Exploring the Cytotoxic Potential of Triterpenoids-enriched Fraction of Bacopa monnieri by Implementing In vitro, In vivo, and In silico Approaches.

BACKGROUND: Bacopa monnieri (BM) is a herbaceous plant traditionally used from time immemorial in Ayurvedic and folklore medicines. We hypothesized that the extract of the whole plant might contain numerous molecules with having antitumor activities that could be very effective in killing of human cancer cells. OBJECTIVES: This work investigated anticancer activity of bioactive fraction of BM. MATERIALS AND METHODS: The hydroalcoholic extract of BM was fractionated with different solvent, namely, hexane, dichloromethane (DCM), acetone, methanol, and water. The in vitro anticancer activity was performed against various Human Cancer Cell lines, namely, Colon (HT29, Colo320, and Caco2), Lung (A549), Cervix (HeLa, SiHa), and Breast (MCF-7, MDAMB-231). Further, DCM fraction was evaluated in vivo for anticancer activity against Ehrlich ascites carcinoma (EAC) tumor-bearing mice since it showed the best cytotoxicity at 72 h (IC50 41.0-60.0 µg/mL). The metabolic fingerprinting of these extract were carried out using high-performance thin-layer chromatography along with quantification of bacoside A, bacoside B, cucurbitacin B, cucurbitacin E, and bittulinic acid. RESULTS: Oral administration of DCM fraction at a dose of 40 mg/kg rendered prominent reduction of tumor regression parameters such as tumor weight, packed cell volume, tumor volume and viable tumor cell count as compared to the untreated mice of the EAC control group. The anticancer activity of DCM fraction may be due to the presence of large amount of bacoside A, B and cucurbitacins. The molecular docking studies of major metabolites with targeted proteins predicted the anticancer activity of DCM fraction which was in support of in vivo activity. CONCLUSION: The in vitro, in vivo, analytical and in silico studies on DCM fraction of Bacopa monieri has proved its great potential for development of anticancer phytopharmaceuticals. SUMMARY: A new HPTLC method has been developed and validated for the qualitative and quantitative analysis of bacoside A, B, cucurbitacin B, D, E and bittulinic acid in Bacopa monnieri extract. Enrichment of active anticancer metabolites was done by polarity based fractionations of hydroalcoholic extract of Bacopa. DCM fraction of a hydroalcoholic extract of Bacopa showed anticancer potential against human cancer cell line (IC50 41.0-60.0 µg/mL) and in EAC treated mice (at a dose of 40 mg/kg body weight). The anticancer activity of Bacopa may be due to the presence of bacosides and cucurbitacin and it was confirmed by in silico screening. Abbreviations used: DBM: DCM fraction of Bacopa monnieri; DCM: Dichloromethane; EAC: Ehrlich ascites carcinoma; HCT: Hematocrit; HGB: Hemoglobin; HPTLC: High performance thin layer chromatography; ICH: International council for Harmonisation; LOD: Limit of detection; LOQ: Limit of quantification; LYM: Lymphocytes; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular haemoglobin concentration (MCHC); MCV: Mean corpuscular volume; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PLT: Platelet; RBC: Red blood cell; RDW: Red blood cell distribution width; RSD: Relative standard deviation; WBC: White blood cells.

Pharmacokinetics and comparative metabolic profiling of iridoid enriched fraction of Picrorhiza kurroa - An Ayurvedic Herb.

ETHNO-PHARMACOLOGICAL RELEVANCE: Picrosides I, II and apocynin are the main active principles present in the roots and rhizomes of Picrorhiza kurroa Royle ex. Benth (Kutki). Ethno-medicinally, the plant is used for the treatment of liver, upper respiratory tract disorders and dyspepsia, since long in Ayurveda. AIM OF THE STUDY: This study attempts to determine the pharmacokinetic profile of picrosides I, II and apocynin in rats after oral administration of iridoid enriched fraction (IRF) and to recognize the pattern of its metabolites as such in IRF and in plasma. MATERIALS AND METHODS: A simple, precise, specific and sensitive RP-HPLC method was developed for simultaneous quantification of picrosides I, II and apocynin in rat plasma and in plant extract. Acetonitrile (ACN) and water was used as a solvent system with a gradient elution for pharmacokinetic studies using HPLC-PDA (Flow rate: 1.0mL/min) and metabolic profiling through UPLC-MS (Flow rate: 0.5mL/min) in selected reaction monitoring. A comparative study was performed in order to recognize the pattern and fate of metabolites in rat plasma up to 24h after single oral administration of IRF. RESULTS: Developed method produced more than 85% recovery of the targeted metabolites in rat plasma. The content of picrosides I, II and apocynin in IRF were found 5.7%, 18.3% and 27.3% w/w, respectively. The mean plasma concentration versus time profiles of picroside I, II and apocynin resulted in peak plasma concentration (Cmax) 244.9, 104.6 and 504.2ng/mL with half-life (t1/2) 14, 8 and 6h, respectively. Other pharmacokinetic parameters such as time to reach Cmax (tmax), area under curve (AUC), absorption (ka) and elimination (ke) constant, volume of distribution (Vd) were also determined. Pattern recognition analysis showed fate of 18 metabolites in rat plasma up to 24h out of 26 present in IRF. CONCLUSION: The information gained from this study postulates the basic pharmacokinetic profiling of picroside I, II and apocynin as well as fate of other metabolites after oral administration of IRF, demonstrating scientific basis of its traditional use in Ayurveda.

Evaluation of anticancer potential of Bacopa monnieri L. against MCF-7 and MDA-MB 231 cell line.

BACKGROUND: The ethanolic extract of Bacopa monnieri contains bacoside A and B, brahmin, cucurbitacins, and betulinic acid. Currently, cucurbitacins have also been reported for their strong anti-tumorigenic and anti-proliferative activity by inducing cell cycle arrest at the G2/M phase and formation of multiplied cells. The present study was carried out to evaluate the in vitro cytotoxic activity of ethanolic extract of dichloromethane (DCM) fraction of B. monnieri on two different cell lines. MATERIALS AND METHODS: The ethanolic extract of B. monnieri was prepared using soxhlet extraction method and different fractions (hexane, DCM, methanol, acetone, and water) of ethanolic extracts were prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of ethanolic extract and of all fractions was carried out on MCF-7 and MDA-MB 231 cell lines. The presence of cucurbitacins and betulinic acid in these fractions was confirmed by high-performance thin layer chromatography. RESULTS: The IC50 values of ethanolic extract of B. monnieri in MCF-7 and MDA-MB 231 cell lines were 72.0 µg/mL and 75.0 µg/mL, respectively. The DCM fraction of B. monnieri showed maximum cytotoxic activity among all fraction upto 72 h and was found to be 57.0 µg/mL and 42.0 µg/mL, respectively. CONCLUSION: The results showed good cytotoxic activity in DCM fraction in both the cell lines may be due to the presence of cucurbitacins and betulinic acid in DCM fraction.

HPTLC Analysis of Bioactivity Guided Anticancer Enriched Fraction of Hydroalcoholic Extract of Picrorhiza kurroa.

OBJECTIVE: Hydroalcoholic extract of Picrorhiza kurroa and its fractions were subjected to in vitro screening for cytotoxicity; further best active fraction (BAF) obtained was tested against Ehrlich ascites carcinoma (EAC) model in Balb/c mice after its quality control analysis. METHODS: Cytotoxicities of all the fractions and mother extract of P. kurroa were determined, using MTT assay on breast cancer (MCF-7, MDA-MB 231) and cervical cancer (HeLa, SiHa) cell lines. Metabolic fingerprinting was developed using HPTLC with quantification of biomarkers (cucurbitacins B and E; betulinic acid; picrosides 1 and 2; and apocynin) in BAF. The EAC tumor-bearing mice were used for in vivo anticancer activity after oral administration (50 mg Kg(-1)) for 10 days. RESULTS: Cytotoxicity assay of mother extract and its fractions over breast cancer and cervix cancer cell lines showed that dichloromethane (DCM) fraction was most cytotoxic (IC50 36.0-51.0 µg mL(-1) at 72 h). Oral administration of DCM fraction showed significant reduction in tumor regression parameters, viable tumor cell count and restoration of hematological parameters may be due to presence of cucurbitacins B and E; betulinic acid; picrosides 1 and 2; and apocynin, as compared to the untreated mice of the control group. CONCLUSION: The DCM fraction of P. kurroa displayed potent anticancer activity and can be further explored for the development of a potential candidate for cancer therapy.

Development of a novel synergistic thermosensitive gel for vaginal candidiasis: an in vitro, in vivo evaluation.

The singular aim of the proposed work is the development of a synergistic thermosensitive gel for vaginal application in subjects prone to recurrent vaginal candidiasis and other microbial infections. The dual loading of Itraconazole and tea tree oil in a single formulation seems promising as it would elaborate the microbial coverage. Despite being low solubility of Itraconazole in tea tree oil, a homogeneous, transparent and stable solution of both was created by co-solvency using chloroform. Complete removal of chloroform was authenticated by GC-MS and the oil solution was used in the development of nanoemulsion which was further translated into a gel bearing thermosensitive properties. In vitro analyses (MTT assay, viscosity measurement, mucoadhesion, ex vivo permeation, etc.) and in vivo studies (bioadhesion, irritation potential and fungal clearance kinetics in rat model) of final formulation were carried out to establish its potential for further clinical evaluation.