Pharmacokinetic and metabolism studies of rohitukine in rats by high performance liquid-chromatography with tandem mass spectrometry.

A sensitive, selective, and rapid high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the quantification of rohitukine in rat plasma. HPLC was performed using a Symmetry-Shield C18 (5 µ, 4.6 × 150 mm) column, and isocratic elution with ammonium acetate buffer (pH4; 10 mM):methanol (08:92, v/v) at a flow rate of 0.6 mL/min. Sample clean-up involved solid phase extraction (SPE) of analyte and internal standard (phenacetin) from 100 µL plasma. The parent→product ion transitions (MRM) for analyte and IS were 306.1→245.1 m/z and 180.1→138.1 m/z respectively, and were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The method was validated across the dynamic concentration range of 5-500 ng/mL for rohitukine, with a fast run time of 4.5 min. The analytical method measured concentrations of rohitukine with accuracy (% bias) of <±10% and precision (% RSD) of <±12%. Rohitukine was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days of storage in a freezer at -70±10°C. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic and in vitro metabolism studies in Sprague-Dawley rat. This method will therefore be highly useful for future preclinical and clinical pharmacokinetic studies of rohitukine.

Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo.

We developed a common feature pharmacophore model using known antiadipogenic compounds (CFPMA). We identified rohitukine, a reported chromone anticancer alkaloid as a potential hit through in silico mapping of the in-house natural product library on CFPMA. Studies were designed to assess the antiadipogenic potential of rohitukine. Rohitukine was isolated from Dysoxylum binacteriferum Hook. to ⬧95% purity. As predicted by CFPMA, rohitukine was indeed found to be an antiadipogenic molecule. Rohitukine inhibited lipid accumulation and adipogenic differentiation in a concentration- and exposure-time-dependent manner in 3T3-L1 and C3H10T1/2 cells. Rohitukine downregulated expression of PPARγ, CCAAT/enhancer binding protein α, adipocyte protein 2 (aP2), FAS, and glucose transporter 4. It also suppressed mRNA expression of LPL, sterol-regulatory element binding protein (SREBP) 1c, FAS, and aP2, the downstream targets of PPARγ. Rohitukine arrests cells in S phase during mitotic clonal expansion. Rohitukine was bioavailable, and 25.7% of orally administered compound reached systemic circulation. We evaluated the effect of rohitukine on dyslipidemia induced by high-fat diet in the hamster model. Rohitukine increased hepatic expression of liver X receptor α and decreased expression of SREBP-2 and associated targets. Rohitukine decreased hepatic and gonadal lipid accumulation and ameliorated dyslipidemia significantly. In summary, our strategy to identify a novel antiadipogenic molecule using CFPMA successfully resulted in identification of rohitukine, which confirmed antiadipogenic activity and also exhibited in vivo antidyslipidemic activity.

Antidyslipidemic effect and antioxidant activity of anthraquinone derivatives from Rheum emodi rhizomes in dyslipidemic rats.

The aim of the present study was to evaluate the antidyslipidemic effect of ethanolic extract of Rheum emodi rhizomes and its constituents in Triton-WR-1339 and high-fat diet (HFD)-induced dyslipidemic rats. In preliminary screening, the ethanolic extract showed significant activity in Triton-treated rats. Bioassay-guided fractionation of the ethanolic extract resulted in the identification of four anthraquinone derivatives, viz. chrysophanol, emodin, chrysophanol 8-O-ß-D-glucopyranoside and emodin 8-O-ß-D-glucopyranoside as active constituents. All these compounds significantly reduced plasma lipid levels. The most active compound emodin showed significant lipid-lowering activity in the HFD-fed model. In addition, these compounds showed significant antioxidant activity. The effect of emodin on enzymes modulating lipid metabolism confirms and supports the efficiency of emodin as a potent antidyslipidemic agent.

Antidyslipidemic and antioxidant effects of novel Lupeol-derived chalcones.

A series of Lupeol-based chalcones have been synthesized aiming to enhance the therapeutic efficacy of parent compound, the novel compounds were evaluated for their antidyslipidemic activity in triton-WR 1339 induced hyperlipidemic rats. Among the ten synthesized chalcones, the most active K4, K8, and K9 reversed the plasma levels of TC by (24, 25, 27 %), phospholipid by (25, 26, 25 %) and triacylglycerol by (27, 24, 24 %) respectively. In addition, the compounds showed significant in vitro antioxidant activity. The lipid lowering activity of these compounds were mediated through lipoprotein lipase activation (12-21 %) and enhanced post-heparin lipolytic activity (15-16 %). The compounds also displayed noteworthy inhibitory effect on 3-hydroxy-3-methyl-glutaryl reductase activity (in vitro). The in vitro effect of the most active compounds on MDI-induced adipogenesis using 3T3-L1 preadipocytes at 10 and 20 µM concentrations showed significant inhibition (20-32 %) of adipogenesis.

Antifilarial activity of marine sponge Haliclona oculata against experimental Brugia malayi infection.

The present study incorporates the findings on in vitro and in vivo antifilarial activity in the marine sponge, Haliclona oculata using an experimental rodent infection of human lymphatic filarial parasite, Brugia malayi. The in vitro antifilarial action was determined on both adult female worms as well as microfilariae using two parameters viz. adverse effect on motility and inhibition in MTT reduction by the treated adult parasite over control worm. The antifilarial activity could be located in the methanol extract and one of its four fractions (chloroform). Bioactivity guided fractionation of chloroform fraction led to localization of in vitro activity in one of its eight chromatographic fractions. Methanol extract, chloroform fraction and one of the chromatographic fractions revealed IC(50) values of 5.00, 1.80, and 1.62µg/ml, respectively when adult B. malayi were exposed to these test samples for 72h at 37°C. Under similar exposure conditions, the IC(50) values for microfilariae were 1.88, 1.72 and 1.19µg/ml, respectively. The active test samples were found to be safe revealing >10 selectivity indices (SI) on the basis of cytotoxicity to Vero cells (monkey kidney cells) and therefore selected for in vivo evaluation against primary (adult B. malayi intraperitoneal transplanted jird) and secondary (subcutaneous infective larvae induced mastomys) screens. In primary jird model, the three test samples at 100mg/kg for five consecutive days by subcutaneous route demonstrated significant macrofilaricidal efficacy to the tune of 51.3%, 64% and 70.7% by methanol extract, chloroform and chromatographic fraction, respectively. The three samples demonstrated 45-50% macrofilaricidal activity with moderate embryostatic effect in secondary model at 5×500, 5×250 and 5×125mg/kg by oral route. Chromatographic fraction possessing highest antifilarial action was primarily found to be a mixture of four alkaloids Mimosamycin, Xestospongin-C, Xestospongin-D and Araguspongin-C in addition to few minor compounds.

Antimalarial activity in Xylocarpus granatum (Koen).

The antimalarial activity of Xylocarpus granatum fruits and their active constituents gedunin and xyloccensin-I were investigated using an in vitro model in this study. The chloroform fraction of X. granatum fruits was found to show promising antimalarial activity using an in vitro model of Plasmodium falciparum. On purification of the active fraction, four pure compounds were isolated and characterised, namely gedunin, photogedunin, xyloccensin-I and palmitic acid. Out of these only gedunin and xyloccensin-I were found to show activity equivalent to the parent active fraction in vitro model.

Antifungal activity of bivittoside-D from Bohadschia vitiensis (Semper).

This study was carried out to investigate the antifungal activity of Bohadschia vitiensis Semper whole body extracts, followed by isolation and characterisation of bioactive molecules. The methanol extract of the B. vitiensis showed promising activity in in vitro models against Candida albicans, Cryptococcus neoformans, Sporothrix schenckii, Trichophyton mentagrophytes, Aspergillus fumigatus and Candida parapsilosis. The antifungal activity was found in aqueous fraction against C. albicans, C. neoformans, S. schenckii, T. mentagrophytes and A. fumigatus. The major compound was purified from the aqueous fraction and was identified as bivittoside-D isolated earlier from the animal. It showed promising results against C. neoformans, C. neoformans, S. schenckii, T. mentagrophytes, A. fumigatus and C. parapsilosis.

Gedunin and photogedunin of Xylocarpus granatum possess antifilarial activity against human lymphatic filarial parasite Brugia malayi in experimental rodent host.

The present study is aimed to evaluate antifilarial activity of Xylocarpus granatum (fruit from Andaman) against human lymphatic filarial parasite Brugia malayi in vivo. The in vitro antifilarial activity has already been reported earlier for this mangrove plant which has traditionally been used against several ailments. Aqueous ethanolic crude extract, four fractions (ethyl acetate fraction, n-butanol fraction, water-soluble fraction and water-insoluble fraction) and pure molecule/s of X. granatum (fruit) were tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active samples were further evaluated in vivo in B. malayi (intraperitoneally) i.p. transplanted in the jird model (Meriones unguiculatus) and Mastomys coucha subcutaneously infected with infective larvae (L3). The crude aqueous ethanolic extract was active in vitro (IC50: adult = 15.46 µg/ml; mf = 13.17 µg/ml) and demonstrated 52.8% and 62.7% adulticidal and embryostatic effect on B. malayi, respectively, in Mastomys at a dose of 5 × 50 mg/kg by oral route. The antifilarial activity was primarily localized in the ethyl acetate-soluble fraction which revealed IC50 of 8.5 and 6.9 µg/ml in adult and mf, respectively. This fraction possessed moderate adulticidal and embryostatic action in vivo in Mastomys. Out of eight pure molecules isolated from the active fraction, two compounds gedunin (IC50 = 0.239 µg/ml, CC50 = 212.5 µg/ml, SI = 889.1) and photogedunin (IC50 = 0.213 µg/ml, CC50 = 262.3 µg/ml, SI = 1231.4) at 5 × 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 80% and 70% transplanted adult B. malayi in the peritoneal cavity of jirds respectively in addition to noticeable microfilaricidalo action on the day of autopsy. The findings reveal that the extract from the fruit X. granatum contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which could be attributed to the presence of two pure compounds gedunin and photogedunin.

In vitro and in vivo antifilarial potential of marine sponge, Haliclona exigua (Kirkpatrick), against human lymphatic filarial parasite Brugia malayi: antifilarial activity of H. exigua.

The present study reports on the antifilarial activity of a marine sponge Haliclona exigua (phylum Porifera). The crude methanol extract and n-butanol-soluble fraction killed adult Brugia malayi at 31.25-microg/ml concentration (both in motility and 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay) while the chloroform fraction was lethal at a lower concentration of 15.6 microg/ml. The activity could be located in a single molecule araguspongin C which brought about mortality of worm at 15.6 microg/ml. In vivo evaluation of the crude extract (5 x 500 mg/kg, orally) and the chloroform fraction (5 x 250 mg/kg, orally) in B. malayi-infected rodent host, Mastomys coucha, did not show any significant microfilaricidal actions; however, microfilarial densities in both the treated groups were significantly much lower than those of untreated group in contrast to standard filaricide diethylcarbamazine which exerted 79% microfilaricidal action on day 8 of treatment. Both these extracts also demonstrated adulticidal (macrofilaricidal) activity which was more pronounced in the chloroform fraction (50.2%). In addition, there was moderate adverse effect on the reproductive potential of female worms (crude extract 46.5%; chloroform 58.6%). The findings suggest that the marine sponge H. exigua possesses adulticidal and embryostatic action against human lymphatic filarial parasite B. malayi in experimental rodent model and this activity could be attributed to the presence of araguspongin C.

Effect of substrates on growth and shelf life of Trichoderma harzianum and its use in biocontrol of diseases.

A study on the effect of different agrowastes on the population density and shelf life of Trichoderma harzianum indicated that all the substrates showed effective propagation of T. harzianum; however, the population count after 30 days was maximum in used tea leaves (8 x 10(8) cfu/g) and shelf life was found to be maximum (2.9 x 10(5) cfu/g after 210 days) in wheat bran-sawdust. The application of these formulations on chickpea and groundnut plants significantly reduced the percent mortality due to chickpea wilt complex and groundnut collar rot disease, respectively.