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Equilibrative nucleoside transporters (ENTs) play a crucial role in the transport of nucleoside and nucleoside analogues, which are important for nucleotide synthesis and chemotherapy. In addition, ENTs regulate extracellular adenosine levels in the vicinity of its receptors and hence influence adenosine-related functions. The clinical applications of ENT inhibitors in the treatment of cardiovascular diseases and cancer therapy have been explored in numerous studies. However, all ENT inhibitors to date are selective for ENT1 but not ENT2. In the present study, we investigated the novel compound 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) as an inhibitor of ENT1 and ENT2. Nucleoside transporter-deficient PK15NTD cells stably expressing ENT1 and ENT2 showed that FPMINT inhibited [3H]uridine and [3H]adenosine transport through both ENT1 and ENT2 in a concentration-dependent manner. The IC50 value of FPMINT for ENT2 was 5-10-fold less than for ENT1, and FPMINT could not be displaced with excess washing. Kinetic studies revealed that FPMINT reduced Vmax of [3H]uridine transport in ENT1 and ENT2 without affecting KM. Therefore, we conclude that FPMINT inhibits ENTs in an irreversible and non-competitive manner. Although already selective for ENT2 over ENT1, further modification of the chemical structure of FPMINT may lead to even better ENT2-selective inhibitors of potential clinical, physiological and pharmacological importance.

Flavonoid dimer FD18 is a new class of dimeric P-gp modulator that can reverse cancer drug resistance. FD18 is a potent (EC50 = 148 nM for paclitaxel), safe (selective index = 574), and selective P-glycoprotein (P-gp) modulator. FD18 can modulate multidrug resistance toward paclitaxel, vinblastine, vincristine, doxorubicin, daunorubicin, and mitoxantrone in human breast cancer LCC6MDR in vitro. FD18 (1 μM) can revert chemosensitivity of LCC6MDR back to parental LCC6 level. FD18 was 11- to 46-fold more potent than verapamil. FD18 (1 μM) can increase accumulation of doxorubicin by 2.7-fold, daunorubicin (2.1-fold), and rhodamine 123 (5.2-fold) in LCC6MDR. FD18 inhibited P-gp-mediated doxorubicin efflux and has no effect on influx. FD18 at 1 μM did not affect the protein expression level of P-gp. Pharmacokinetics studies indicated that intraperitoneal administration of 45 mg/kg FD18 was enough to maintain a plasma level above EC50 (148 nM) for more than 600 min. Toxicity studies with FD18 (90 mg/kg, i.p. for 12 times in 22 days) with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) revealed no obvious toxicity or death in mice. In vivo efficacy studies indicated that FD18 (45 mg/kg, i.p. for 12 times in 22 days) together with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) resulted in a 46% reduction in LCC6MDR xenograft volume (n = 11; 648 ± 84 mm(3)) compared to paclitaxel control (n = 8; 1201 ± 118 mm(3)). There were no animal deaths or significant drop in body weight and vital organ wet weight. FD18 can increase paclitaxel accumulation in LCC6MDR xenograft by 1.8- to 2.2-fold. The present study suggests that FD18 represents a new class of safe and potent P-gp modulator in vivo.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural polyphenolic compound that exists in Polygonum cuspidatum, grapes, peanuts and berries, as well as their manufactured products, especially red wine. Resveratrol is a pharmacologically active compound that interacts with multiple targets in a variety of cardiovascular disease models to exert protective effects or induce a reduction in cardiovascular risks parameters. This review attempts to primarily serve to summarize the current research findings regarding the putative cardioprotective effects of resveratrol and the molecular pathways underlying these effects. One intent is to hopefully provide a relatively comprehensive resource for clues that may prompt ideas for additional mechanistic studies which might further elucidate and strengthen the role of the stilbene family of compounds in cardiovascular disease and cardioprotection. Model systems that incorporate a significant functional association with tissues outside of the cardiovascular system proper, such as adipose (cell culture, obesity models) and pancreatic (diabetes) tissues, were reviewed, and the molecular pathways and/or targets related to these models and influenced by resveratrol are discussed. Because the body of work encompassing the stilbenes and other phytochemicals in the context of longevity and the ability to presumably mitigate a plethora of afflictions is replete with conflicting information and controversy, especially so with respect to the human response, we tried to remain as neutral as possible in compiling and presenting the more current data with minimal commentary, permitting the reader free reign to extract the knowledge most helpful to their own investigations.

Hemorrhagic stroke occurs when a weakened vessel ruptures and bleeds into the surrounding brain, leading to high rates of death and disability worldwide. A series of complex pathophysiological cascades contribute to the risk of hemorrhagic stroke, and no therapies have proven effective to prevent hemorrhagic stroke. Stabilization of vascular integrity has been considered as a potential therapeutic target for hemorrhagic stroke. ROCKs, which belong to the serine/threonine protein kinase family and participate in the organization of actin cytoskeleton, have become attractive targets for the treatment of strokes. In this study, in vitro enzyme-based assays revealed that a new compound (FPND) with a novel scaffold identified by docking-based virtual screening could inhibit ROCK1 specifically at low micromolar concentration. Molecular modeling showed that FPND preferentially interacted with ROCK1, and the difference between the binding affinity of FPND toward ROCK1 and ROCK2 primarily resulted from non-polar contributions. Furthermore, FPND significantly prevented statin-induced cerebral hemorrhage in a zebrafish model. In addition, in vitro studies using the xCELLigence RTCA system, immunofluorescence and western blotting revealed that FPND prevented statin-induced cerebral hemorrhage by enhancing endothelial cell–cell junctions through inhibiting the ROCK-mediated VE-cadherin signaling pathway. As indicated by the extremely low toxicity of FPND against mice, it is safe and can potentially prevent vascular integrity loss-related diseases, such as hemorrhagic stroke.

Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4 μM, which effectively kill zebrafish larvae in less than 10 min. With non-lethal concentrations (

Ethnopharmacological relevance Fritillariae Bulbus (Beimu in Chinese) is derived from the bulbus of many Fritillaria species (family Liliaceae), which has been used as an antitussive herb in traditional Chinese medicine for more than 2000 years. Due to the complexity of plant origins and significant variations in chemical profiles, the characterization of the profile of the major bioactive constituents and its association with pharmacological activity are important for the quality control of Beimu herbs from different origins. Aim of the study This study aims to investigate the distribution of major bioactive isosteroidal alkaloids in Beimu herbs of different origins and its correlation with the tracheobronchial relaxant activity. Methods Quantification of 7 main bioactive 5α-cevanine isosteroidal alkaloids, including ebeiedine, ebeiedinone, hupehenine, isoverticine, verticine, verticinone and imperialine, in 23 Fritillaria species was performed using gas chromatography. The relaxant effect of different extracts of 4 commonly used Beimu herbs, namely Zhe-Beimu (F. thunbergii Miq.), Chuan-Beimu (F. cirrhosa D. Don), Hubei-Beimu (F. hupehensis Hsiao et K. C. Hsia) and Yi-Beimu (F. pallidiflora Schrenk), was evaluated using rat isolated tracheal and bronchial preparations pre-contracted with carbachol, the well established in vitro antitussive model. Results Amongst 23 Fritillaria species detected, significant variations of the types and quantities of 7 major isosteroidal alkaloids were determined, which served as an important indicator for the classification of different Beimu herbs with distinct geographic distributions. Based on the type and quantity of these alkaloids, different origins of Beimu could be clearly clustered into several subgroups by principal component analysis. Furthermore, both crude alkaloid and water extracts of all 4 Beimu herbs showed a dose-dependent tracheobronchial relaxation with different potencies. The total content of alkaloids (weight adjusted based on the activity of individual alkaloids) in Beimu extracts significantly correlated with their tracheobronchial relaxation effects (r2 > 0.9, p Conclusions The results demonstrated that the differences in chemical profile of major bioactive isosteroidal alkaloids and pharmacological activity of Beimu could be incorporated into a simple and unified method for quality control and potential prediction of activity of Beimu herbs from different origins.

Ethnopharmacological relevance Semen Astragali Complanati (SAC), the dried ripe seed of Flatstem Milkvetch (Astragalus complanatus Bunge) (Leguminosae), is commonly used in traditional Chinese medicine (TCM) for treating muscle, liver, kidney, blood, skin and reproductive system diseases. Materials and methods Relevant information about SAC was gathered via “Google Scholar”, “ISI Web of Knowledge”, “PubMed”, “ScienceDirect”, “Medline Plus”, “ACS”, “CNKI” and “Wiley Online Library” and from books in local libraries. Results The major contents of SAC include fatty acids, amino acids, polysaccharides, flavonoids, triterpene glycosides and trace elements. Previous scientific studies have reported that SAC exhibits a number of therapeutic effects on chronic diseases such as cardiovascular diseases, diabetes mellitus and cancers. It has been found that flavonoids are the main bioactive component in SAC. However most of the previous studies have shown the effects brought by the total flavonoid fraction extracted from SAC only; further studies are warranted for the biological effects produced by individual components. There are only a few studies on the toxicity of SAC and the overall results show that its toxicity is quite low or even non-existent. Conclusions SAC is a valuable TCM herb with multiple pharmacological effects for treating some chronic diseases. More studies on SAC will help us to have a better understanding of its pharmacological mechanisms so as to provide more scientific evidence to explain its traditional uses, identify its therapeutic potential on other diseases and understand its possible harmful effects. Based on previous studies, it is easy to identify that antioxidant effect of SAC might play an important role on its pharmacological effects. Studying the effects of SAC on handling intracellular reactive oxygen species may be a potential direction to help understanding the molecular mechanisms of SAC on preventing and/or treating chronic diseases.

Ergothioneine is a thiourea derivative of histidine found in food, especially mushrooms. Experiments in cell-free systems and chemical assays identified this compound as a powerful antioxidant. Experiments were designed to test the ability of endothelial cells to take up ergothioneine and hence benefit from protection against oxidative stress. Reverse-transcription polymerase chain reaction and Western blotting demonstrated transcription and translation of an ergothioneine transporter in human brain microvascular endothelial cells (HBMECs). Uptake of [(3)H]ergothioneine occurred by the organic cation transporter novel type-1 (OCTN-1), was sodium-dependent, and was reduced when expression of OCTN-1 was silenced by small interfering RNA (siRNA). The effect of ergothioneine on the production of reactive oxygen species (ROS) in HBMECs was measured using dichlorodihydrofluorescein and lucigenin, and the effect on cell viability was studied using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. ROS production and cell death induced by pyrogallol, xanthine oxidase plus xanthine, and high glucose were suppressed by ergothioneine. The antioxidant and cytoprotective effects of ergothioneine were abolished when OCTN-1 was silenced using siRNA. The expression of NADPH oxidase 1 was decreased, and those of glutathione reductase, catalase, and superoxide dismutase enhanced by the compound. In isolated rat basilar arteries, ergothioneine attenuated the reduction in acetylcholine-induced relaxation caused by pyrogallol, xanthine oxidase plus xanthine, or incubation in high glucose. Chronic treatment with the compound improved the response to acetylcholine in arteries of rats with streptozotocin-induced diabetes. In summary, ergothioneine is taken up by endothelial cells via OCTN-1, where the compound then protects against oxidative stress, curtailing endothelial dysfunction.

The incidence of cardiovascular diseases (CVDs) is high in both developed and developing countries. It has a high global rate of mortality and causes heavy social burden. Drugs are available for managing or treating CVDs and its complications. Consumption of dietary supplements or functional foods for reducing the risk of CVDs has also gained wide recognition by the general public. Piceatannol, an analog and metabolite of resveratrol, is a natural stilbene commonly found in the skin of grapes and wine. Piceatannol is believed to be a potent compound with certain cardiovascular therapeutic effects, such as the prevention of hypercholesterolemia, arrhythmia, atherosclerosis, and angiogenesis. It also has vasorelaxation and antioxidant activities. A comprehensive review of piceatannol concludes that piceatannol has the potential to be developed into health products for the cardiovascular system to help modern society reduce the high CVD incidence. However, further investigations are warranted in order to increase the bioavailability and understand the biological mechanisms and safety of using piceatannol.

This study investigated the mechanism of the cytotoxic effect of emodin, an active anthraquinone, on human lung adenocarcinoma A549 cells. In vitro growth inhibition and suppression on colony forming were used to evaluate the effects of emodin on A549 cells. Emodin's ability in changing the expressions of apoptosis-related genes was studied by real-time RT-PCR. Emodin could significantly inhibit the growth of A549 cells with IC50 = 16.85 μg/ml (~60 μM). It also concentration dependently inhibited the colony-forming ability of A549 cells with IC50 = 7.60 μg/ml (~30 μM). Hallmarks of apoptosis, such as single-strand DNA breakage and DNA fragmentation, were observed in A549 cells treated with emodin. Emodin (72 h) treatment could up-regulate the gene expression of FASL (p < 0.05) and down-regulate the gene expression of C-MYC (p < 0.01), but induce no significant changes in the gene expressions of MCL1, GAPDH, BAX and CCND1. These results suggest that emodin could induce growth inhibition and apoptosis in A549 cells through modifying the extrinsic apoptotic pathways and the induction of cell cycle arrest.

Gui-ling-gao (GLG), also known as turtle jelly, is a popular medicinal Chinese health/functional food prepared from several traditional Chinese medicinal herbs. The present study aimed at examining and explaining the anti-inflammatory properties of GLG by using lipopolysaccharide (LPS)-induced inflammation in splenocytes isolated from BALB/c mice. Additionally, the effects of GLG on mRNA and protein expressions of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines in LPS-stimulated splenocyte proliferation were evaluated by real-time RT-PCR and Western blot assays. We demonstrated that GLG significantly inhibited LPS-induced splenocyte proliferation in a concentration-dependent manner. LPS-mediated up-regulations in the gene and protein expressions of iNOS, interleukin-1β (IL-1β) and tumour necrosis factor-alpha (TNF-α) were suppressed by GLG. These findings suggest for the first time that GLG exerts anti-inflammatory effects by inhibiting the expression of iNOS and pro-inflammatory cytokines such as IL-1β and TNF-α. The current study provides strong scientific evidence for the health-beneficial claim that consumption of GLG could help the body to modulate the immune system.

The dried ripe seed ofRaphanus sativusL., commonly known as radish seed (or Raphani Semen), is used as traditional Chinese medicine (TCM) to treat constipation, chronic tracheitis, and hypertension. The major active compounds in Raphani Semen are alkaloids, glucosinolates, brassinosteroids, and flavonoids. Fatty acids are its main nutritional contents. Raphani Semen has been demonstrated to have beneficial effects on hypertension, obesity, diabetes mellitus, constipation, and cough. So far, there is no report about the adverse/toxic effects of this herb on humans. However, Raphani Semen processed by roasting was reported to exhibit some adverse effects on mice. Additionally, erucic acid, the main fatty acid in Raphani Semen, was shown to enhance the toxicity of doxorubicin. Thus, Raphani Semen has a potential risk of causing toxicity and drug interaction. In summary, Raphani Semen is a valuable TCM herb with multiple pharmacological effects. More studies on Raphani Semen could help better understand its pharmacological mechanisms so as to provide clear scientific evidence to explain its traditional uses, to identify its therapeutic potential on other diseases, and to understand its possible harmful effects.

Some of the major components of Danshen (Salvia miltiorrhiza), a widely used Chinese herbal medicine rich in phenolic acids, are thermosensitive and may degrade to other phenolic acids during extractions with heating. The chemical profiles of Danshen water-extract may vary with different heat water extraction at different temperatures, affecting the composition and bioactivity of the extracts. In this study, six water-extracts of Danshen obtained from heat reflux water extraction and microwave-assisted extraction with water (MAE-W) at different temperatures were tested for their composition and pharmacological effects. Among these extracts, the third-round MAE-W (100°C) extract had the highest phenolic acids and tanshinones contents, with the strongest antioxidant activity in 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay and ferric reducing/antioxidant potential (FRAP) assay. This extract also showed the strongest inhibitory effects on 2,2'-azobis-2-amidinopropane (AAPH)-induced hemolysis in human red blood cells, hydrogen peroxide-induced apoptosis in rat heart H9c2 cells and the highest relaxation effects on rat basilar artery. The antioxidant effects of Danshen water-extracts linearly correlated to their relaxation effects (r=0.895-0.977). Through multiple linear regression analysis, danshensu was found to be the most significant marker in the antioxidant and vasodilation effects of Danshen water-extract, while tanshinone IIA as the marker on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells. Danshensu is, therefore, a useful marker for the quality control of Danshen water-extracts in antioxidant and vasodilation, while tanshinone IIA for anti-apoptotic potential of different extracts.

Background Cycloartane triterpenoids exhibited anticancer effects. This study aims to identify any potential novel anticancer cycloartane triterpenoids from Cimicifuga foetida L. rhizome (Sheng ma) and the mode of actions. Methods Cycloartane triterpenoids were isolated from the C. foetida rhizome by a series of column chromatography and identified by IR, MS and NMR. Their anticancer effects on several human cancer cell lines, MCF-7, HepG2, HepG2/ADM, HeLa, and PC3, and normal human mammary epithelial cells MCF10A were investigated by colony formation and MTT assays. Morphological analysis of apoptosis induction was performed by acridine orange/ethidium bromide dual-staining and Hoechst 33258 nuclear staining. The cell-cycle profile and annexin V staining were evaluated by flow cytometry. Apoptosis were investigated by measuring changes in mitochondrial membrane potential and analyzing expression of cell cycle- and apoptosis-related proteins in MCF-7 cells by Western blotting. Results A novel cycloartane triterpenoid, 25-O-acetyl-7,8-didehydrocimigenol-3-O-β-d-(2-acetyl)xylopyranoside (ADHC-AXpn), together with the known 7,8-didehydrocimigenol-3-O-β-d-xylopyranoside (DHC-Xpn) were isolated. MCF-7 growth was significantly inhibited by ADHC-AXpn in a dose- and time-dependent manner (IC50: 27.81 µM at 48 h; P = 0.004 vs. control at 25 μM for 48 h treatment), and ADHC-AXpn was selectively cytotoxic for cancerous cells (MCF-7, HepG2/ADM, HepG2 and HELA cells) based on its higher IC50 values for normal cells MCF10A (IC50: 78.63 µM at 48 h) than for tumor cells. In MCF-7 cells, ADHC-AXpn induced G2/M cell cycle arrest by mediating cyclin-B1, and CDK1 and its phosphorylation; and induced apoptosis through the mitochondrial-mediated apoptotic pathway, with inhibition of Akt activation. As ADHC-AXpn suppressed phosphorylation of ERK1/2, Raf and Akt proteins in MCF-7 cells, its apoptotic effect might be associated with Raf/MEK/ERK signaling and Akt activation. Conclusions ADHC-AXpn significantly suppressed the growth of MCF-7 cells, induced mitochondrial apoptosis and cell-cycle arrest, and inhibited Raf/MEK/ERK signaling pathway and Akt phosphorylation.

Cardiovascular disease (CVD) is a category of chronic noncommunicable diseases causing high global mortality and has been a heavy social burden in many countries. In the search of chemicals that arise from natural food source, allicin is one such ingredient from garlic that was discovered with the potential to provide beneficial effects to the cardiovascular system. From the pharmacokinetic studies, allicin is known to be hydrophobic and can be readily absorbed through the cell membrane without inducing any damage to the phospholipid bilayer and then rapidly metabolized to exert pharmacological effects that are important to the cardiovascular system. It was found to provide cardio-protective effects by inducing vasorelaxation and alleviating various pathological conditions of CVD, including cardiac hypertrophy, angiogenesis, platelet aggregation, hyperlipidemia and hyperglycemia. Allicin was also discovered to further protect the cardiovascular system by enhancing the antioxidant status by lowering the level of reactive oxygen species and stimulating the production of glutathione. Other pharmacological benefits such as anticancer and antimicrobial activities were also discussed. It is concluded that allicin can be potentially developed into a health product for the cardiovascular system.

Hypercholesterolemia is a major risk factor for the development of cardiovascular disease and nonalcoholic fatty liver disease. Natural compounds have been proved to be useful in lowering serum cholesterol to slow down the progression of cardiovascular disease and nonalcoholic fatty liver disease. In the present study, the hypocholesterolemic and hepatoprotective effects of the dietary consumption of chlorogenic acid were investigated by monitoring plasma lipid profile (total cholesterol, triglycerides, high-density lipoprotein and low-density lipoprotein) in Sprague-Dawley rats fed with a normal diet, a high-cholesterol diet or a high-cholesterol diet supplemented with chlorogenic acid (1 or 10 mg/kg/day p.o.) for 28 days. Chlorogenic acid markedly altered the increased plasma total cholesterol and low-density lipoprotein but decreased high-density lipoprotein induced by a hypercholesterolemic diet with a dose-dependent improvement on both atherogenic index and cardiac risk factor. Lipid depositions in liver were attenuated significantly in hypercholesterolemic animals supplemented with chlorogenic acid. It is postulated that hypocholesterolemic effect is the primary beneficial effect given by chlorogenic acid, which leads to other secondary beneficial effects such as atheroscleroprotective, cardioprotective and hepatoprotective functions. The hypocholesterolemic functions of chlorogenic acid are probably due to the increase in fatty acids unitization in liver via the up-regulation of peroxisome proliferation-activated receptor α mRNA.

The anticancer effects of traditional Chinese medicine (TCM) have attracted the attention of the public vis-à-vis existing cancer therapies with various side effects. Lycium barbarum fruit, commonly known as Gou Qi Zi in China, is a potential anticancer agent/adjuvant. Its major active ingredients, L. barbarum polysaccharides (LBP), scopoletin and 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), are found to have apoptotic and antiproliferative effects on cancer cell lines. Moreover, LBP also contributes to body's immunomodulatory effects and enhances effects of other cancer therapies. It is not known whether there are any undesirable effects. Further studies on its pharmacological mechanisms and toxicology could facilitate a safe usage of this TCM herb.

Arctium lappa, commonly known as burdock, is being promoted/recommended as a healthy and nutritive food in Chinese societies. Burdock has been used therapeutically in Europe, North America and Asia for hundreds of years. The roots, seeds and leaves of burdock have been investigated in view of its popular uses in traditional Chinese medicine (TCM). In this review, the reported therapeutic effects of the active compounds present in the different botanical parts of burdock are summarized. In the root, the active ingredients have been found to "detoxify" blood in terms of TCM and promote blood circulation to the skin surface, improving the skin quality/texture and curing skin diseases like eczema. Antioxidants and antidiabetic compounds have also been found in the root. In the seeds, some active compounds possess anti-inflammatory effects and potent inhibitory effects on the growth of tumors such as pancreatic carcinoma. In the leaf extract, the active compounds isolated can inhibit the growth of micro-organisms in the oral cavity. The medicinal uses of burdock in treating chronic diseases such as cancers, diabetes and AIDS have been reported. However, it is also essential to be aware of the side effects of burdock including contact dermatitis and other allergic/inflammatory responses that might be evoked by burdock.

BACKGROUND AND PURPOSE We evaluated the role(s) of monoamine oxidase (MAO)-mediated H2O2 generation on 5-hydroxytryptamine (5-HT)-induced tension development of isolated basilar artery of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. EXPERIMENTAL APPROACH Basilar artery (endothelium-denuded) was isolated for tension measurement and Western blots. Enzymically dissociated single myocytes from basilar arteries were used for patch-clamp electrophysiological and confocal microscopic studies. KEY RESULTS Under resting tension, 5-HT elicited a concentration-dependent tension development with a greater sensitivity (with unchanged maximum tension development) in SHR compared with WKY (EC50: 28.4 ± 4.1 nM vs. 98.2 ± 9.4 nM). The exaggerated component of 5-HT-induced tension development in SHR was eradicated by polyethylene glycol-catalase, clorgyline and citalopram whereas exogenously applied H2O2 enhanced the 5-HT-elicited tension development in WKY. A greater protein expression of MAO-A was detected in basilar arteries from SHR than in those from WKY. In single myocytes and the entire basilar artery, 5-HT generated (clorgyline-sensitive) a greater amount of H2O2 in SHR compared with WKY. Whole-cell iberiotoxin-sensitive Ca2+-activated K+ (BKCa) amplitude measured in myocytes of SHR was approximately threefold greater than that in WKY (at +60 mV: 7.61 ± 0.89 pA·pF−1 vs. 2.61 ± 0.66 pA·pF−1). In SHR myocytes, 5-HT caused a greater inhibition (clorgyline-, polyethylene glycol-catalase- and reduced glutathione-sensitive) of BKCa amplitude than in those from WKY. CONCLUSIONS AND IMPLICATIONS 5-HT caused an increased generation of mitochondrial H2O2 via MAO-A-mediated 5-HT metabolism, which caused a greater inhibition of BKCa gating in basilar artery myocytes, leading to exaggerated basilar artery tension development in SHR.

Hypercholesterolemia is a major risk factor for the development and progression of cardiovascular diseases including atherosclerosis. A major ac tive ingredient, scutellarin, from the plant Erigeron breviscapus was investigated for its hypocholesterolemic and atheroscleroprotective effects (30 and 100 mg/kg/day, p. o.). The serum lipid profile (total cholesterol, triglycerides, high density lipoprotein cholesterol and low density lipoprotein cholesterol) was monitored and aortic functions in Sprague-Dawley rats fed with normal diet, atherogenic diet or atherogenic diet plus oral administration of either scutellarin or simvastatin (a positive control) were tested. It was found that scutellarin markedly attenuated the increased serum total cholesterol induced by atherogenic diet. It caused a significant reduction in the atherogenic index. In addition, scutellarin administration could significantly enhance acetylcholine-induced nitrate/nitrite production, increase the gene expression of endothelial nitric oxide synthase and improve acetylcholine-induced endothelium-dependent vasorelaxation in rat isolated aortas. These data revealed that scutellarin could reduce the atherogenic properties of dietary cholesterol in rats. However, whether scutellarin's atheroscleroprotective potential targets endothelial function directly or indirectly on its antioxidative activity remains to be determined.

Scutellaria baicalensis Georgi (Labiatae) (“huang qin”), Ligusticum chuanxiong Hort. (Umbelliferae) (“chuang xiong”), Panax notoginseng (Burk.) F.H. Chen (Araliaceae) (“san qi”), Uncaria rhynchophylla (Miq.) Jackson. (Rubiaceae) (“gou teng”), Rhokiola rosea L. (Crassulaceae) (“hong jing tian”) and Stephania tetrandra S. Moore (Menispermaceae) (“fang ji”) are commonly used traditional Chinese medicine (TCM) for hypertensive patients. The pharmacologically active compounds found in these TCMs are baicalin, ligustrazine, notoginsenoside R1, rhynchophylline, salidroside and tetrandrine, respectively, which possess antihypertensive properties with diverse cellular mechanisms. In this study, we attempted to evaluate a possible correlation of the antioxidative activities (using the DPPH assay) and the vaso-relaxation effects (using rat isolated thoracic aorta) of these compounds. In the antioxidative study, a relative order of free radical scavenging capacity (SR%) of baicalin ≥ tetradrine >> salidroside ≥ ligus...

We investigated the role(s) of monoamine oxidases (MAOs) on the altered 5-hydroxytryptamine (5-HT, serotonin)-induced tension development of the isolated umbilical artery of preeclamptic pregnancy of Chinese women. An enhanced 5-HT-induced tension development of the umbilical artery of preeclamptic pregnancy was observed when compared with that of normal pregnancy. The enhanced component of 5-HT-induced tension development was eradicated by clorgyline (a MAO-A inhibitor). Blockade of eNOS (endothelial isoform nitric oxide synthase) (N(omega)-nitro-L-arginine methyl ester), 5-HT transporter (citalopram), 5-HT receptor subtypes (5HT2B, SB 204741; 5-HT2C, RS 102221; 5-HT7, SB 269970), and endothelium denudation of the umbilical artery of normal pregnancy mimicked the enhanced 5-HT-induced tension development as observed in the preeclamptic tissues. In contrast, no apparent changes in 5-HT-induced tension development of the umbilical artery of preeclamptic pregnancy were observed with the same pharmacological manipulations. A decreased protein expression levels of MAO-A and eNOS (no iNOS and MAO-B expression was detected) and no change in caveolin-1 and 5-HT transporter expression were demonstrated in the umbilical artery (endothelium intact) lysate of preeclamptic pregnancy, compared to that of the umbilical artery of normal pregnancy. Thus, in the umbilical artery of preeclamptic pregnancy, a decrease of MAO-A and eNOS protein expression levels are probably associated with, or responsible for, the exaggerated 5-HT-induced tension development.

Sequential extraction with petroleum ether, chloroform–ethyl acetate, methanol, and water was carried out on 40 commonly used Chinese medicinal herbs. The antioxidant power of different fraction extracts was measured with both the DPPH assay and the FRAP assay. Ascorbic acid and butylated hydroxytoluene were used as two reference antioxidants. The total phenolic content of the extracts was measured by Folin-Ciocalteu method. The fraction extracts showed different antioxidant activity in the two assays; however, there was a significant positive correlation between the DPPH assay and the FRAP assay. Methanol fraction extracts generally demonstrated stronger antioxidant power and higher total phenolic content than did the others. The results suggest that Radix Sanguisorbae, Cortex cinnamomi, Herba taxilli, Semen arecae, and L., Cinnamomum cassia Presl ., Taxillus sutchuenensis (Lecomte) Danser, Areca catechu Linn. and Scutellaria baicalensis Georgi contain relatively high values in antioxidant power ...

In this study, we determined the in vitro effect of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation on the aortic relaxation, lipolysis and insulin-induced [(3)H]-glucose uptake of the abdominal (omental) adipocytes of the non-diabetic (+db/+m) and obese/diabetic (+db/+db) mice. The expression of PPAR-gamma (mRNA and protein) in aorta and adipose tissues was evaluated and compared. Cumulative application of ciglitazone, pioglitazone and troglitazone (PPAR-gamma agonists) caused a concentration-dependent aortic relaxation (sensitive to 2-chloro-5-nitro-N-phenylbenzamide (GW9662) (1 microM, a selective PPAR-gamma antagonist) and N(omega)-nitro-l-arginine methyl ester (l-NAME) (20 microM, a nitric oxide synthase inhibitor)) with a maximum relaxation of approximately 30% (3 microM) in +db/+m mice, whereas no relaxation was observed in +db/+db mice. All PPAR-gamma agonists examined did not alter the basal lipolysis of both species, but forskolin caused a concentration-dependent lipolysis, with a greater magnitude observed in +db/+m mice. Insulin (0.1 and 1 microM) caused an enhancement of [(3)H]-glucose uptake into adipocytes with a greater magnitude in +db/+m mice. In contrast, none of the PPAR-gamma agonists tested (0.1, 1 and 10 microM) altered the basal and the insulin (0.1 microM)-induced [(3)H]-glucose uptake into adipocytes of both species. In addition, there was no difference in PPAR-gamma expression (mRNA and protein) in the aorta and adipose tissues between the species. In conclusion, our results demonstrate that PPAR-gamma is present in the abdominal (omental) adipose tissue and thoracic aorta. An acute activation of PPAR-gamma produced a small ( approximately 30%) aortic relaxation (nitric oxide/endothelium-dependent) of +db/+m mice. However, all PPAR-gamma agonists examined have no acute effect on lipolysis and the insulin-induced glucose uptake into adipocytes of both +db/+m and +db/+db mice.

We evaluated the acute effect of homocysteine on the iberiotoxin-sensitive, Ca 2+ -activated K + (BK Ca ) channels of the porcine coronary artery smooth muscle cells. NS 1619 (1 to 30 μM) caused a concentration-dependent enhancement of the BK Ca amplitude (recorded using the whole-cell, membrane-rupture configuration) only with an elevated [Ca 2+ ] i of ∼ 444 nM, but not with [Ca 2+ ] i of ∼ 100 nM. Homocysteine (30 μM) caused a small inhibition (∼ 16%) of the BK Ca amplitude ([Ca 2+ ] i = ∼ 444 nM), and a greater inhibition (∼ 77%) was observed with 100 μM NADH present in the pipette solution. The inhibition persisted after washing. With NADPH (100 μM), a smaller magnitude of inhibition (∼ 34%) of the BK Ca amplitude was recorded. The NS 1619-mediated enhancement of the BK Ca amplitude (with elevated [Ca 2+ ] i plus NADH in the pipette) was attenuated by homocysteine. The homocysteine-mediated inhibition of the BK Ca amplitude was suppressed by Tiron (10 mM) or diphenylene iodonium (30 nM), applied alone, but not by superoxide dismutase (500 U/ml) and catalase (500 U/ml). Generation of superoxide ( O 2 − ) of the smooth muscle cells (with NADH presence), measured using the lucigenin-enhanced chemiluminescence, was markedly increased by angiotensin II (100 nM) and homocysteine (30 μM). The chemiluminescence signal was sensitive to apocynin (300 μM) or Tiron, applied alone, but not to superoxide dismutase and catalase. In conclusion, our results demonstrate that acute homocysteine application inhibits the iberiotoxin-sensitive BK Ca channels (with elevated [Ca 2+ ] i and NADH present) which is probably caused by the NADH oxidase activation and the concomitant generation of intracellular superoxide.

Lobeliae chinensis. Herba (“ban bian lian”), Rheum officinale. Baill. (“da huang”), Sanguisorba officinalis. Linn. (“di yu”), Agrimonia pilosa. Ledeb. (“xian he cao”), and Paris polyphylla. Smith (“zhi hua tou”) are well-known traditional Chinese medicines. They are commonly used in traditional Chinese medicine formulae against cancer. In this study, the antioxidant and anticancer effects of water extracts of these herbs were investigated. In the antioxidant and anticancer studies, water extracts of di yu, xian he cao, and da huang were show to be the most antioxidative and had the highest growth inhibitory effect on human lung adenocarcinoma A549 cell and human breast cancer MCF-7 cell. By comparing their percentage free radical scavenging capacity (SR%) and percentage growth inhibition on A549 and MCF-7 cells, a positive linear relationship between antioxidant activity and anticancer effect of the five herbal water extracts was found. This suggested that the antioxidants of the herbal water extr...

In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K2 (10 nM) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (+/+) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K2 (10 nM) and 1,25(OH)2D3 (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K2 and 1,25(OH)2D3 co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K2 plus 1,25(OH)2D3 treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K2 plus D3 combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis.

Natural products are good sources of natural dietary antioxidants that are believed to protect the body against hepatotoxic effect induced by oxidative stress. Hedyotis diffusa Willd (Rubiaceae) (HDW) is a traditional Chinese medicinal herb that has been shown to possess a variety of antioxidant properties.The present study examines and explains the cell protective property of HDW water extract (WEHDW).2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay was used to measure the free radical scavenging property of WEHDW (0.001-10 mg/mL). The protective effect of WEHDW (0.3-10 mg/mL 2 h pretreatment) against hydrogen peroxide (H2O2, 200 μM for 6 h) induced cytotoxicity in human hepatic cells, LO2, was evaluated using cell viability assay and nuclear staining. The molecular pathway of WEHDW's effect was investigated by using Western blot assay.WEHDW had a 50% scavenging concentration (SC50) at 0.153 mg/mL in the DPPH assay. Exposure of LO2 cells to H2O2 resulted in apoptosis which could be markedly attenuated by pre-treating WEHDW in a concentration-dependent manner (0.5, 1, 3, 5, or 10 mg/mL) (all with p 0.001, versus control). Moreover, Hoechst (nuclear) staining showed that 1 mg/mL WEHDW could protect LO2 cells by attenuating apoptotic cell death mediated by H2O2. It was found that WEHDW reversed H2O2-induced activation of MEK/ERK pathway and H2O2-induced inhibition of P13-K/AKT/GSK3β pathway in LO2 cells.WEHDW may help to improve the antioxidant defense system, resulting in prevention of oxidative stress-related fatty liver diseases.

Ethnopharmacological relevance The fruit of Schisandra chinensis (Turcz.) Baill, has been traditionally used in management of liver diseases and ageing associated neurodegeneration. The bioactive compound from this medicinal plant would be valuable for its potential use in prevention and treatment of Parkinson׳s disease. Aim of the study The overall objective of the present study was to understand the neuroprotective effect of schisantherin A, a dibenzocyclooctadiene lignan from the fruit of S. chinensis (Turcz.) Baill, and to elucidate its underlying mechanism of action. Material and methods This study investigated the protective effect of schisantherin A against selective dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced neural damage in human neuroblastoma SH-SY5Y cells and zebrafish models. Oxidative stress and related signaling pathways underlying the neuroprotective effect were determined by multiple biochemical assays and Western blot. Results Pretreatment with schisantherin A offered neuroprotection against 6-OHDA-induced SH-SY5Y cytotoxicity. Moreover, schisantherin A could prevent 6-OHDA-stimulated dopaminergic neuron loss in zebrafish. Our mechanistic study showed that schisantherin A can regulate intracellular ROS accumulation, and inhibit NO overproduction by down-regulating the over-expression of iNOS in 6-OHDA treated SH-SY5Y cells. Schisantherin A also protects against 6-OHDA-mediated activation of MAPKs, PI3K/Akt and GSK3β. Conclusion These findings demonstrate that schisantherin A may have potential therapeutic value for neurodegenerative diseases associated with abnormal oxidative stress such as Parkinson׳s disease.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder having close relationship with oxidative stress induced by reactive oxygen species (ROS). Cortex Fraxini (QP) is a kind of traditional Chinese medicinal herb with antioxidant properties. It may be a potential candidate for preventing the development of chronic neurodegenerative diseases. Thus, the key objective of the current study was to investigate the neuroprotective effect of QP water extract on 6-hydroxydopamine (6-OHDA) induced apoptosis in rat pheochromocytoma (PC12) cells. It was found that QP water extract possesses strong antioxidant property with SC50= 0.15 mg/mL. Total phenolic content of QP water extract was found to be 200.78 ± 2.65 mg GAE/g. QP water extract’s free radical scavenging capacity was demonstrated by reversing the increased level of intracellular ROS induced by 6-OHDA, using 2′,7′-dichlorodihydrofluorescein diacetate. Moreover, QP water extract (0.5 mg/mL) could remarkably increase the viability of PC12 cells treated with 6-OHDA. The protective effect of QP water extract was found to be via inhibiting MEK/ERK pathway and reversing PI3-K/Akt/GSK3βpathway. The current results suggest that QP might be a potential candidate for preventing the development of neurodegenerative diseases, such as PD.

Danshen (Radix Salviae miltiorrhizae) and ChuanXiong (Ligusticum wallichii) are two traditional herbal medicines commonly used in China for the treatment of cardiovascular diseases. The active components in Danshen and ChuanXiong are Danshensu (DSS, (R)-3, 4-dihydroxyphenyllactic acid) and tetramethylpyrazine (TMP), respectively. In the present study, a new compound named ADTM, which is a conjugation of DSS and TMP, was synthesized and its effect on the contractility of rat mesenteric arteries was examined. The relaxation effect of ADTM on rat mesenteric arteries was studied using myography. The effects of ADTM on Ca(2+) channels were measured by Ca(2+) imaging and patch-clamp techniques. The results showed that ADTM caused a concentration-dependent relaxation of rat mesenteric arteries. This relaxation effect was not affected by the removal of endothelium or inhibitors of nitric oxide synthase, cyclooxygenase, guanylyl cyclase and adenylyl cyclase. Potassium channel blockers including tetraethylammonium, iberiotoxin, apamin, 4-aminopyridine, BaCl2 and glibenclamide also failed to inhibit the relaxation response to ADTM. ADTM inhibited CaCl2-induced contractions and reduced the Ca(2+) influx in isolated mesenteric arterial muscle cells. Our results suggest that ADTM may be a novel relaxing agent. Its mechanism of action involves the direct blockade of voltage-gated Ca(2+) channels in vascular smooth muscle cells, resulting in a decrease in Ca(2+) influx into the cells.

Background Centipeda minima (Ebushicao) has been used for the treatment of various diseases, such as nasal allergies, rhinitis and sinusitis, nasopharyngeal carcinoma, cough, and headache. This study aims to investigate the anticancer activities of Centipeda minima ethanol extracts (CME) against nasopharyngeal carcinoma cell CNE-1 and their underlying mechanism. Methods CNE-1 cells were treated with different concentrations (15–50 μg/mL) of CME for different time intervals (24, 48, and 72 h). Cytotoxicity of CME was determined by MTT assay. Cell morphological changes were observed by fluorescence microscopy after HO 33258 staining. Cell cycle status was evaluated by flow cytometry following propidium iodide staining. Apoptosis was detected by flow cytometry following annexin V-FITC/PI staining. The levels of apoptosis-associated and PI3K-Akt-mTOR signaling related proteins were measured by western blotting analysis. Results CME (15–50 μg/mL) significantly inhibited the proliferation of CNE-1 in a dose- and time-dependent manner (P = 0.026 for 15 μg/mL, P

Sichuan pepper, a well-known traditional Chinese spice, is popularly used in Chinese cuisine. In this study, the antioxidant activities of Sichuan pepper water extract (SP) were evaluated both chemically [in 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay] and biologically [in 2,2′-azobis-(2-amidinopropane) (AAPH)-induced haemolysis assay]. The protective effect of SP against H2O2-induced cytotoxicity in primary Sprague Dawley rat hepatocytes was investigated by using cell viability assay, nuclear staining, intracellular reactive oxygen species (ROS) measurement and flow cytometry. It was found to possess high total phenolic content as assessed by the Folin–Ciocalteu method. The antiapoptotic effect of SP may be mediated, in part, by significantly inhibiting intracellular ROS generation and depletion of the mitochondrial membrane potential. SP was also found to enhance the cellular antioxidant defense system through up-regulating heme oxygenase-1 expression. SP is an effective antioxidant that can be consumed regularly as a functional food for the prevention of oxidative stress-related liver diseases. Practical applications The present study demonstrated that Sichuan pepper water extract (SP) could enhance the cellular antioxidant ability in hepatocytes. SP is an effective antioxidant that can be consumed regularly as a functional food for the prevention of oxidative stress-related liver diseases.

Gui-ling-gao (GLG), also known as turtle/tortoise jelly, has been traditionally used as a functional food in southern China, Singapore, and Malaysia to regulate the immune system. In the present study, the antiinflammatory effects of GLG against Concanavalin A (Con A)-induced inflammation in BALB/c mice isolated splenocytes were evaluated. GLG significantly (p

The rhizome of Ligusticum chuanxiong Hort. (LC), also known as chuanxiong, is a very common herb widely used to treat cardiovascular and cerebrovascular diseases. It is also used as a major ingredient in soups for regular consumption to promote good health. To study the protective effect of LC ethanolic extract (LCEE, 600 mg per kg per day, p.o.) on the integrity of the vascular system, ovariectomized (OVX) rats were fed with a high-fat diet (HFD) plus LCEE for 12 weeks. The animal model was used to mimic the dyslipidemic condition seen in postmenopausal women. LCEE was found significantly to reduce the body weight gain, improve serum lipid profiles (by lowering total cholesterol and low density lipoprotein cholesterol but raising high density lipoprotein cholesterol) and protect vascular endothelium in the HFD-fed OVX rats. It is postulated that LCEE could exert its vascular protective effect through multiple targets by (1) improving serum lipid profiles to reduce the detrimental effects of cholesterol; (2) reducing the ROS level in the body via enhancing the hepatic anti-oxidative activity or antioxidant level to scavenge the reactive oxygen species generated in the postmenopausal hypercholesterolemic condition; (3) stimulating eNOS-derived nitric oxide production; (4) counteracting the up-regulation of inflammatory cytokine (TNF-α, VCAM-1 and ICAM-1) expressions so as to reduce endothelium damage.

In ischemic disorders such as chronic wounds and myocardial ischemia, there is inadequate tissue perfusion due to vascular insufficiency. Besides, it has been observed that prolonged use of anti-angiogenic agents in cancer therapy produces cardiovascular toxicity caused by impaired vessel integrity and regeneration. In the present study, we used VEGFR tyrosine kinase inhibitor II (VRI) to chemically induce vascular insufficiency in zebrafish in vivo and human umbilical vein endothelial cells (HUVEC) in vitro to further study the mechanisms of vascular morphogenesis in these pathological conditions. We also explored the possibility of treating vascular insufficiency by enhancing vascular regeneration and repair with pharmacological intervention. We observed that pretreatment of VRI induced blood vessel loss in developing zebrafish by inhibiting angiogenesis and increasing endothelial cell apoptosis, accompanied by down-regulation of kdr, kdrl and flt-1 genes expression. The VRI-induced blood vessel loss in zebrafish could be restored by post-treatment of calycosin, a cardiovascular protective isoflavone. Similarly, VRI induced cytotoxicity and apoptosis in HUVEC which could be rescued by calycosin post-treatment. Further investigation of the underlying mechanisms showed that the PI3K/AKT/Bad cell survival pathway was a main contributor of the vascular regenerative effect of calycosin. These findings indicated that the cardiovascular toxicity in anti-angiogenic therapy was mainly caused by insufficient endothelial cell survival, suggesting its essential role in vascular integrity, repair and regeneration. In addition, we showed that VRI-induced blood vessel loss in zebrafish represented a simple and effective in vivo model for studying vascular insufficiency and evaluating cancer drug vascular toxicities.

The king of herbs,Panax ginseng, has been used widely as a therapeutic agent vis-à-vis its active pharmacological and physiological effects. Based on Chinese pharmacopeiaBen Cao Gang Muand various pieces of literature,Panax ginsengwas believed to exert active vascular protective effects through its antiobesity and anti-inflammation properties. We investigated the vascular protective effects of ginseng by administrating ginseng extracts to rats after the induction of diabetes. We found thatPanax ginsengcan restore diabetes-induced impaired vasorelaxation and can reduce serum triglyceride but not cholesterol level in the diabetic rats. The ginseng extracts also suppressed the expression of atherosclerosis-related genes and altered the expression of lipid-related genes. The results provide evidence thatPanax ginsengimproves vascular dysfunction induced by diabetes and the protective effects may possibly be due to the downregulation of atherosclerosis-related genes and altered lipid metabolism, which help to restore normal endothelium functions.

The poor prognostic outcome of breast cancer is largely due to its resistance to cancer therapies. Development of therapeutic agents that can inhibit growth and induce apoptosis in breast cancer cells can help solve the problem. Emodin is an active anthraquinone that has been reported to have diverse biological effects.In this study, the anticancer effects of emodin on growth inhibition, apoptosis induction and the expression of apoptosis-related genes in MCF-7 cells were investigated.Growth inhibition induced by emodin was investigated by the MTS assay and the colony formation assay; while emodin-induced apoptosis was determined by the COMET assay and DNA fragmentation detection. Emodin (35 μM)-induced alterations in the expression of apoptotic-related genes were detected by using real-time PCR.Emodin had significant growth inhibitory effects on MCF-7 cells with IC₅₀ = 7.22 µg/ml (∼30 μM). It also exerted a concentration-dependant inhibitory effect on the colony-forming ability of MCF-7 cells with IC₅₀ = 7.60 µg/ml (∼30 µM). Hallmarks of apoptosis, such as single-strand DNA breakage and DNA fragmentation, were observed in emodin-treated MCF-7 cells. The gene expression of Fas ligand (FASL) was up-regulated (p 0.01) but those of MCL1, CCND1 and C-MYC were down-regulated (p 0.05) in emodin-treated MCF-7 cells.This study indicated that emodin could induce growth inhibition and apoptosis in MCF-7 cells through the modulation of the expression of apoptosis-related genes. The growth inhibitory effects of emodin might involve both the intrinsic and the extrinsic apoptotic pathways and cell cycle arrest.

The serotonin (5-HT) uptake system is supposed to play a crucial part in vascular functions by "fine-tuning" the local concentration of 5-HT in the vicinity of 5-HT(2) receptors in vascular smooth muscle cells. In this study, the mechanism of 5-HT uptake in human brain vascular smooth muscle cells (HBVSMCs) was investigated. [(3)H]5-HT uptake in HBVSMCs was Na(+)-independent. Kinetic analyses of [(3)H]5-HT uptake in HBVSMCs revealed a K(m) of 50.36 ± 10.2 mM and a V(max) of 1033.61 ± 98.86 pmol/mg protein/min. The specific serotonin re-uptake transporter (SERT) inhibitor citalopram, the specific norepinephrine transporter (NET) inhibitor desipramine, and the dopamine transporter (DAT) inhibitor GBR12935 inhibited 5-HT uptake in HBVSMCs with IC(50) values of 97.03 ± 40.10, 10.49 ± 5.98, and 2.80 ± 1.04 μM, respectively. These IC(50) values were 100-fold higher than data reported by other authors, suggesting that those inhibitors were not blocking their corresponding transporters. Reverse transcription-polymerase chain reaction results demonstrated the presence of mRNA for organic cation transporter (OCT)-3 and plasma membrane monoamine transporter (PMAT), but the absence of OCT-1, OCT-2, SERT, NET, and DAT. siRNA knockdown of OCT-3 and PMAT specifically attenuated 5-HT uptake in HBVSMCs. It is concluded that 5-HT uptake in HBVSMCs was mediated predominantly by a low-affinity and Na(+)-independent mechanism. The most probable candidates are OCT-3 and PMAT, but not the SERT.

This letter describes the preparation of quinoline derivatives and their cytotoxic potentials toward human carcinoma cell lines. Among the selected compounds, 8-hydroxy-2-quinolinecarbaldehyde (3) showed the best in vitro cytotoxicity against the human cancer cell lines, including MDA231, T-47D, Hs578t, SaoS2, K562, SKHep1 (with a MTS50 range of 12.5–25 μg/mL) and Hep3B (with a MTS50 range of 6.25±0.034 μg/mL). The in vivo antitumor activity of compound 3 on subcutenaous Hep3B hepatocellular carcinoma xenograft in athymic nude mice was then studied. The results showed that the dose of 10 mg/kg/day of compound 3 with intraperitoneal injection for 9 days totally abolished the growth of the xenograft tumor of Hep3B with no histological damage on vital organs as compared with the control. The experimental results suggested that compound 3 has a good potential as an antitumor agent.

Apoptotic neuronal cell death plays an important role in Parkinson's disease (PD), a progressive neurodegenerative disorder. Luteolin, a flavonoid, has been shown to possess various pharmacological properties including strong antioxidant capacity.This study investigated the neuroprotective effect of luteolin against cytotoxicity induced by 6-hydroxy-dopamine (6-OHDA) (250 µM) in rat pheochromocytoma (PC12) cell line.The neuroprotective effect of luteolin against 6-OHDA-induced cytotoxicity in PC12 was evaluated by using cell viability test, nuclear staining and flow cytometry. In addition, the apoptotic role of luteolin was unveiled by monitoring mRNA expression of proapoptotic and anti-apoptotic genes.Pretreatment with luteolin (3.13, 6.25, 12.5, 25 or 50 µM) could markedly attenuate 6-OHDA-induced PC12 cell viability loss in a concentration-dependent manner. Cell morphologic analysis and nuclear staining assays showed that luteolin (3.13, 12.5 or 50 µM) protected the cells from 6-OHDA-induced damage. As shown in the flow cytometry assay, the increased apoptotic rate induced by 6-OHDA could be significantly (p0.001) suppressed by luteolin (12.5 or 50 µM) pretreatment. The protection of luteolin (50 µM) against 6-OHDA-induced cell damage was shown to be through suppressing the over-expression of Bax gene (p0.01), inhibiting the reduction of Bcl-2 gene expression (p0.05) and markedly depressing the enhanced Bax/Bcl-2 ratio. Luteolin also downregulated the gene expression level of p53.Luteolin has protective effects against 6-OHDA-induced cell apoptosis and might be a potential nutritional supplement which could be used to prevent neurodegenerative diseases such as PD.

Background Panax notoginseng is commonly used for the treatment of cardiovascular diseases in China. The present study investigates the effects of three different saponin fractions (ie total saponins, PNS; protopanaxadiol-type saponin, PDS; and protopanaxatriol-type saponin, PTS) and two major individual ingredients (ie ginsenoside Rg1 and Rb1) from P. notoginseng on the endothelial inflammatory response in vitro and in vivo. Methods Recombinant human tumor necrosis factor-α (TNF-α) was added to the culture medium of human coronary artery endothelial cells (HCAECs) to induce an inflammatory response. A cell adhesion assay was used to determine the effect of the P. notoginseng saponin fractions on endothelial-monocyte interaction. The cell adhesion molecule (CAMs) expression, including ICAM-1 and VCAM-1, in the protein level on the surface of endothelial cells were measured by cellular ELISA. CAMs expression in mRNA level was also assayed by qRT-PCR in the HCAECs and the aorta of rat fed with high cholesterol diet (HCD). Western blotting was used to detect effect of the saponin fractions on CAMs protein expression in HCAECs. In addition, nuclear translocation of p65, a surrogate marker for NF-κB activation, was measured by immunostaining. Results Three saponin fractions and two individual ginsenosides exhibited the inhibitory effects on monocyte adhesion on TNF-α-activated HCAECs and expression of ICAM-1 and VCAM-1 at both mRNA and protein levels in vitro. The saponin fractions exhibited a similar trend of the inhibitory effects on the mRNA expression of CAMs in the aorta of HCD-fed rat in vivo. These inhibitory effect of saponin fractions maybe attribute partially to the suppression of the TNF-α-induced NF-κB activation. Conclusion Our data demonstrate that saponin fractions (ie PNS, PDS and PTS) and major individual ginsenosides (ie Rg1 and Rb1) have potential anti-atherogenic effects. Among the tested saponin fractions, PDS is the most potent saponin fraction against TNF-α-induced monocyte adhesion as well as the expression of adhesion molecules in vitro and in vivo.

Although zebrafish has become a popular animal model for drug discovery and screening, drug metabolism in zebrafish remains largely unknown. In this study, we probed the metabolic capability of zebrafish larvae with calycosin, one of the major isoflavone constituents of Radix Astragali that was previously demonstrated to be angiogenic in the zebrafish model. The metabolism of calycosin and accumulation of its metabolites in zebrafish larvae were determined using an LC-MS/MS method. Calycosin showed a slow but steady decrease from the culture medium as well as a steady accumulation in zebrafish larvae. Calycosin underwent major conjugation and minor oxidation in zebrafish larvae. A total of ten calycosin metabolites formed from glucuronidation, glucosylation, sulfation, oxidation or a combination of two of these metabolisms were identified, most of which were reported for the first time. Most metabolites increased steadily in the larvae over 24-h experimental period. The dominant phase II conjugation of calycosin in zebrafish larvae matched well with existing knowledge of isoflavone metabolism in mammalians. The findings shed a light in certain degree of similarity of phase II drug metabolism between zebrafish larvae and mammals and warrant further investigation on feasibility of adopting the zebrafish larvae as a whole-organism model for examining drug metabolism.

Grifola frondosa (Polyporaceae), maitake, is a widely consumed edible mushroom in some Asian countries. The fruit bodies and mycelia of maitake have shown different bioactive compounds with anticancer and other therapeutic properties.This study evaluated three chemically modified maitake polysaccharide-peptides' (MPSP) adjuvant effect (in vivo) and anticancer activity (in vitro growth inhibitory effect) compared with crude MPSP from G. frondosa.We investigated the possibility of enhancing the adjuvant effect and anticancer effect of crude MPSP by using simple chemical modification methods to convert crude MPSP to phosphorylated, acetylated or esterified MPSPs. The adjuvant effect and growth inhibitory effect were evaluated by C6 cell inoculated rat model with cyclophosphamide (CPA) treatment and in vitro cell viability assay, respectively.All four tested MPSPs showed significant adjuvant effect to CPA treatment on rats inoculated with C6 cancer cells. In addition, an obvious growth inhibitory effect was observed in C6 cancer cells but not in normal brain cells treated with various forms of MPSPs. Only phosphorylation could significantly (p 0.05) improve the adjuvant effect (in vivo) and growth inhibitory effect. A same rank order (phosphorylated MPSPesterified MPSP ≥ acetylated MPSP ≥ crude MPSP) of efficacy was observed in both the in vivo and in vitro assays.This study showed chemical phosphorylation could markedly enhance both adjuvant effects and growth inhibitory effects. This study demonstrated the feasibility of enhancing the efficacy of MPSP by using a simple chemical modification method, and this provides a foundation for future study in this area.

To investigate the involvement of Cl⁻ channels in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in rat mesenteric arteries.Cl⁻ channel and K(ir) channel activities were studied using whole-cell patch clamping in rat mesenteric arterial smooth muscle cells. Isometric tension of arterial rings was measured in organ chambers.The volume-activated Cl⁻ current in rat mesenteric arterial smooth muscle cells was abolished by Cl⁻ channel blockers NPPB or DIDS. The EDHF-mediated vasorelaxation was potentiated by NPPB and DIDS. The EDHF response was diminished by a combination of apamin and charybdotoxin, which agreed with the hypothesis that EDHF response involves the release of K(+) via the Ca²(+)-activated K(+) channels in endothelial cells. The elevation of K(+) concentration in bathing solution from 1.2 mmol/L to 11.2 mmol/L induced an arterial relaxation, which was abolished by the combination of BaCl₂ and ouabain. It is consistent to the hypothesis that K(+) activates K(+)/Na(+)-ATPase and inward rectifier K(+) (K(ir)) channels, leading to the hyperpolarization and relaxation of vascular smooth muscle. The K(+)-induced relaxation was augmented by NPPB, DIDS, or withdrawal of Cl⁻ from the bathing solution, which could be reversed by BaCl₂, but not ouabain. The potentiating effect of Cl⁻ channel blockers on K(+)-induced relaxation was probably due to the interaction between Cl⁻ channels and K(ir) channels. Moreover, the K(+)-induced relaxation was potentiated when the arteries were incubated in hyperosmotic solution, which is known to inhibit volume-activated Cl⁻ channels.The inhibition of Cl⁻ channels, particularly the volume-activated Cl⁻ channels, may potentiate the EDHF-induced vasorelaxation through the K(ir) channels.

Background: Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.Methodology: Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 μM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 μM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 mM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting. Conclusion: Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERa and ERb. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways. © 2010 Tang et al., published_or_final_version

Epoxyeicosatrienoic acids (EETs) induce vasorelaxation, probably through G protein-coupled receptors. The identity of these receptors is unclear, but it has been reported that EETs may bind to peroxisome proliferator activated receptors (PPARs) and E-prostanoid (EP) receptors. Therefore, we studied whether PPARs or EP receptors were involved in 14,15-EET-induced vasorelaxation. Isometric tensions of rat mesenteric arteries were measured. The vasorelaxant effect of 14,15-EET was inhibited by NF449 (G(s)-protein inhibitor), Rp-cAMP (cAMP antagonist) and KT5720 (PKA inhibitor), suggesting that the effect of 14,15-EET was mediated through G(s) protein-coupled receptors which were linked to the cAMP/PKA-dependent pathway. Pretreatments with MK886 (PPAR(alpha) antagonist) and GW9662 (PPAR(gamma) antagonist) did not influence 14,15-EET-induced vasorelaxation. The vasorelaxant effect of 14,15-EET was inhibited by AH6809 (EP(2) receptor antagonist), whereas SC19220 (EP(1) receptor antagonist), L798106 (EP(3) receptor antagonist) and GW627368X (EP(4) receptor antagonist) had no effect. The effect of 14,15-EET and the mechanism involved was mimicked by prostaglandin E(2) (an EP(2) receptor agonist). The 14,15-EET-induced relaxation was slightly potentiated in the presence of indomethacin (cyclooxygenase inhibitor which block PGE(2) synthesis). Binding study showed that the amount of 14,15-EET bound to the cell membrane of rat mesenteric arterial smooth muscle cells was much higher than that bound to the nuclear membrane. The binding of 14,15-EET to the cell membrane was attenuated by AH6809 and siRNA against EP(2) receptors. In conclusion, our study has demonstrated that 14,15-EET exerts relaxant effects on rat mesenteric arteries, at least partly via the stimulation of EP(2) receptors. This subsequently leads to activation of cAMP/PKA-dependent pathway in vascular smooth muscle cells.