin integrity, suggesting a counterbalance between Top2 and these DXR factors in conferring doxorubicin resistance. CONCLUSION The findings reported here show a functional interaction between Top2 and factors that confer genomic stability at centromeric chromatin under doxorubicin condition. Overall, this molecular basis for mitotic catastrophe associated with doxorubicin treatment will help to facilitate drug combinatorial usage in Doxorubicin-related chemotherapeutic regimens.

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Medicinal plants and malaria

Woon-Chien TENG, Hwee-Ling KOH

2015, 29(S1): 116-116.

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OBJECTIVE To carry out a literature survey on medicinal plants documented for use in malaria, and to create a comprehensive database documenting the usage and preparation of these medicinal plants for malaria. METHODS A search was done through Scopus, ScienceDirect, and PubMed, on all ethnobotanical surveys that were specifically done on medicinal plants used in malaria using the keywords "ethnobotanical", "survey", "ethnopharmacological", and "malaria". In addition, Dr Duke's Phytochemical and Ethnobotanical Database, and books on medicinal plants from the Library of Botany and Horticulture, Singapore Botanic Gardens, and the Medical and Science Libraries, National University of Singapore, were used in the search. Plants used specifically as quinine substitutes were also included. Plants that were reported to be used solely for fever (other than malarial fever), external application, or insect repelling property, were excluded. Data collected were analyzed according to family, genus, location of use, method of preparation, part used, and indication (treatment and/or prevention). RESULTS A total of 1739 plants from 185 families and 973 genera were reportedly used for malaria globally, with 59 plantsused in three or more continents. Of these, 11 were used in four continents for malaria, and 7 of these can be found in Singapore. Anti-malarial plants from the family Fabaceae and the genus Vernoniawere the most commonly reported. Most of the plants are prepared as decoctions, followed by infusions. Leaves were most frequently used, followed by roots, and bark. 97.8% of the plants are used solely for curative purposes, 1.8% of the plants are both curative and prophylactic, while 0.4% are solely prophylactic. Priority of plants for further research could either focus on geographical extent of use, plant family, or genus. CONCLUSION An extensive database documenting the medicinal plants used for malaria has been compiled. Sustained interest in anti-malarial medicinal plant research is evident over the past decade. Promising plants for further research is presented.

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Potency of Urena lobata leaves extract on the inhibition of hepatic complication on diabetic rats

Amila MUFIDA, Rista Eka SUCIWULANSARI, Karina WINDA BESSUFI, Yudi PURNOMO

2015, 29(S1): 117-117.

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OBJECTIVE To investigate the potency of Urena lobata leaves extract on the inhibition of hepatic complication on diabetic rats. METHODS This study uses control group post test only with male Sprague dawley rats. Diabetic rats was induced by high fructose diet (HFD) and single dose streptozotocin 25 mg·kg^-1 bw intra peritoneal. The rat was administrated orally with water extract of U. lobata leaves in concentrations of 250, 500 and 1000 mg·kg^-1 bw for 4 weeks. After scarifying, liver organ and blood were collected and then superoxyde dismutase (SOD) hepar level, malondialdehyda(MDA), tumor necrosis factor-alpha (TNF-α), serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic piruvic transaminase (SGPT) were examined. The data was analyzed using ANOVA test continued with LSD test (P<0,05). RESULTS The oral administration of U. lobata leaves extract 250, 500 and 1000 mg·kg^-1 bw were able to increase SOD hepar level about 90%, 100% and 120% respectively compared to diabetic group (P<0.05), while the MDA hepar level was decreased by 40%, 50% and 70% respectively (P<0.05), whereas the TNF-α hepar level was decreased by 30%, 50% and 70% respectively (P<0.05). The supplementation of water extract from U. lobata in dose of 250, 500 and 1000 mg·kg^-1 bw decrease SGOT level approximately 10%, 30% and 50% compared to control group(P<0.05), while the SGPT level was decreased by 10%, 20% and 40% respectively(P<0.05). In diabetic groups, SOD heparlevel was decreased compared to normal group (P<0.05) whereas the MDA and TNF-α were increased (P<0.05). Meanwhile SGOT level and SGPT were increased in diabetic group (P<0.05). CONCLUSION U. lobata leaves extract could inhibit hepatic complication on diabetic rats by increasing of SOD hepar level, decreasing of MDA hepar level, TNF-α, SGOT and SGPT. This effect may be related to active compounds that act as an antioxidant and anti-inflammatory in U. lobata extract.

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Effect of Urena lobata leaves extract on the improvements of lipid profiles on diabetic rats

Triari NIZUAR, Eko BAGUS, Yudi PURNOMO

2015, 29(S1): 117-118.

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OBJECTIVE To investigate effect of U. lobata leaves extract on the improvement of lipid profiles on diabetic rats. METHODS This study uses control group post test only with male Sprague dawley rats. Diabetic rats was induced by high fructose diet (HFD) and single dose streptozotocin 25 mg·kg^-1 bw intra peritoneal. The rat was administrated orally with water extract of U. lobata leaves in dose of 250, 500 and 1000 mg·kg^-1 bw for 4 weeks. After scarifying, blood sample was collected and then total cholesterol (TC) serum level, triglyceride(TG), low density lippoprotein (LDL) and high density lipoprotein (HDL) were examined. The data was analyzed using ANOVA test continued with LSD test (P<0.05). RESULTS The supplementation of water extract from U. lobata in dose of 250, 500 and 1000 mg·kg^-1 bw decrease TC serum level approximately 15%, 25% and 35% compared to diabetic group (P<0.05), whereas the TG was decreased by 10%, 20% and 30%(P<0.05) respectively. The oral administration of U. lobata leaves extract 250, 500 and 1000 mg·kg^-1 bw also were able to decrease LDL serum level about 30%, 60% and 90% respectively compared to diabetic group (P<0.05), while the HDL serum level was increased by 40%, 80% and 100% (P<0.05) respectively. In diabetic groups, HDL level serum was decreased compared to normal group (P<0.05) while the TC, TG and HDL were increased (P<0.05). CONCLUSION U.lobata leaves extract could repair lipid profiles of diabetic rats by increasing of HDL serum level, decreasing of TC serum level, TG and LDL. This potency may be related to active substances which act as an anti-cholesterolemia and antioxidant in U. lobata extract.

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Evaluation of antihyperlipidemic effect of Polygonum minus, a South East Asian salad plant in poloxamer 407-induced acute hyperlipidemic rats

Parayil Varghese CHRISTAPHER, Josephine Maria Arokiaswamy CHRISTINA, Mohd. Zaini ASMAWI, Vikneswaran MURUGAIYAH

2015, 29(S1): 118-118.

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OBJECTIVE To investigate the antihyperlipidemic effect of methanol and aqueous extracts of the leaves of Polygonum minus in acute hyperlipidemic rat model. METHODS Acute hyperlipidemia was chemically induced in Sprague Dawley rats by using poloxamer 407 (500 mg·kg^-1 of body weight; intraperitoneal). Increase in total cholesterol was confirmed after six hours of induction. The normal and hyperlipidemic control groups were administered with 1 mL carboxymethylcellulose(CMC), the two test groups received aqueous and methanol extract of leaves of P. minus respectively (1000 mg·kg^-1; orally; suspended in 1% CMC) whereas reference standard treated group received atorvastatin (60 mg·kg^-1; orally; suspended in 1% CMC) once daily for 3 consecutive days. Blood samples were collected at 10th and 24th hour of the study for total cholesterol and triglycerides determination, while terminal blood samples were collected at 58th hour for full lipids profile analysis. RESULTS In the present study, both methanol and aqueous extracts lowered the serum total cholesterol and triglycerides significantly (P<0.001 and P<0.05, respectively) when compared with the hyperlipidemic control, similar to the standard drug atorvastatin. Varying effects were observed for both extracts on the other lipid parameter studied. Methanol extract showed significant reduction in LDL (P<0.05), VLDL (P<0.01) and atherogenic index (AI; P<0.001) and it showed a significant elevation in HDL levels (P<0.05). On the other hand, the aqueous extract showed significant reduction only in VLDL and AI (P<0.05) but no increase in HDL levels. CONCLUSION Present study confirmed the antihyperlipidemic effect of leaves of P. minus in acute hyperlipidemic rat model. The study also suggested that the methanol extract possess higher antihyperlipidemic effect than aqueous extract. Currently a study is ongoing to evaluate the antihyperlipidemic effect of the methanol extract in high-fat diet-induced chronic hyperlipidemic rat model.

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Cardioprotective effect of Urena lobata leaves extract on diabetic rats

Chandra Analis, Ahmat Baihaki, Elok Zakiyyatu Sholehah, Yudi Purnomo

2015, 29(S1): 118-119.

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OBJECTIVE To know cardioprotective effect of U. lobata leaves extract on diabetic rat. METHODS This study uses control group post test only with male sprague dawley rats. Diabetic rats was induced by high fructose diet (HFD) and single dose streptozotocin 25 mg·kg^-1 bw intra peritoneal. The rat was administrated orally with water extract of U. lobata leaves in concentrations of 250, 500 and 1000 mg·kg^-1 bw for 4 weeks. After scarifying, heart organ were collected and then superoxyde dismutase (SOD) heart level, malondialdehyda(MDA) and tumor necrosis factor-alpha (TNF-α) were examined. The data was analyzed using ANOVA test continued with LSD test (P<0.05). RESULTS The oral administration of U. lobata leaves extract 250, 500, and 1000 mg·kg^-1 bw were able to increase SOD heart level about 40%, 50% and 70% respectively compared to diabetic group (P<0.05), while the MDA heart level was decreased by 60%, 90% and 110% (P<0.05) respectively. The supplementation of water extract from U. lobata in dose of 250, 500 and 1000 mg·kg^-1 bw were also decrease TNF-α heart level approximately 20%, 40% and 60% compared to control group (P<0.05). In diabetic groups, SOD heart level was decreased compared to normal group (P<0.05) while the MDA and TNF-α were increased (P<0.05). CONCLUSION U. lobata leaves extract acts as cardioprotector on diabetic rats by increasing of SOD heart level, decreasing of MDA heart level and TNF-α. This effect may be related to active compounds that act as an antioxidant and anti-inflammatory in U. lobata extract.

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Effect of Urena lobata leaves extract on the inhibition of nephropathy diabetic complication

Pinta Surya KINANTI, Miranti SASTRANINGRUM, Yudi PURNOMO

2015, 29(S1): 119-119.

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OBJECTIVE To investigate the effect of Urena lobata leaves extract on the inhibition of nephropathy diabetic complication. METHODS This study uses control group post test only with male Sprague dawley rats. Diabetic rats was induced by high fructose diet (HFD) and single dose streptozotocin 25 mg·kg^-1 bw intra peritoneal. The rat was administrated orally with water extract of U. lobata leaves in concentrations of 250, 500 and 1000 mg·kg^-1 bw for 4 weeks. After scarifying, kidney organ were collected and then superoxyde dismutase (SOD) kidney level, malondialdehyda(MDA) and tumor necrosis factor-alpha (TNF-α) were examined. The data was analyzed using ANOVA test continued with LSD test (P<0.05). RESULTS The oral administration of U. lobata leaves extract 250, 500 and 1000 mg·kg^-1 bw were able to increase SOD kidney level about 30%, 60% and 90% respectively compared to diabetic group (P<0.05), while the MDA kidney level was decreased by 30%, 60% and 70% (P<0.05) respectively. The supplementation of water extract from U. lobata in dose of 250, 500 and 1000 mg·kg^-1 bw were also decrease TNF-α kidney level approximately 30%, 40% and 60% compared to control group(P<0.05). In diabetic groups, SOD kidney level was decreased compared to normal group (P<0.05) while the MDA and TNF-α were increased (P<0.05). CONCLUSION U. lobata leaves extract could inhibit nephropathy diabetic complication by increasing of SOD kidney level, decreasing of MDA kidney level, and TNF-α. This effect may be related to active compounds that act as an antioxidant and anti-inflammatory in U. lobata extract.

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Potency of Urena lobata leaves extract on the inhibition of vasculopathy on diabetic rats

Dimas Bintoro KRESNA, Alfien ARIPASHA, Eka SUKMA BUDI, Yudi PURNOMO

2015, 29(S1): 120-120.

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OBJECTIVE To know the potency of Urena lobata leaves extract on the vasculopathy inhibition of diabetic rats. METHODS This study used control group post test only with male Sprague dawley rats. Diabetic rats were induced by high fructose diet (HFD) and single dose streptozotocin 25 mg·kg^-1 bw intra peritoneal. The rat was administrated orally with water extract of U. lobata leaves in dose of 250, 500 and 1000 mg·kg^-1 bw for 4 weeks. After scarifying, blood sample were collected and then superoxyde dismutase (SOD) serum level, malondialdehyda(MDA), tumor necrosis factor-alpha (TNF-α) and circulating endothelial cells (CECs) were examined. The data was analyzed using ANOVA test continued with LSD test (P<0.05). RESULTS The oral administration of U. lobata leaves extract 250, 500 and 1000 mg·kg^-1 bw were able to increase SOD serum level about 40%, 70% and 100% respectively compared to diabetic group (P<0.05), while the MDA serum level was decreased by 20%, 40% and 50% (P<0.05) respectively. The supplementation of water extract from U. lobata in dose of 250, 500 and 1000 mg·kg^-1 bw also decrease TNF-α serum level approximately 40%, 60% and 80% compared to control group (P<0.05), whereas the CECs level was decreased by 30%, 50% and 70% (P<0.05) respectively. In diabetic groups, SOD serum level was decreased compared to normal group (P<0.05) while the MDA, TNF-α and CECs were increased (P<0.05). CONCLUSION U. lobata leaves extract could inhibit vasculopathy on diabetic rats by increasing of SOD serum level, decreasing of MDA serum level, TNF-α and CECs. This potency may be related to active substances which act as an anti-inflammatory and antioxidant in U. lobata extract.

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