Ring A structural modified derivatives of withaferin A and the evaluation of their cytotoxic potential.

Regio-/stereoselective Michael addition to ring A of withaferin-A was performed using an optimized reaction procedure to synthesise a library of 2,3-dihydro,3-ß-substituted withaferin-A derivatives. The analogues thus obtained were evaluated for in vitro cytotoxicity against various human cancer cell lines. 3-Azido analogue exhibited 35-fold increase (IC(50)=0.02-1.9 µM) in cytotoxicity against almost the entire cell lines tested when compared to the parent molecule. However, further modifications of 3-azido analogue with various alkynes under Husigen's cycloaddition conditions generated a variety of triazole derivatives with reduced cytotoxicity.

Inhibition in radiation mediated cellular toxicity by minimizing free radical flux: one of the possible mechanisms of biological protection against lethal ionizing radiation by a sub- fraction of Podophyllum hexandrum.

The study has focused on exploring the mechanism of action of Podophyllum hexandrum sub-fraction (G-001M) exhibiting >90% protection in lethally irradiated mice. Currently, G-001M was assessed for antioxidant characteristics by evaluating DPPH, superoxide and hydrogen peroxide radical formation, anti-lipid per oxidation, metal chelation and total flavonoid content. To affirm cytoprotective efficacy of G-001M, plasmid DNA protection, blood WBC counts, marker for lipid peroxidation (MDA) and antioxidant status (GSH) in mice splenocytes and thymocytes were studied. G-001M, having high amount of total phenolic contents (200±10mg, w/w), exhibited dose dependent inhibition in DPPH and superoxide radical formation. Hydrogen peroxide radical scavenging was higher than standards. With pre-treatment of G-001M, plasmid DNA was also maximally restored to supercoiled form. Radiation modulated MDA and GSH values in splenocytes and thymocytes of mice altered significantly after 24 hrs and at later intervals, values were close to the controls. Radiation mediated losses in WBC counts were significantly regained (p<0.001) in G-001M pre-treated irradiated mice. The above findings explicitly conveyed that G-001M has successfully minimized radiation inflicted free radicals generation and their multiplication. This activity of G-001M could be undoubtedly among one of the major modes of action in extending whole body survival in lethally irradiated mice.

Studies on novel D-ring substituted steroidal pyrazolines as potential anticancer agents.

An efficient and facile synthesis of 17-pyrazolinyl derivatives of pregnenolone and their evaluation as potential anticancer agents against various human cancer cell lines are reported. The scheme involves the transformation of the starting pregnenolone acetate into pregnenolone, conversion of pregnenolone to the corresponding benzylidine derivatives and finally the conversion of this derivative to the stable steroidal 17-pyrazoline. Various compounds 4b, 4c, 4e, 4f, 4h and 4j showed significant cytotoxic activity especially against HT-29, HCT-15, 502713 cell lines.

Modulation of P-glycoprotein ATPase activity by some phytoconstituents.

In the present investigation 16 phytoconstituents, which are active moieties found in several medicinal herbs, have been evaluated for their P-glycoprotein (P-gp) stimulation/inhibition profiles using a P-gp-dependent ATPase assay in rat jejunal membrane (in vitro). Acteoside, agnuside, catechin, chlorogenic acid, picroside -II and santonin showed an inhibitory effect. Negundoside, picroside -I and oleanolic acid caused a stimulatory effect. Andrographolide, apocyanin, berberine, glycyrrhizin, magniferin and piperine produced a biphasic response (stimulation at low concentration and inhibition at high concentration). The results suggested that a possible interaction of these phytoconstituents at the level of P-gp, could be an important parameter in determining their role in several key pharmacodynamic events.

Production dynamics of Withaferin A in Withania somnifera (L.) Dunal complex.

Withaferin A (WA) is an important withanolide holding promise in cancer treatment and as a relatively safe radiosensitive/chemotherapeutic agent, which is present in traces in all parts of Withania somnifera except the leaves, where as it is reported to be present in only two non-Indian chemotypes (South African chemotype/Israel chemotype 1). The present studies have marked its presence in all Indian populations (wild/cultivated), as well as two identified Indian chemotypes (AGB002 and AGB025). The quantitative dynamics of WA production in Indian populations and interchemotypic hybrids developed at our institute have been studied, and the results were compared with five previously reported chemotypes from Israel, South Africa and India. An analysis on inheritance characteristics based on presence/absence of WA in hybrid plants and their respective parents is given for future studies on the chemogenetics of this complex species in greater detail. Further, the production potential of WA in vitro propagated plants of elite varieties developed at our institute is discussed, in view of maintaining chemotypic fidelity and stability from a production point of view. Also, evidence-based clues suggesting the leaves as the site of the synthesis of WA is provided.

Immunomodulatory activity of Asparagus racemosus on systemic Th1/Th2 immunity: implications for immunoadjuvant potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Roots of Asparagus racemosus Willd (Shatavari in vernacular) are widely used in Ayurveda as Rasayana for immunostimulation, galactogogue as also in treatment of conditions like ulcers and cancer. Various studies have indicated immunomodulatory properties of Shatavari root extracts and formulations. AIM OF THE STUDY: To study the effect of standardized Asparagus racemosus root aqueous extract (ARE) on systemic Th1/Th2 immunity of SRBC sensitized animals. MATERIALS AND METHODS: We used HPTLC to quantify steroidal saponins (Shatavarin IV, Immunoside) and flow cytometry to study effects of ARE on Th1/Th2 immunity. SRBC specific antibody titres and DTH responses were also monitored as markers of Th2 and Th1 responses, respectively. We also studied lymphocyte proliferation. Cyclosporin, cyclophosphamide and levamisole were used as controls. RESULTS: Treatment with ARE (100mg/(kg b.w.p.o.)) resulted in significant increase of CD3(+) and CD4/CD8(+) percentages suggesting its effect on T cell activation. ARE treated animals showed significant up-regulation of Th1 (IL-2, IFN-g) and Th2 (IL-4) cytokines suggesting its mixed Th1/Th2 adjuvant activity. Consistent to this, ARE also showed higher antibody titres and DTH responses. ARE, in combination with LPS, Con A or SRBC, produced a significant proliferation suggesting effect on activated lymphocytes. CONCLUSION: The study suggests mixed Th1/Th2 activity of ARE supports its immunoadjuvant potential.

A new vaccine adjuvant (BOS 2000) a potent enhancer mixed Th1/Th2 immune responses in mice immunized with HBsAg.

Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component. In search of a potent vaccine adjuvant, the water-soluble biopolymeric fraction BOS 2000 from Boswellia serrata was evaluated for desired activity. We investigated the ability of BOS 2000 to enhance HBsAg specific immune responses. The effect was determined in the form of protective anti-HBsAg titers, neutralizing antibodies (IgG1 and IgG2a), spleen cell lymphocyte proliferation by using MTT assay, Th1 (IFN-gamma and TNF-alpha) and Th2 (IL-4) cytokines as well as T-lymphocyte subsets (CD4/CD8) and intracellular cytokines (IFN-gamma/IL-4), these responses were highest in BOS 2000 immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of adjuvant by 18.1-fold in comparison to alum, total IgG and its subtypes (IgG1 and IgG2a) antibodies titer in serum was significantly enhanced. Analysis of HBsAg specific cytokines revealed that alum was associated with a predominantly IL-4 response. In contrast, BOS 2000 was associated with production of both IFN-gamma and IL-4. We conclude that BOS 2000 is a potent enhancer of antigen-specific Th1 and Th2 immune responses in comparison to alum with Th2 limitation and is a promising adjuvant for vaccine applications.

Hepatoprotective potential of Aloe barbadensis Mill. against carbon tetrachloride induced hepatotoxicity.

Aloe barbadensis Mill. Syn. Aloe vera Tourn. ex Linn.(Liliaceae) has been used in variety of diseases in traditional Indian system of medicine in India and its use for hepatic ailments is also documented. In the present study an attempt has been made to validate its hepatoprotective activity. The shade dried aerial parts of Aloe barbadensis were extracted with petroleum ether (AB-1), chloroform (AB-2) and methanol (AB-3). The plant marc was extracted with distilled water (AB-4). All the extracts were evaluated for hepatoprotective activity on limited test models as hexobarbitone sleep time, zoxazolamine paralysis time and marker biochemical parameters. AB-1 and AB-2 were observed to be devoid of any hepatoprotective activity. Out of two active extracts (AB-3 and AB-4), the most active AB-4 was studied in detail. AB-4 showed significant hepatoprotective activity against CCl4 induced hepatotoxicity as evident by restoration of serum transaminases, alkaline phosphatase, bilirubin and triglycerides. Hepatoprotective potential was confirmed by the restoration of lipid peroxidation, glutathione, glucose-6-phosphatase and microsomal aniline hydroxylase and amidopyrine N-demethylase towards near normal. Histopathology of the liver tissue further supports the biochemical findings confirming the hepatoprotective potential of AB-4. The present study shows that the aqueous extract of Aloe barbadensis is significantly capable of restoring integrity of hepatocytes indicated by improvement in physiological parameters, excretory capacity (BSP retention) of hepatocytes and also by stimulation of bile flow secretion. AB-4 did not show any sign of toxicity up to oral dose of 2 g/kg in mice.

Immunomodulatory activity of biopolymeric fraction RLJ-NE-205 from Picrorhiza kurroa.

In the last three decades, numerous biopolymeric fractions have been isolated from medicinal plants and used as a source of therapeutic agents. The most promising biopharmacological activities of these biopolymers are their immunomodulatory effects. The biopolymeric fraction RLJ-NE-205 was isolated and purified from the rhizomes of Picrorhiza kurroa. We evaluated the effects of biopolymeric fraction RLJ-NE-205 from P. kurroa on the in vivo immune function of the mouse. Balb/c mice were treated with the biopolymeric fraction RLJ-NE-205 (12.5, 25 and 50 mg/kg body weight) for 14 days with sheep red blood cells (SRBC) as an antigen. Haemagglutination antibody (HA) titre, plaque forming cell (PFC) assay, delayed type hypersensitivity (DTH) reaction, phagocytic index, proliferation of lymphocytes, analysis of cytokines in serum and CD4/CD8 population in spleen (determined by flowcytometry) were studied. At the dose of 50 mg/kg, significant increases in the proliferation of lymphocytes (p<0.001) and cytokine levels (IL-4 and IFN-gamma) in serum (p<0.001) were observed. A dose dependent increase was demonstrated in HA titre (p<0.05), DTH (p<0.01), PFC (p<0.05), phagocytic index (p<0.05) and CD4/CD8 (p<0.01) population. This suggests that the biopolymeric fraction RLJ-NE-205 improves the immune system and might be regarded as a biological response modifier.

Amelioration of adjuvant-induced arthritis by ursolic acid through altered Th1/Th2 cytokine production.

The objective of the study was to investigate the activity of ursolic acid (UA) on proinflammatory (Th1) and anti-inflammatory (Th2) cytokines in the peripheral blood of arthritic balb/c mice. Ursolic acid is ubiquitous in the plant kingdom and is a constituent of numerous plants which are having diversified phylogenetic origin and taxonomic position. We applied Cytometric bead array (CBA) technology for simultaneously measurement of these cytokines in adjuvant inflammatory arthritis induced mice treated with ursolic acid in graded oral doses. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate five cytokines in a 50 microl sample volume. The T-helper (Th1) deviated cells produce detectable level of tumor necrosis factor (TNF-alpha), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), while the Th2 deviated cells produce significant amount of interleukin-4 (IL-4) and interleukin-5 (IL-5). Oral administration of UA at doses of 10, 20, 40, 80 and 160 mg kg(-1) per oral dose inhibited the presence of IL-2, IFN-gamma and TNF-alpha in the peripheral blood.

Anti-inflammatory activity of the hydrosoluble fraction of Euphorbia royleana latex.

The hydrosoluble fraction of Euphorbia royleana latex (AER), administered by gavage at doses of 50-200 mg/kg, showed dose-dependent anti-inflammatory and anti-arthritic effects in different acute and chronic test models in rats and mice. It reduced the exudate volume and the migration of leukocytes and showed a poor inhibitory effect on the granuloma formation induced by cotton pellets, while it had a low ulcerogenic score. The oral LD(50) was more than 1500 mg/kg in both rats and mice.