Deoxycholylglycine, a conjugated secondary bile acid, reduces vascular tone by attenuating Ca2+ sensitivity via rho kinase pathway.

Patients with cirrhosis have reduced systemic vascular resistance and elevated circulating bile acids (BAs). Previously, we showed that secondary conjugated BAs impair vascular tone by reducing vascular smooth muscle cell (VSMC) Ca2+ influx. In this study, we investigated the effect of deoxycholylglycine (DCG), on Ca2+ sensitivity in reducing vascular tone. First, we evaluated the effects of DCG on U46619- and phorbol-myristate-acetate (PMA)-induced vasoconstriction. DCG reduced U46619-induced vascular tone but failed to reduce PMA-induced vasoconstriction. Then, by utilizing varied combinations of diltiazem (voltage-dependent Ca2+ channel [VDCC] inhibitor), Y27632 (RhoA kinase [ROCK] inhibitor) and chelerythrine (PKC inhibitor) for the effect of DCG on U46619-induced vasoconstriction, we ascertained that DCG inhibits VDCC and ROCK pathway with no effect on PKC. We further assessed the effect of DCG on ROCK pathway. In ß-escin-permeabilized arteries, DCG reduced high-dose Ca2+- and GTPγS (a ROCK activator)-induced vasoconstriction. In rat vascular smooth muscle cells (VSMCs), DCG reduced U46619-induced phosphorylation of myosin light chain subunit (MLC20) and myosin phosphatase target subunit-1 (MYPT1). In permeabilized VSMCs, DCG reduced Ca2+- and GTPγS-mediated MLC20 and MYPT1 phosphorylation, and further, reduced GTPγS-mediated membrane translocation of RhoA. In VSMCs, long-term treatment with DCG had no effect on ROCK2 and RhoA expression. In conclusion, DCG attenuates vascular Ca2+ sensitivity and tone via inhibiting ROCK pathway. These results enhance our understanding of BAs-mediated regulation of vascular tone and provide a platform to develop new treatment strategies to reduce arterial dysfunction in cirrhosis.

Characterization of pressure-mediated vascular tone in resistance arteries from bile duct-ligated rats.

In cirrhosis, changes in pressure-mediated vascular tone, a key determinant of systemic vascular resistance (SVR), are unknown. To address this gap in knowledge, we assessed ex vivo dynamics of pressurized mesenteric resistance arteries (diameter ~ 260 µm) from bile duct-ligated (BDL) and sham-operated (SHAM) rats and determined the underlying mechanisms. At isobaric intraluminal pressure (70 mmHg) as well as with step-wise increase in pressure (10-110 mmHg), arteries from SHAM-rats constricted more than BDL-rats, and had reduced luminal area. In both groups, incubation with LNAME (a NOS inhibitor) had no effect on pressure-mediated tone, and expression of NOS isoforms were similar. TEA, which enhances Ca2+ influx, augmented arterial tone only in SHAM-rats, with minimal effect in those from BDL-rats that was associated with reduced expression of Ca2+ channel TRPC6. In permeabilized arteries, high-dose Ca2+ and γGTP enhanced the vascular tone, which remained lower in BDL-rats that was associated with reduced ROCK2 and pMLC expression. Further, compared to SHAM-rats, in BDL-rats, arteries had reduced collagen expression which was associated with increased expression and activity of MMP-9. BDL-rats also had increased plasma reactive oxygen species (ROS). In vascular smooth muscle cells in vitro, peroxynitrite enhanced MMP-9 activity and reduced ROCK2 expression. These data provide evidence that in cirrhosis, pressure-mediated tone is reduced in resistance arteries, and suggest that circulating ROS play a role in reducing Ca2+ sensitivity and enhancing elasticity to induce arterial adaptations. These findings provide insights into mechanisms underlying attenuated SVR in cirrhosis.

Sunflower oil mediated biomimetic synthesis and cytotoxicity of monodisperse hexagonal silver nanoparticles.

In this work, sunflower oil was utilized for the biomimetic synthesis of silver (Ag) nanoparticles (NPs), leading to highly mono-dispersed hexagonal-shaped silver nanoparticles (NPs) at various concentrations. It was found that the biomolecules of the oil not only have the capability to reduce silver ions, due to its extended phenolic system, but also appear to recognize and affect the Ag nanocrystal growth on the (110) face, leading to hexagonal growth of the NPs of 50 nm size. Initially, some spherical AgNPs of less than 10nm diameter were observed; however, over a longer period of time, a majority of hexagonal-shaped nanocrystals were formed. The one step synthesis can be extended for other metals. The as prepared sunflower oil capped AgNPs being completely free of toxic chemicals can be directly utilized for in vitro studies and offer a more rational approach for cellular applications. The NP solution exhibited dose-dependent cytotoxicity in human lung carcinoma cells and physiologically relevant cell model (3T3L1 cells).

Herbal medicines for the treatment of nonalcoholic steatohepatitis: current scenario and future prospects.

Nonalcoholic steatohepatitis (NASH) is a multifactorial disease and has close correlations with other metabolic disorders. This makes its treatment difficult using a single pharmacological drug. Use of plant extract/decoction or polyherbal formulation to treat various liver diseases is very well mentioned in various traditional systems of medicine (Ayurveda, Japanese or traditional Chinese Medicine, and Kampo medicine). Medicinal herbs are known for their multifaceted implications and thus can form an effective treatment schedule against NASH. Till date, several plant extracts, polyherbal formulations, and phytochemicals have been evaluated for their possible therapeutic potential in preventing onset and progression of NASH in experimental models, but clinical studies using the same are sparse. Herbal extracts with antioxidants, antidiabetic, and antihyperlipidemic properties have been shown to ameliorate symptoms of NASH. This review article is a meticulous compilation of our current knowledge on the role of natural products in alleviating NASH and possible lacunae in research that needs to be addressed.

Minimizing human infection from Escherichia coli O157:H7 using GUMBOS.

OBJECTIVES: Reduction in faecal shedding of Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) in food-producing animals is a viable strategy to minimize human disease initiated by exposure to these microorganisms. To this end, an intervention strategy involving the electrostatic hybridization of two commonly used anti-infective agents for veterinary practice (i.e. chlorhexidine and ampicillin) was evaluated to curtail EHEC-transmitted disease from ruminant sources. Chlorhexidine di-ampicillin is a novel group of uniform material based on organic salts (GUMBOS) with inherent in vitro antibacterial activity that comes from its parent antimicrobial ions, chlorhexidine and ampicillin. METHODS: Antibacterial activities for chlorhexidine diacetate, sodium ampicillin, chlorhexidine di-ampicillin and stoichiometrically equivalent 1 : 2 chlorhexidine diacetate : sodium ampicillin were assessed using the serial 2-fold dilution method and time-kill studies against seven isolates of E. coli O157:H7 and one non-pathogenic E. coli 25922. Further studies to investigate synergistic interactions of reacted and stoichiometrically equivalent unreacted antimicrobial agents at MICs and possible mechanisms were also investigated. RESULTS: Synergism and in vitro antibacterial activities against EHEC were observed in this study, which suggests chlorhexidine di-ampicillin could be a useful reagent in reducing EHEC transmission and minimizing EHEC-associated infections. Likewise, chlorhexidine di-ampicillin reduced HeLa cell toxicity as compared with chlorhexidine diacetate or the stoichiometric combination of antimicrobial agents. Further results suggest that the mechanisms of action of chlorhexidine di-ampicillin and chlorhexidine diacetate against E. coli O157:H7 are similar. CONCLUSIONS: Reacting antimicrobial GUMBOS as indicated in this study may enhance the approach to current combination drug therapeutic strategies for EHEC disease control and prevention.

Protective role of standardized Feronia limonia stem bark methanolic extract against carbon tetrachloride induced hepatotoxicity.

OBJECTIVE: This study evaluates hepatoprotective potential of Feronia limonia stem bark (FSB) extracts and fractions using experimental models. MATERIALS AND METHODS: Activity levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and cell viability were evaluated in HepG2 cells treated with carbon tetrachloride (CCl4) in presence or absence of FL extracts or fractions. Also, plasma markers of hepatic damage, hepatic antioxidants, lipid peroxidation and histopathological alterations were assessed in rats treated with CCl4 alone or in combination with 200 or 400 mg/kg bodyweight (BW) of FSB-7 or 25 mg/kg BW of silymarin. RESULTS: In vitro co-supplementation of FSB extracts or fractions recorded varying degree of hepatoprotective potentials. Also, pre-supplementation of FSB methanolic extract (FSB-7) followed by CCl4 treatment significantly prevented hepatic damage and depletion of cellular antioxidants. Also, CCl4+ FSB-7 group showed minimal distortion in the histoarchitecture of liver and results were comparable to that of CCl4+ silymarin treated rats. CONCLUSION: This inventory is the first scientific report on hepatoprotective potential of FSB methanolic extract.

Hepatoprotective potential of Tecomella undulata stem bark is partially due to the presence of betulinic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Tecomella undulata (TU;` Family Bignoniaceae) is used in Indian Ayurvedic system of medicine for treating various diseases including hepatic ailments. It is also incorporated in various marketed hepatoprotective polyherbal formulations. AIM: The present study was aimed at evaluating possible hepatoprotective role of isolated compounds from TU stem bark (TSB) using in vitro and in vivo experimental models. METHODS: In vitro cytotoxicity and hepatoprotective potential of various extract, fractions and isolated compounds from TU stem bark were evaluated using HepG2 cells. Rats were pre-treated with TU methanolic extract (TSB-7) or betulinic acid (MS-2) or silymarin for 7 days followed by a single dose of CCl(4) (0.5 ml/kg, i.p.). Plasma markers of hepatic damage, hepatic antioxidants and indices of lipid peroxidation along with microscopic evaluation of liver were assessed in control and treatment groups. RESULTS: TSB-2 and MS-1 accounted for significant cell death whereas; TSB-1, TBS-7, TSB-9, TSB-10 and, MS-2 did not register significant cytotoxicity. Further, non-cytotoxic components exhibited ascending grade of hepatoprotection in vitro (TSB-10

Hepatoprotective activity of Feronia limonia root.

OBJECTIVES: The aim of this study was to evaluate the hepatoprotective potential of a methanolic extract and of marmesin isolated from the root bark of Feronia limonia. METHODS: Activity levels of aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT), cell viability and cell death were evaluated in HepG2 cells (human liver hepatoma cells) treated with CCl4 in the presence or absence of F. limonia extract or marmesin. Plasma activity levels of AST, ALT, bilirubin, alkaline phosphatase, protein, hepatic antioxidants, lipid peroxidation and histopathological evaluations were carried out in rats treated with CCl4 alone or co-supplemented with F. limonia extract or marmesin in a dose-dependent manner. KEY FINDINGS: In-vitro co-supplementation of F. limonia methanolic extract or marmesin significantly minimized alteration in levels of AST and ALT and improved cell viability. Oral administration of F. limonia methanolic extract or marmesin significantly prevented CCl4-induced elevation in the plasma markers of hepatic damage and hepatic lipid peroxidation and a decrease in hepatic antioxidants. In-vivo hepatoprotective potential of F. limonia methanolic extract and marmesin was evident from the minimal alterations in the histoarchitecture of liver. CONCLUSIONS: This has been the first scientific report on the hepatoprotective potential of F. limonia root bark methanolic extract and marmesin.

Sida rhomboidea.Roxb aqueous extract down-regulates in vivo expression of vascular cell adhesion molecules in atherogenic rats and inhibits in vitro macrophage differentiation and foam cell formation.

The present study evaluates efficacy of Sida rhomboidea.Roxb (SR) leaves extract in ameliorating experimental atherosclerosis using in vitro and in vivo experimental models. Atherogenic (ATH) diet fed rats recorded significant increment in the serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), very LDL (VLDL), autoantibody against oxidized LDL (Ox-LDL), markers of LDL oxidation and decrement in high-density lipoprotein (HDL) along with increment in aortic TC and TG. The ex vivo LDL oxidation assay revealed an increased susceptibility of LDL isolated from ATH rats to undergo copper mediated oxidation. These set of changes were minimized by simultaneous co-supplementation of SR extract to ATH diet fed rats. Histopathology of aorta and immunolocalization studies recorded pronounced atheromatous plaque formation, vascular calcification, significant elastin derangements and higher expression of macrophage surface marker (F4/80), vascular cell adhesion molecule-1 (VCAM-1) and p-selectin in ATH rats. Whereas, ATH+SR rats depicted minimal evidence of atheromatous plaque formation, calcium deposition, distortion/defragmentation of elastin and accumulation of macrophages along with lowered expression of VCAM-1 and P-selectin compared to ATH rats. Further, monocyte to macrophage differentiation and in vitro foam cell formation were significantly attenuated in presence of SR extract. In conclusion, SR extract has the potency of controlling experimental atherosclerosis and can be used as promising herbal supplement in combating atherosclerosis.

Anthocyanin-rich red cabbage (Brassica oleracea L.) extract attenuates cardiac and hepatic oxidative stress in rats fed an atherogenic diet.

BACKGROUND: Oxidative stress induced by reactive oxygen species plays an important role in the aetiology of several diseases including atherosclerosis and coronary heart disease. Anthocyanin-rich extracts have been shown to possess a variety of therapeutic roles, including antioxidant, cardioprotective and hepatoprotective properties. The present inventory was undertaken to evaluate the protective role of anthocyanin-rich red cabbage extract (ARCE) on an atherogenic (ATH) diet-induced hypercholesterolaemia and related cardiac and, hepatic oxidative stress in rats. RESULTS: ARCE (100 mg kg(-1) body weight) treatment of rats fed the ATH diet significantly prevented elevation in serum and tissue lipids, circulating levels of cardiac and hepatic damage markers, and resulted in excretion of lipids through faeces. Also, the ARCE extract significantly attenuated alterations in the cardiac and hepatic antioxidants and lipid peroxidation, and histopathological changes in cardiac and hepatic tissue. CONCLUSION: Thus, the present study provides the first scientific evidence for a protective role of ARCE against ATH diet-induced hypercholesterolaemia and cardiac and hepatic oxidative stress.

Sida rhomboidea.Roxb extract alleviates pathophysiological changes in experimental in vivo and in vitro models of high fat diet/fatty acid induced non-alcoholic steatohepatitis.

The present study was aim to evaluate protective role of Sida rhomboidea.Roxb (SR) extract against high fat diet/fatty acid induced pathophysiological alterations in experimental model of non-alcoholic steatohepatitis (NASH). Effect of SR extract on plasma levels of markers of hepatic damage, plasma and hepatic lipids, mitochondrial oxidative stress, status of enzymatic and non-enzymatic antioxidants and histopathological changes in liver tissue were evaluated in high fat diet fed C57BL/6J mice. Also, the effect of SR supplementation on lipid accumulation, lipid peroxidation, cytotoxicity and cell viability were evaluated in oleic acid treated HepG2 cells. Supplementation of NASH mice with SR extract prevented high fat diet induced elevation in plasma marker enzymes of liver damage, plasma and hepatic lipids, mitochondrial oxidative stress and compromised enzymatic and non-enzymatic antioxidant status. Further, addition of SR extract to in vitro HepG2 cells minimized oleic acid induced lipid accumulation, higher lipid peroxidation, cytotoxicity and reduced cell viability. These in vivo and in vitro studies suggest that SR extract has the potential of preventing high fat/fatty acid induced NASH mainly due to its hypolipidemic and antioxidant activities.

Traditional uses, phytochemistry and pharmacology of Clerodendron glandulosum Coleb--a review.

Present review for the first time provides a complete botanical description and information on ethnomedicinal uses of Clerodendron glandulosum.Coleb (CG; Fam, Verbenaceae). Recent studies conducted from our laboratory provide pharmacological evidence for its anti-hypertensive, anti-diabetic and anti-obesity potentials. Further, its beneficial potential in preventing in vitro and in vivo non-alcoholic steatohepatitis and atherosclerosis and potent hepatoprotective and free radical scavenging abilities along with its acute and sub-chronic toxicological evaluations are also reported from our laboratory. In keeping with its traditional uses, CG extract was capable of ameliorating experimentally induced hypertension, diabetes and obesity. Its beneficial potential against NASH induced oxidative stress and atherosclerosis can be attributed to its potent free radical scavenging potential. Non-toxic nature of CG leaf extract further provides added merit to its reported pharmacological properties. The present review summarizes the pioneering scientific evidence for the pharmacological effects of CG against related metabolic disorders like hypertension, diabetes and obesity along with anti oxidant potential and beneficial effects against non alcoholic steatohepatitis.

Clerodendron glandulosum.Coleb leaf extract attenuates in vitro macrophage differentiation and expression of VCAM-1 and P-selectin in thoracic aorta of atherogenic diet fed rats.

Present inventory evaluates the anti-atherogenic potential of C. glandulosum.Coleb leaf extract (CG) using in vivo and in vitro experimental models. Serum markers of low density lipoprotein (LDL-C) oxidation, cholesterol, triglycerides, lipoproteins, auto-antibody titer, ex vivo LDL-C oxidation, LDL-C aggregation, aortic lipids, histopathological evaluations and immunolocalization of macrophage surface marker (F4/80), vascular cell adhesion molecule-1 (VCAM-1) and P-selectin were performed in CON [rats treated with single dose of saline (i.p.) and fed with laboratory chow], ATH [rats treated with single dose of vitamin D3 (600,000 IU, i.p) and fed with atherogenic diet] and ATH+CG [rats treated with single dose of vitamin D3 (600,000 IU, i.p.) and fed with atherogenic diet and simultaneously treated with 200 mg/kg CG extract, p.o.] for 8 weeks. CG extract supplementation to atherogenic diet fed rats significantly prevented increment in serum cholesterol, triglycerides, and lipoproteins, markers of LDL-C oxidation, auto-antibody titer and aortic lipids. Also, LDL-C isolated from ATH+CG rats recorded mimimal aggregation and susceptibility to undergo ex vivo LDL-C oxidation. Microscopic evaluation of thoracic aorta of ATH+CG rats reveled prevention of atheromatous plaque formation, accumulation of lipid laden macrophages, calcium deposition, distortion/defragmentation of elastin, accumulation of macrophages and, down regulation of cell adhesion molecules (VCAM-1 and P-selectin) expression. Further, in vitro monocyte to macrophage differentiation was significantly attenuated in presence of CG extract (200 µg/mL). It can be concluded from the present study that, CG extract is capable of controlling induction of experimental atherosclerosis and warrants further scrutiny at the clinical level as a possible therapeutic agent.

Standardized flavonoid-rich Eugenia jambolana seed extract retards in vitro and in vivo LDL oxidation and expression of VCAM-1 and P-selectin in atherogenic rats.

The present inventory evaluates anti-atherogenic potential of flavonoid-rich Eugenia jambolana seed extract (EJSE) against in vitro low-density lipoprotein (LDL) oxidation, foam cell formation, and atherogenic (ATH) diet-induced experimental atherosclerosis in rats. EJSE was able to prevent in vitro LDL oxidation and oxidized LDL-induced macrophage foam cell formation. Also, EJSE supplementation to ATH rats significantly minimized increment in serum markers of LDL oxidation. The ex vivo oxidation indices were also minimized in LDL of EJSE-treated animals. Microscopic evaluation of thoracic aorta of ATH + EJSE rats recorded minimal evidence of atheromatous plaque formation, accumulation of lipid laden macrophages, calcium deposition, and expression of cell adhesion molecules (vascular cell adhesion molecule-1 and P-selectin). This is the first scientific report that demonstrates anti-atherogenic potential of EJSE and warrants further evaluation at clinical level.

Sida rhomboidea. Roxb leaf extract down-regulates expression of PPARγ2 and leptin genes in high fat diet fed C57BL/6J Mice and retards in vitro 3T3L1 pre-adipocyte differentiation.

Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity.

Safety evaluations on ethanolic extract of red cabbage (Brassica oleracea L.) in mice.

The present study has carried out safety evaluations on an ethanolic extract of red cabbage (RC) leaves in terms of acute and subchronic oral toxicity tests as per Organisation for Economic Cooperation and Development (OECD) guidelines in Swiss albino mice. Single-dose administration of RC extract (1000, 2000, 3000, 4000, or 5000 mg/kg body weight) to Swiss albino mice did not manifest toxicity or any significant adverse behavioral alterations. Chronic administration of RC extract (1000, 2000, and 3000 mg/kg body weight) for 28 d also did not register any significant alterations in fluid intake, organ weights, plasma lipid profile, plasma creatine kinase-MB, lactate dehydrogenase, aspartate transaminase, alanine transaminase, creatinine, electrolytes, and calcium levels, and the total blood count showed a nonsignificant change. However, significant reduction in body-weight gain, food intake, red blood cell count, and hemoglobin content along with higher alkaline phosphatase, bilirubin, and urea levels was observed in mice treated with 3000 mg/kg body weight for 28 d. Since there was no mortality up to a dose of 5000 mg/kg body weight, 50% lethal dose (LD(50)) could not be determined, and hence, it can be assumed that, LD(50) of RC extract is >5000 mg/kg. No observable adverse effect level dose of the RC extract was found to be 2000 mg/kg body weight. Hence, consumption of RC extract for various medicinal purposes is safe. Practical Application: RC is a popularly consumed foodstuff that has been ubiquitously reported to exert medicinal properties. It is mandatory to understand the highest permissible consumption limit of any food supplement to avoid toxicity. This study establishes the safe dose of RC. These results can be of relevance for the scientific fraternity as well as laymen who consume this vegetable or its phytochemical preparation.