Evaluation of Cholesterol-lowering Activity of Standardized Extract of Mangifera indica in Albino Wistar Rats.

INTRODUCTION: Cholesterol lowering activity of Mangifera indica L. has been determined by earlier researchers and kernel, leaf and bark have shown significant activity. However, the specific cholesterol lowering activity of leaf methanol extract has not been determined. MATERIALS AND METHODS: The present study involved evaluation of cholesterol lowering potential of methanol extract of M. indica leaves using high cholesterol diet model in albino Wistar rats. The acute oral toxicity at a dose of 5000 mg/ kg body weight was also determined in female albino Wistar rats. Phytoconstituents Iriflophenone 3-C-ß-D-glucoside and mangiferin were quantified in methanol extracts of different varieties of mango leaves using high performance liquid chromatography. RESULTS AND DISCUSSION: Significant cholesterol lowering activity was observed with methanol extract of M. indica leaves, at dose of 90 mg/kg body weight in rats and it was also found to be safe at dose of 5000 mg/kg rat body. Iriflophenone 3-C-ß-D-glucoside and mangiferin were found to be in the range of 1.2 to 2.8% w/w and 3.9 to 4.6% w/w, respectively which along with 3 ß taraxerol and other sterols could be contributing to the cholesterol lowering activity of mango leaves extract. CONCLUSIONS: The phytosterols rich extract of Mangifera indica leaves is a good source of nutraceutical ingredient that have the potential to lower serum cholesterol levels. SUMMARY: The Mangifera indica leaves methanolic extract showed significant cholesterol lowering activity in high cholesterol diet induced hypercholesterolaemia model in rats when evaluated at a dose of 90 mg/kg rat body weight. The extract was found to contain Iriflophenone 3-C-ß-D-glucoside and mangiferin which along with 3 ß taraxerol and other sterols could be contributing to the cholesterol lowering activity.

Effect of Flavonoid-Rich Extract of Glycyrrhiza glabra on Gut-Friendly Microorganisms, Commercial Probiotic Preparations, and Digestive Enzymes.

Flavonoid-rich extract prepared from Glycyrrhiza glabra has been found to be beneficial in patients with functional dyspepsia and was reported to possess some gut health-promoting properties such as antioxidant, anti-inflammatory and anti-Helicobacter pylori activities. In the present study, the flavonoid-rich extract of Glycyrrhiza glabra was evaluated for its compatibility with probiotic strains (Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, and Streptococcus thermophilus), commercial probiotic drinks, and digestive enzymes (pancreatic α-amylase, α-glucosidase, phytase, xylanase, and pancreatic lipase). Results of this study indicated that the flavonoid-rich extract of Glycyrrhiza glabra is compatible with the tested probiotic strains, probiotic drinks and digestive enzymes.

Elucidation of Molecular Mechanism(s) of Cognition Enhancing Activity of Bacomind®: A Standardized Extract of Bacopa Monnieri.

BACKGROUND: Bacopa monnieri (L.) Wettst., commonly known as Brahmi, is renowned in Indian traditional system for its potent memory enhancing activity, which has been validated by various scientific studies. OBJECTIVE: The objective of this study was to understand the molecular mechanism of memory enhancing activity of BacoMind® (BM), a standardized extract of B. monnieri. MATERIALS AND METHODS: BM was screened in vitro in a panel of cell-free and receptor-transfected cell assays. The purified enzymes/membrane homogenates/cells were incubated with substrate/standard ligand in the absence or presence of the test compound. The IC50 values and EC50 values were determined by nonlinear regression analysis of the concentration-response curves generated with mean replicate values using Hill equation curve fitting. RESULTS: BM was found to inhibit three enzymes; Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also had an antagonistic effect on serotonin 6 and 2A (5-HT6 and 5-HT2A) receptors, known to influence the different neurological pathways, associated with memory and learning disorders, age-associated memory impairment. CONCLUSION: BM was found to inhibit three enzymes namely, Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also exhibited an antagonistic effect on 5-HT6 and 5-HT2A receptors. SUMMARY: This study was conducted to understand the molecular mechanism of memory enhancing activity of a standardized extract of B. monnieri by was screening it in vitro in a panel of cell-free and receptor-transfected cell assays. The purified enzymes/membrane homogenates/cells were incubated with substrate/standard ligand in the absence or presence of the test compound. BM was found to inhibit three enzymes; Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also had an antagonistic effect on serotonin6 and2A (5-HT6 and 5-HT2A) receptors, known to influence the different neurological pathways, associated with memory and learning disorders, age-associated memory impairment. Abbreviations used: HTRF: Homogenous time resolved fluorescence, cAMP: Cyclic adenosine monophosphate, CHO: Chinese hamster ovary, RFU: Relative fluorescence unit, pNP: Para nitro phenol, AMC: 7-amino-4-methylcoumarin, ELISA: Enzyme linked immunosorbent assay, Z-Pro-Pro-CHO: Z-prolyl-prolinal, HEK: Human embryonic kidney, TE: Trolox equivalent.

Erectogenic and Aphrodisiac Property of Moringa oleifera: Involvement of Soluble Epoxide Hydrolase Enzyme.

Soluble epoxide hydrolase (sEH) inhibitors have been reported to improve penile erection; therefore, sEH could be useful for management of erectile dysfunction. Methanolic and aqueous extracts of 30 Indian medicinal plants were screened for their sEH inhibition potential. Fifteen extracts showed >50% inhibition when screened at 50 µg/mL in sEH inhibition assay. Methanolic extract of Moringa oleifera Lam. (Moringaceae) seeds (MEMO) was most potent with IC50 1.7 ± 0.1 µg/mL and was selected for in vitro studies on isolated rat corpus cavernosum smooth muscle and in vivo sexual behaviour studies on healthy and diabetic rats. Rats were divided into five groups, each containing six animals and treated orally with either water, vehicle (1% Tween-20), MEMO (45 and 90 mg/kg/day for 21 days), and standard drug, sildenafil (5 mg/kg/day for 7 days). An equal number of female rats were used, and the effect of MEMO and sildenafil was compared with that of vehicle. MEMO significantly relaxed isolated rat corpus cavernosum smooth muscle at 0.1-100 µg/mL in vitro and significantly increased (p < 0.05) sexual activity, intracavernous pressure/mean arterial pressure in normal and diabetic rats. The increase in erectile function of rats by MEMO could be because of its sEH inhibitory activity. Copyright © 2016 John Wiley & Sons, Ltd.

Aldose reductase and protein tyrosine phosphatase 1B inhibitory active compounds from Syzygium cumini seeds.

CONTEXT: Syzygium cumini (L.) Skeels (Myrtaceae), commonly known as jamun, is an Indian plant, traditionally well known for its medicinal properties including antidiabetic activity. OBJECTIVE: To isolate the antidiabetic compounds from Syzygium cumini seeds and evaluate their activity using aldose reductase (AR) and protein-tyrosine phosphatase 1B (PTP1B) inhibition assays. MATERIALS AND METHODS: The dried seeds were extracted with methanol and partitioned with ethyl acetate, butanol, and water. The extracts were screened for antidiabetic activity at a concentration of 100 µg/mL using in vitro AR and PTP 1B inhibition assays. RESULTS AND DISCUSSION: The highly enriched fractions obtained from broad ethyl acetate fraction yielded maslinic acid (1), 5-(hydroxymethyl) furfural (2), gallic acid (3), valoneic acid dilactone (4), rubuphenol (5), and ellagic acid (6). Structures were elucidated by (1)H-NMR and (13)C-NMR. The initial ethyl acetate fraction showed AR inhibitory activity with the IC50 value of 2.50 µg/mL and PTP1B enzyme inhibition with the IC50 value of 26.36 µg/mL. Compounds 3, 4, 5, and 6 were found to inhibit AR with IC50 values of 0.77, 0.075, 0.165, and 0.12 µg/mL while the compounds 4, 5, and 6 inhibited PTP1B with IC50 values of 9.37, 28.14, and 25.96 µg/mL, respectively. CONCLUSION: The results of this study demonstrate that the isolated constituents show promising in vitro antidiabetic activity and, therefore, can be candidates for in vivo biological screening using relevant models to ascertain their antidiabetic activity.

Effect of Cinnamomum cassia methanol extract and sildenafil on arginase and sexual function of young male Wistar rats.

INTRODUCTION: Herbs have been used as an aphrodisiac since ages. Cinnamomum cassia is an important ingredient of many Ayurvedic formulations to treat male sexual disorder including erectile dysfunction (ED). AIM: The objective of the present study was to evaluate erectogenic and aphrodisiac activity of methanol extract of C. cassia bark in young male rats. METHODS: Methanol extract of C. cassia was screened in vitro for arginase inhibition potential and IC50 was determined. Effect of the extract was observed in vitro on phenylephrine pre-contracted isolated rat corpus cavernosum smooth muscle (CCSM) at 0.1, 1, 10, and 100 µg/mL. Young male Wistar rats were dosed with extract at 100 mg/kg body weight for 28 days and its effects on sexual behavior and penile smooth muscle : collagen level were observed. MAIN OUTCOME MEASURE: Effect of C. cassia was studied on arginase activity in vitro and sexual behavior of young male rats. RESULTS: C. cassia inhibited arginase activity in vitro with an IC50 of 61.72 ± 2.20 µg/mL. The extract relaxed phenylephrine pre-contracted isolated rat CCSM up to 43% and significantly increased (P < 0.05) sexual function of young male rats. Treatment with the extract also increased smooth muscle level and decreased collagen level in rat penile tissue. CONCLUSION: The study proves usefulness of methanol extract of C. cassia bark for increasing sexual function.

Use of in vitro bioassays for assessing botanicals.

In vitro bio assays can play a vital role in evaluating botanicals ranging from comparative screening, interaction studies, bio-activity guided fractionation, biological characterization, assisting in stability studies to studying mechanism of actions. In this review, we present some challenges and common pitfalls of using bioassays for assessing botanicals including guidance to overcome them. The overall objective of this review is to improve the success of botanicals products by incorporating robust bioassays during various stages of research and development.

Cholesterol esterase inhibitory activity of bioactives from leaves of Mangifera indica L.

BACKGROUND: In the earlier studies, methanolic extract of Mangifera indica L leaf was exhibited hypocholesterol activity. However, the bioactive compounds responsible for the same are not reported so far. OBJECTIVE: To isolate the bioactive compounds with hypocholesterol activity from the leaf extract using cholesterol esterase inhibition assay which can be used for the standardization of extract. MATERIALS AND METHODS: The leaf methanolic extract of M. indica (Sindoora variety) was partitioned with ethyl acetate and chromatographed on silica gel to yield twelve fractions and the activity was monitored by using cholesterol esterase inhibition assay. Active fractions were re-chromatographed to yield individual compounds. RESULTS AND DISCUSSION: A major compound mangiferin present in the extract was screened along with other varieties of mango leaves for cholesterol esterase inhibition assay. However, the result indicates that compounds other than mangiferin may be active in the extract. Invitro pancreatic cholesterol esterase inhibition assay was used for bioactivity guided fractionation (BAGF) to yield bioactive compound for standardization of extract. Bioactivity guided fractionation afford the active fraction containing 3b-taraxerol with an IC50 value of 0.86µg/ml. CONCLUSION: This study demonstrates that M. indica methanol extract of leaf have significant hypocholesterol activity which is standardized with 3b-taraxerol, a standardized extract for hypocholesterol activity resulted in development of dietary supplement from leaves of Mangifera indica.

Erectogenic and Aphrodisiac Effects of Butea frondosa Koenig ex Roxb. in Rats: Involvement of Enzyme Inhibition.

Butea frondosa Koenig ex Roxb. (BF) is traditionally used to manage male sexual disorders including erectile dysfunction (ED). Methanol extract of BF (bark) inhibited Rho-kinase 2 (ROCK-II) enzyme activity in vitro with an IC50 of 20.29 ± 1.83 µ g/mL. The relaxant effect of methanol extract of BF (MEBF) was studied on phenylephrine precontracted corpus cavernosum smooth muscle (CCSM) isolated from young rats. The effect of MEBF treatment on sexual behaviour of both young (5 month) and aged (24 month) rats was also studied in addition to the influence on smooth muscle, collagen (collagen-I and -III) level in penis, and sperm characteristics of young and aged rats. MEBF relaxed CCSM up to 21.77 ± 2.57% and increased sexual behavior of young and aged rats. This increase in sexual function could be attributed to ROCK-II inhibition and increase in ratio of smooth muscle to collagen level in rat penile tissue. Increased sperm production and decreased defective sperms in young and aged rats corroborate the usefulness of Butea frondosa in male infertility in addition to ED.

Clitoria ternatea ameliorated the intracerebroventricularly injected streptozotocin induced cognitive impairment in rats: behavioral and biochemical evidence.

RATIONALE: Clitoria ternatea, commonly known as Aparajita, is used as Medhya rasayana in Ayurveda. The role of C. ternatea in experimental models of cognitive impairment is yet to be explored. OBJECTIVES: The present study was designed to study the effect of aqueous and hydroalcoholic extracts of C. ternatea on biochemical and behavioral parameters related to cognitive impairment in in vitro and in vivo studies. METHODS: In vitro free radical scavenging and enzyme-inhibitory (cholinesterase, glycogen synthase kinase-3-ß, rho kinase, prolyl endopeptidase, catechol-O-methyl transferase, and lipoxygenase) activities of aqueous and hydroalcoholic extracts of C. ternatea plant were evaluated. Based on in vitro results, hydroalcoholic extract of C. ternatea (100, 300, and 500 mg/kg, p.o.) was selected for evaluation in intracerebroventricularly injected streptozotocin (STZ)-induced cognitive impairment in male Wistar rats. Behavioral assessment was performed at baseline and on the 14th, 21st, and 28th days after STZ injection using elevated plus maze, passive avoidance, Morris water maze, and photoactometer. Oxidative stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels, and superoxide dismutase activity), cholinesterase activity, and rho kinase (ROCK II) expression were studied in cerebral cortex and hippocampus of rats' brain at the end of the study. RESULTS: The hydroalcoholic extract possessed significantly more in vitro antioxidant and enzyme-inhibitory activities as compared to aqueous extract. The hydroalcoholic extract of C. ternatea prevented STZ-induced cognitive impairment dose dependently by reducing oxidative stress, cholinesterase activity, and ROCK II expression. CONCLUSION: In vitro and in vivo results suggest the potential of hydroalcoholic extract of C. ternatea for treatment of cognitive deficit in neurological disorders.

In vitro anti-Helicobacter pylori activity of a flavonoid rich extract of Glycyrrhiza glabra and its probable mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza glabra Linn. is regarded useful for peptic ulcer in traditional systems of medicine in India and Helicobacter pylori has been considered as one of the causative factors for peptic ulcer. Aim of the present study is to evaluate the anti-Helicobacter pylori action of GutGard(®), a flavonoid rich extract of Glycyrrhiza glabra and further to elucidate the possible mechanisms of its anti-Helicobacter pylori action. MATERIALS AND METHODS: Agar dilution and microbroth dilution methods were used to determine the minimum inhibitory concentration of GutGard(®) against Helicobacter pylori. Protein synthesis, DNA gyrase, dihydrofolate reductase assays and anti-adhesion assay in human gastric mucosal cell line were performed to understand the mechanisms of anti-Helicobacter pylori activity of GutGard(®). RESULTS: GutGard(®) exhibited anti-Helicobacter pylori activity in both agar dilution and microbroth dilution methods. Glabridin, the major flavonoid present in GutGard(®) exhibited superior activity against Helicobacter pylori while glycyrrhizin did not show activity even at 250 µg/ml concentration. In protein synthesis assay, GutGard(®) showed a significant time dependent inhibition as witnessed by the reduction in (35)S methionine incorporation into Helicobacter pylori ATCC 700392 strain. Additionally, GutGard(®) showed a potent inhibitory effect on DNA gyrase and dihydrofolate reductase with IC(50) value of 4.40 µg/ml and 3.33 µg/ml respectively. However, the extract did not show significant inhibition on the adhesion of Helicobacter pylori to human gastric mucosal cell line at the tested concentrations. CONCLUSION: The present study shows that, GutGard(®) acts against Helicobacter pylori possibly by inhibiting protein synthesis, DNA gyrase and dihydrofolate reductase.

Efficacy of Cinnamomum cassia Blume. in age induced sexual dysfunction of rats.

OBJECTIVE: Cinnamomum cassia has been suggested in Ayurveda for the management of sexual dysfunction. This research work was conducted to shed some light on the mechanism of action of the extract, and evaluate the efficacy of its methanol extract in age induced sexual dysfunction in male Wistar rats. Secondary objective of the project was to study the effect of treatment on sperm parameters and smooth muscle:collagen level in rat penile tissue. METHODS: Young and aged male rats were treated with methanol extract of Cinnamomum cassia at a dose of 100 mg/kg and sexual behavior was observed on 28th day in presence of female rats in estrous phase. Sildenafil was used as standard medicine. Effect of treatment was studied on epididymal sperm parameters, and Massons trichrome staining of rat penile tissues was performed to know the level of smooth muscle:collagen. RESULTS: The treatment significantly increased sexual function in aged rats that had decreased in comparison to young rats, but did not have any significant effect on sperm count, live and defective sperm percentage. However, treatment induced an increase in smooth muscle level and a decrease in collagen level in the aged rat penile tissue in comparison to that of age matched control. CONCLUSION: Based on our studies, we found that Cinnamomum cassia extract was effective in management of sexual dysfunction in aged rats and hence we propose a possible mechanism of action for Cinnamomum cassia which could be responsible for restoring sexual activity in aged rat.

Screening for Rho-kinase 2 inhibitory potential of Indian medicinal plants used in management of erectile dysfunction.

THE AIM OF THE STUDY: Activation of Rho-kinase 2 (ROCK-II) results in contraction of corpus cavernosum smooth muscle and ROCK-II inhibitors relax corpus cavernosum in vitro and in vivo hence, plant extracts capable of inhibiting ROCK-II enzyme may be useful in management of erectile dysfunction (ED). The aim of the study was to screen selected Indian medicinal plants, having similar ethnopharmacological use for ROCK-II inhibition. MATERIALS AND METHODS: Some Indian medicinal plants reported as aphrodisiacs in Ayurveda and modern scientific literature were collected, authenticated and extracted. Direct methanol and successive aqueous extracts of these plants were screened for ROCK-II inhibitory activity using HTRF(®)KinEASE™ STK S2 Kit (Cisbio Bioassays). Relaxant effect of potent extract was recorded on isolated rat corpus cavernosum. RESULTS: Methanolic and successive aqueous extracts of 30 plants were screened for ROCK-II inhibition and 15 herbal extracts showed inhibition ranging between 50 and 88% at 50 µg/mL. While IC(50) of Y-27632, a standard ROCK-II inhibitor, was found to be 163.8 ± 1.2 nM. The Methanolic extract of Terminalia chebula (METC) with IC(50) value of 6.09 ± 0.17 µg/mL was found to be most potent and relaxed isolated rat corpus cavernosum significantly (p<0.01). Chebulagic and chebulinic acid of METC were found to inhibit ROCK-II and might be responsible for the inhibitory potential of the extract. The traditional use of plants like Butea frondosa, Syzygium aromaticum, Butea superba, Chlorophytum borivilianum and Mucuna pruriens, as aphrodisiacs and for male sexual disorder (MSD) might be in part due to the ROCK II inhibitory potential of these plants. CONCLUSION: Some of the Indian medicinal plants have ROCK-II inhibitory potential and those deserve further investigation.

Amelioration of intracerebroventricular streptozotocin induced cognitive impairment by Evolvulus alsinoides in rats: in vitro and in vivo evidence.

Evolvulus alsinoides, also known as Shankpushapi, is a commonly used traditional medicine for enhancing memory. We evaluated the in vitro free radical scavenging and enzymes [acetylcholinesterase, butyrylcholinestrase, glycogen synthase kinase-3-ß (GSK-3-ß), rho kinase (ROCK II), prolyl endopeptidase (PEP), catechol-O-methyl transferase (COMT) and lipoxygenase (LOX)] inhibitory activities of aqueous and hydro-alcoholic extracts of E. alsinoides. Hydro-alcoholic extract of E. alsinoides demonstrated more free radical scavenging activity as compared to aqueous extract. Hydro-alcoholic extract also showed higher cholinesterase, GSK-3-ß, ROCK II, PEP, COMT and LOX enzyme inhibitory activities as compared to aqueous extract. Phytochemical analysis revealed more flavanoids in hydro-alcoholic extract as compared to aqueous extract but no significant difference in phenolic content of the two extracts was observed. Based on in vitro data, hydro-alcoholic extract (100, 300 and 500mg/kg, p.o.) was selected for in vivo study in intracerebroventricularly injected streptozotocin (STZ) induced cognitive impairment in male Wistar rats. Elevated plus maze, passive avoidance and Morris water maze were used for assessment of cognitive function on 14th, 21st and 28th day after STZ injection. Oxidative stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels and superoxide dismutase activity), cholinergic dysfunction and rho kinase (ROCK II) expression were studied in cerebral cortex and hippocampus of rat brain at the end of the study. Hydro-alcoholic extract of E. alsinoides dose dependently prevented STZ induced cognitive impairment by reducing the oxidative stress, improving cholinergic function and preventing the increase in rho kinase expression. The results suggest an anti-Alzheimer potential of hydro-alcoholic extract of E. alsinoides.

Hepatoprotective and antioxidant activity of standardized herbal extracts.

BACKGROUND: Phyllanthus emblica, Camellia sinensis, Mangifera indica, Punica granatum, and Acacia catechu have been shown to possess widespread pharmacological application against multitude of diseases namely cancer, diabetes, liver disorders, and oxidative stress. OBJECTIVE: We evaluated the hepatoprotective activity of the standardized herbal extracts against tert-butyl hydroperoxide (t-BH) induced toxicity and their mechanism of hepatoprotective action in human hepatocarcinoma cells (HepG2 cell line). MATERIALS AND METHODS: The hepatoprotective activity was studied by observing the effect of these herbal extracts on t-BH induced reduction in cell viability of HepG2 cells. In addition, the reducing power of the extracts and their ability to scavenge free radicals were evaluated using two antioxidant assay systems: cell free [oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)] (ABTS)] and cell based [cellular antioxidant activity (CAA)]. RESULTS AND DISCUSSION: The results obtained showed that these extracts possess significant hepatoprotective activity. This may indicate that the plant extracts contain compounds, which can remove toxic metabolites following t-BH induced toxicity. The extracts exhibited significant antioxidant property as evident by the Trolox values and effective scavenging of DPPH and ABTS radicals. The extracts also demonstrated inhibition of AAPH-induced fluorescence in HepG2 cells. These results indicate the ability of the plant extracts to protect the liver cells from chemical-induced damage, which might be correlated to their radical scavenging potential. CONCLUSION: This study demonstrates that these extracts have potential hepatoprotective activity which is mainly attributed to the antioxidant potential, which might occur by reduction of lipid peroxidation and cellular damage.