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Immunomodulatory activity of biopolymeric fraction RLJ-NE-205 from Picrorhiza kurroa
No full textIn 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
Isolation, structure elucidation andIn Vivo hepatoprotective potential oftrans-tetracos-15-enoic acid from Indigofera tinctoria Linn.
No full textThe bioassay guided fractionation of the dried aerial part of Indigofera tinctoria Linn. led to the identification
of an active fraction labelled as indigotin. On further chemical analysis, a compound isolated from indigotin
was identified and characterized as trans-tetracos-15-enoic acid (TCA). The chemical structure of this compound
was established on the basis of physical properties and spectral data, including NMR. It afforded significant hepatoprotection against carbon tetrachloride and paracetamol induced hepatotoxicity in experimental
models. Silymarin, a well known plant based hepatoprotective agent, and N-acetylcysteine, which has
proven efficacy as a replenisher of sulfhydryls, were used for relative efficacy. TCA was found to reverse the altered hepatic parameters in experimental liver damage. In the safety evaluation study the oral LD50 was found to be more than 2000 mg/ kg, with no signs of abnormalities or any mortality for the 15 day period of observation after administration of a single dose of drug in mice. The studies revealed significant and concentration dependent hepatoprotective potential of TCA as it reversed the majority of the altered hepatic parameters in experimental liver damage in rats and mice and may be useful in the management of liver disorders
Molecular cloning of carboxylesterase gene and biochemical characterization of encoded protein from Bacillus subtilis (RRL BB1)
No full textAn isolated strain of Bacillus subtilis identified by 16S rDNA sequence analysis produces an enantioselective ester hydrolase.Whole cells of B. subtilis (RRL BB1) and enzyme derived from it was capable of enantioselective hydrolysis of several racemates including drug intermediates with moderate to high enantioselectivity as already reported by us. In this communication, we describe cloning of the gene encoding the enantioselective esterase designated as estBB1. The primary structure of the enzyme determined from the nucleotide sequence indicated that esterase estBB1 has Mw ∼52 kDa and pI ∼5.2 and belongs to the family of type B carboxylesterases with 50–60% similarity at amino acid level. Alignment studies of sequences of the estBB1 and Pnb
esterase 56C8 from B. subtilis showed that estBB1 has an �/� hydrolase fold with catalytic triad formed by Ser190, Glu305 and His394 at active site and Ser190 is located in the conserved motif –G–X–S–X–G–
Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker—an endangered medicinal herb
No full textAn efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4 x 10(-6) M Kn and 2 x 10(-6) MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1 x 10(-5) M Kinetin (Kn) and 5 x 10(-6) M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm +/- 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5 x 10(-6) M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb
Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny inSalvia sclarea
No full textChanges in the essential oil concentration, composition and glandular morphology of Salvia sclarea L. were studied at different stages of inflorescence maturity. The chemical composition of oil was determined by GC–MS, NMR and the peak enrichment technique. The oil yield at bud stage on a fresh basis was minimum (0.08%), peaked at full bloom stage (0.18%) and was followed by sharp decline on maturation (0.07%). The main components of the oil were linalool (36.6–41.9%) and linalyl acetate (13.2–19.2%). The maximum percentage composition of various constituents was coincident with full bloom stage. β -Humulene (6.4–8.9%), α -cadinene (t–1.5%), β -caryophyllene (t–1.4%), β -
caryophyllene oxide (0.4–1.4%) and sclareol (0.3–1.8%) present in the oil showed gradual increase in percentage over the different stages of maturity, with no significant turnover losses at maturation stage. Turnover of essential oil (61.1% loss) and monoterpenes (23.6% loss) occurred late in development at full maturity. Scanning electron microscopy (SEM) was used to follow the changes in the oil secretory glands over different temporal phases of maturation. The decline in oil concentration and monoterpene constituents compared very well with the observed deterioration and lyses of secretory
glandular system. An abrupt fall in oil concentration apparently appears due to differential evaporation of the more volatile constituents, rather than as a dynamic balance between biosynthetic and catabolic processes
6α,7α-epoxy-5α,17α,dihydroxy-1-oxo-22R-witha-2,24-dienolide in leaves of Withania somnifera: Isolation and its crystal structure
No full text6α,7α-epoxy-5α,17α,dihydroxy-1-oxo-22R-witha-2,24-dienolide (C28H38O6) was isolated from Withania somnifera leaves. The structure of the withanolide was established by spectral analysis and X-ray diffraction studies as withanone. The compound crystallizes in the orthorhombic space group P212121 with unit cell parameters: a=9.191(10) A° ,
b=12.858(6) A° , c=21.400(16) A° , Z=4. The crystal structure was solved by direct methods and refined to R=0.0603 for 1742 observed reflections. There is positional disorder of the H atom in a hydroxy group (O5), resulting in two possible hydrogen-bond linkages. All the rings of the steroid skeleton are trans connected. Ring A exists in a half-chair conformation, ring B is intermediate between a half-chair and a sofa, ring C a distorted chair, and five-membered ring D is intermediate between a half-chair and an envelope. The δ-lactone ring E adopts a sofa conformation. The twist along the length of the steroid nucleus is negligible [C19–C10. . .C13–C18=1.8◦]. Both the hydroxy groups are involved
in hydrogen bonding
A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells
No full textAP9-cd, a standardized lignan composition from Cedrus deodara consisting of (¡)-wikstromal, (¡)-matairesinol, and dibenzyl butyrolactol,showed cytotoxicity in several human cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC50 of »15 �g/ml,
increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced DNA ladder formation. Flow cytometric analysis of annexinV-FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic necrosis. All these biological end-points indicated cell death by apoptosis. Further, initial events involved massive nitric oxide (NO) formation within 4 h with subsequent late appearance of peroxides in cells; measured by Xow cytometry using speciWc Xuorescent probes. Persistently high levels of NO and peroxide appeared to decrease mitochondrial membrane potential (�mt) which was recovered by cyclosporin A in Molt-4 cells. AP9-
cd caused 2-fold activation of caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced caspases activities indicating a pro-oxidant eVect of AP9-cd. Further, caspase-3 activation correlated
with NO generation that was partially impaired by nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of prooxidant species in caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to caspases activation, peroxide generation, and mitochondrial
depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of leukemia cells by AP9-c
Enantioselectivity modulation through immobilization of Arthrobacter sp. lipase: Kinetic resolution of fluoxetine intermediate
No full textArthrobacter sp. lipase (ABL, MTCC no. 5125) has been identified for its excellent performance in kinetic resolution of a number of drug intermediates. ABL free enzyme provided product II and V (ee < 95%) from racemic fluoxetine intermediate (I and IV) compared to its cell
biomass in naturally immobilized state (ee < 98%). To overcome this problem and obtaining high enantioselectivity (R isomer ee 99%), ABL enzyme was modulated by immobilization using various methods vis-`a-vis substrate modification (Scheme 2). Immobilized enzyme obtained by
hydrophobic binding provided 6710–7720 U/g, covalent binding 200 U/g, and sol–gel entrapment 65–110 U/g activity. Substantial improvement in enantioselectivity was obtained using acylates of ethyl 3-hydroxy 3-phenylpropanoate a fluoxetine drug intermediate (R isomer ee from 93 to 99% and E from 43 to 127–473) at 29–45% conversion in fixed time period of 21 h, indicating thereby some change in conformation of ABL immobilized enzyme. The ABL immobilized by covalent binding and sol–gel entrapment has demonstrated reasonable superiority over the free
ABL in enantioselectivity as well as over all rate of hydrolysis. Immobilized enzymes prepared by covalent and entrapment methods have shown excellent operational stability and used for 10 cycles without loss in activity and the technique can be upscaled for process development
Phytochemical and genetic analysis in selected chemotypes of Withania somnifera
No full textThe main active components and genetic profile of 15 selected accessions of Withania somnifera Dunal. were analysed. Ethanolic extract of the dried roots/leaves of the plant was concentrated under pressure at 50 ± 5 �C and was analysed for main compounds (withanolides and withaferin A) by HPLC. All the main components were found to be present in accessions (AGB 002, AGB 009, RSS 009,
RSS 033). Correlation of these main components with their genetic factors, was undertaken using AFLP (amplified fragment length polymorphism)markers. Among 64 primers 7 yielded optimum polymorphism. A total of 913 polymorphic peaks were generated using these primers. Jaccard’s similarity coefficient indicated that accessions having almost the same active compounds clustered together. The present study demonstrates that AFLP can be successfully used to resolve the correlation of AFLP data with the presence of secondary metabolites
Selective Th1 up-regulating activity of Withania somnifera aqueous extract in an experimental system using flow cytometry
No full textWithania somnifera (Ashwagandha) is reported to be immunoprotective and immunoadjuvant. We studied its roots aqueous extract on T helper(Th) immunity using flowcytometry. This extractwas standardized with six withanolides as marker compounds using HPLC. Once daily dose ranging from 25 to 400 mg/kg/p.o. was used to study effect on Th1: IFN-gamma, IL-2 and Th2: IL-4 cytokine modulation. We also studied effect on CD4 and CD8 in normal and immunesuppressed mice. The results indicate that extract at 100 mg/kg resulted significant selective up-regulation of Th1 response.Treatment with extract showed significant increase inCD4andCD8counts as compared to control and cyclopsorin A, with a faster recovery
of CD4+ T cells in immunesuppressed animals. Under immunesuppressed conditions, potentiation of cellular and humoral immune responses of extract was comparable to levamisole. This study indicates the selective Th1 up-regulating effect of extract and suggests its use for selective Th1/Th2 modulation