Comparative study of various processes used for removal of bitterness from kinnow pomace and kinnow pulp residue.

The current study was focused on new approaches for debittering of by-products like kinnow pomace and kinnow pulp residue by using various food grade mild chemical methods, such as alkali treatment, acid treatment, and solventogenesis. Whereas in the studied various chemical treatments, the solventogenesis method with acetone resulted in maximum extraction of naringin and limonene from kinnow pomace and pulp residue and showed high acceptability for food product development. The acetone treatment was further optimized by RSM for the maximum extraction of naringin and limonene. Under optimized conditions, the maximum amount of naringin and limonene extracted were found to be 8.955, 2.122 mg/g from kinnow pomace and 9.971, 3.838 mg/g from pulp residue, respectively. This process can not only result in the effective utilization of agro-industrial by-product but also provide a sustainable solution to the environmental pollution caused by kinnow juice industry.

Alginate-pectin co-encapsulation of dextransucrase and dextranase for oligosaccharide production from sucrose feedstocks.

The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497, respectively. Heterologous expression of genes was performed in Escherichia coli. The purified enzyme fractions were entrapped in the alginate-pectin beads. A high immobilization yield of dextransucrase (~ 96%), and dextranase (~ 85%) was achieved. Alginate-pectin immobilization did not affect the optimum temperature and pH of the enzymes; rather, the thermal tolerance and storage stability of the enzymes was improved. The repetitive batch experiments suggested substantially good operational stability of the co-immobilized enzyme system. The synergistic catalytic reactions of alginate-pectin co-entrapped enzyme system were able to produce 7-10 g L-1 oligosaccharides of a high degree of polymerization (DP 3-9) from sucrose (~ 20 g L-1) containing feedstocks, e.g., table sugar and cane molasses. The alginate-pectin-based co-immobilized enzyme system is a useful catalytic tool to bioprocess the agro-industrial bio-resource for the production of prebiotic biomolecules.

Interspecies comparative features of trichomes in Ocimum reveal insights for biosynthesis of specialized essential oil metabolites.

Ocimum species commonly referred to as "Tulsi" are well-known for their distinct medicinal and aromatic properties. The characteristic aroma of Ocimum species and cultivars is attributed to their specific combination of volatile phytochemicals mainly belonging to terpenoid and/or phenylpropanoid classes in their essential oils. The essential oil constituents are synthesized and sequestered in specialized epidermal secretory structures called as glandular trichomes. In this comparative study, inter- and intra-species diversity in structural attributes and profiles of expression of selected genes related to terpenoid and phenylpropanoid biosynthetic pathways have been investigated. This is performed to seek relationship of variations in the yield and phytochemical composition of the essential oils. Microscopic analysis of trichomes of O. basilicum, O. gratissimum, O. kilimandscharicum, and O. tenuiflorum (green and purple cultivars) revealed substantial variations in density, size, and relative proportions of peltate and capitate trichomes among them. The essential oil yield has been observed to be controlled by the population, dominance, and size of peltate and capitate glandular trichomes. The essential oil sequestration in leaf is controlled by the dominance of peltate glandular trichome size over its number and is also affected by the capitate glandular trichome size/number with variations in leaf area albeit at lower proportions. Comprehension and comparison of results of GC-MS analysis of essential oils showed that most of the Ocimum (O. basilicum, O. tenuiflorum, and O. gratissimum) species produce phenylpropanoids (eugenol, methyl chavicol) as major volatiles except O. kilimandscharicum, which is discrete in being monoterpenoid-rich species. Among the phenylpropanoid-enriched Ocimum (O. basilicum, O. gratissimum, O. tenuiflorum purple, O. tenuiflorum green) as well, terpenoids were important constituents in imparting characteristic aroma. Further, comparative abundance of transcripts of key genes of phenylpropanoid (PAL, C4H, 4CL, CAD, COMT, and ES) and terpenoid (DXS and HMGR) biosynthetic pathways was evaluated vis-à-vis volatile oil constituents. Transcript abundance demonstrated that richness of their essential oils with specific constituent(s) of a chemical group/subgroup was manifested by the predominant upregulation of phenylpropanoid/terpenoid pathway genes. The study provides trichomes as well as biosynthetic pathway-based knowledge for genetic improvement in Ocimum species for essential oil yield and quality.

Transcriptome mining and in silico structural and functional analysis of ascorbic acid and tartaric acid biosynthesis pathway enzymes in rose-scanted geranium.

Rose-scented geranium (Pelargonium sp.) is widely known as aromatic and medicinal herb, accumulating specialized metabolites of high economic importance, such as essential oils, ascorbic acid, and tartaric acid. Ascorbic acid and tartaric acid are multifunctional metabolites of human value to be used as vital antioxidants and flavor enhancing agents in food products. No information is available related to the structural and functional properties of the enzymes involved in ascorbic acid and tartaric acid biosynthesis in rose-scented geranium. In the present study, transcriptome mining was done to identify full-length genes, followed by their bioinformatic and molecular modeling investigations and understanding of in silico structural and functional properties of these enzymes. Evolutionary conserved domains were identified in the pathway enzymes. In silico physicochemical characterization of the catalytic enzymes revealed isoelectric point (pI), instability index, aliphatic index, and grand average hydropathy (GRAVY) values of the enzymes. Secondary structural prediction revealed abundant proportion of alpha helix and random coil confirmations in the pathway enzymes. Three-dimensional homology models were developed for these enzymes. The predicted structures showed significant structural similarity with their respective templates in root mean square deviation analysis. Ramachandran plot analysis of the modeled enzymes revealed that more than 84% of the amino acid residues were within the favored regions. Further, functionally important residues were identified corresponding to catalytic sites located in the enzymes. To, our best knowledge, this is the first report which provides a foundation on functional annotation and structural determination of ascorbic acid and tartaric acid pathway enzymes in rose-scanted geranium.

Effect of cadmium stress on inductive enzymatic and nonenzymatic responses of ROS and sugar metabolism in multiple shoot cultures of Ashwagandha (Withania somnifera Dunal).

Withania somnifera is one of the most important medicinal plant and is credited with various pharmacological activities. In this study, in vitro multiple shoot cultures were exposed to different concentrations (5-300 µM) of cadmium (Cd) as cadmium sulphate to explore its ability to accumulate the heavy metal ion and its impact on the metabolic status and adaptive responses. The results showed that supplemental exposure to Cd interfered with N, P, and K uptake creating N, P, and K deficiency at higher doses of Cd that also caused stunting of growth, chlorosis, and necrosis. The study showed that in vitro shoots could markedly accumulate Cd in a concentration-dependent manner. Enzymatic activities and isozymic pattern of catalase, ascorbate peroxidase, guaiacol peroxidase, peroxidase, glutathione-S-transferase, glutathione peroxidase, monodehydroascorbate reductase, and dehydroascorbate reductase were altered substantially under Cd exposure. Sugar metabolism was also markedly modulated under Cd stress. Various other parameters including contents of photosynthetic pigments, phenolics, tocopherol, flavonoids, reduced glutathione, nonprotein thiol, ascorbate, and proline displayed major inductive responses reflecting their protective role. The results showed that interplay of enzymatic as well as nonenzymatic responses constituted a system endeavor of tolerance of Cd accumulation and an efficient scavenging strategy of its stress implications.

Comparative transcripts profiling of fruit mesocarp and endocarp relevant to secondary metabolism by suppression subtractive hybridization in Azadirachta indica (neem).

Azadirachta indica (neem) is a medicinally important plant that is valued for its bioactive secondary metabolites. Higher levels of the bioactive phytochemicals are accumulated in fruits than in other tissues. In the present study, a total of 387 and 512 ESTs, respectively, from endocarp and mesocarp of neem fruits were isolated and analyzed. Out of them 318 ESTs (82.17%) clones from endocarp and 418 ESTs (81.64%) from mesocarp encoded putative proteins that could be classified into three major gene ontology categories: biological process, molecular function and cellular component. From the analyses of contigs, 73 unigenes from the forward subtracted library and 35 unigenes from the reverse subtracted library were obtained. The ESTs from mesocarp encoded cytochrome P450 enzymes, which indicated hydroxylation to be a major metabolic event and that biogeneration of hydroxylated neem fruit phytochemicals was differentially regulated with developmental stage-specificity of synthesis. Through this study, we present the first report of any gene expression data in neem tissues. Neem hydroxy-methyl glutaryl-coenzyme A reductase (NHMGR) gene was used as expressing control vis-a-vis subtracted tissues. NHMGR was present in fruit, endocarp and mesocarp tissues, but absent in subtractive libraries, revealing that it was successfully eliminated during subtraction. Eight genes of interest from subtracted libraries were profiled for their expression in fruit, mesocarp and endocarp. Expression profiles validated the quality of the libraries and functional diversity of the tissues. The subtractive cDNA library and EST database described in this study represent a valuable transcript sequence resource for future research aimed at improving the economically important medicinal plant.

Effect of prolonged water stress on specialized secondary metabolites, peltate glandular trichomes, and pathway gene expression in Artemisia annua L.

Artemisia annua L. accumulates substantial quantities of unique sesquiternoid artemisinin and related phytomolecules and characteristic essential oil in glandular trichomes, present on its leaves and inflorescence. Water stress is a major concern in controlling plant growth and productivity. In this study, our aim was to find out the modulation of artemisinin and essential oil constituents in plants grown under prolonged water stress conditions. A. annua CIM-Arogya plants grown in pots were subjected to mild (60% ± 5) and moderate (40% ± 5) water stress treatment and continued during entire developmental period. Results revealed that artemisinin, arteannuin-B, artemisinic acid, dihydroartemisinic acid and essential oil content were positively controlled by the growth and development however negatively modulated by water deficit stress. Interestingly, some of minor monoterpenes, all sesquiterpenes and other low molecular weight volatiles of essential oil components were induced by water deficit treatment. Camphor which is the major essential oil constituents did not alter much while 1, 8 cineole was modulated during development of plant as well as under water stress conditions. Water deficit stress induces a decrease in glandular trichome density and size as well. The dynamics of various secondary metabolites is discussed in the light of growth responses, trichomes and pathway gene expression in plants grown under two levels of prolonged water stress conditions.

De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural, functional and regulatory genes with special reference to secondary metabolism.

Centella asiatica (L.) Urban is an important medicinal plant and has been used since ancient times in traditional systems of medicine. C. asiatica mainly contains ursane skeleton based triterpenoid sapogenins and saponins predominantly in its leaves. This investigation employed Illumina next generation sequencing (NGS) strategy on a pool of three cDNAs from expanding leaf of C. asiatica and developed an assembled transcriptome sequence resource of the plant. The short transcript reads (STRs) generated and assembled into contigs and singletons, representing majority of the genes expressed in C. asiatica, were termed as 'tentative unique transcripts' (TUTs). The TUT dataset was analyzed with the objectives of (i) development of a transcriptome assembly of C. asiatica, and (ii) classification/characterization of the genes into categories like structural, functional, regulatory etc. based on their function. Overall, 68.49% of the 46,171,131 reads generated in the NGS process could be assembled into a total of 79,041 contigs. Gene ontology and functional annotation of sequences resulted into the identification of genes related to different sets of cellular functions including identification of genes related to primary and secondary metabolism. The wet lab validation of seventeen assembled gene sequences identified to be involved in secondary metabolic pathways and control of reactive oxygen species (ROS) was established by semi-quantitative and real time PCR (qRT-PCR). The validation also included sequencing/size matching of a set of semi-quantitative PCR amplicons with their in silico assembled contig/gene. This confirmed the appropriateness of assembling the reads and contigs. Thus, the present study constitutes the largest report to date on C. asiatica transcriptome based gene resource that may contribute substantially to the understanding of the basal biological functions and biochemical pathways of secondary metabolites as well as the transcriptional regulatory elements and genetic markers. This work sets the stage for multi-faceted future improvement of the plant, through discovery of new genes, marker-assisted breeding or genetic engineering, on this species as well as for other species of Apiaceae and triterpene producing medicinal plants.

Effect of gibberellic acid and calliterpenone on plant growth attributes, trichomes, essential oil biosynthesis and pathway gene expression in differential manner in Mentha arvensis L.

Extensive research is going on throughout the world to find out new molecules from natural sources to be used as plant growth promoter. Mentha arvensis L. is the main source of menthol rich essential oil used commercially in various food, pharmaceutical and other preparations. Experiments were conducted on field grown plants for understanding the effect of calliterpenone (CA), a stereo-isomer of abbeokutone, in comparison to gibberellic acid (GA3) on growth attributes, trichomes, essential oil biosynthesis and expression of some oil biosynthetic pathway genes. The exogenous application of CA (1 µM, 10 µM and 100 µM) was found to be better in improving plant biomass and stolon yield, leaf area, branching and leaf stem ratio than with counterpart GA3 at the same concentrations. CA treated plants showed higher glandular trichome number, density and diameter and also correlated with enhanced oil biogenetic capacity as revealed by feeding labeled (14)C-sucrose for 72 h to excised shoots. Semi-quantitative PCR analysis of key pathway genes revealed differential up regulation under CA treatments. Transcript level of menthol dehydrogenase/menthone reductase was found highly up regulated in CA treated plants with increased content of menthone and menthol in oil. These findings demonstrate that CA positively regulated the yields by enhanced branching and higher density of trichomes resulting into higher accumulation of essential oil. The results suggest CA as a novel plant derived diterpenoid with growth promoting action and opens up new possibilities for improving the crop yields and essential oil biosynthesis in qualitative and quantitative manner.

Biochemical characteristics of a novel vegetative tissue geraniol acetyltransferase from a monoterpene oil grass (Palmarosa, Cymbopogon martinii var. Motia) leaf.

Plants synthesize volatile alcohol esters on environmental insult or as metabolic induction during flower/fruit development. However, essential oil plants constitutively produce them as the oil constituents. Their synthesis is catalyzed by BAHD family enzymes called alcohol acyltransferases (AATs). However, no AAT has been characterized from plant foliage synthesizing acyclic monoterpenoids containing essential oils. Therefore, we have purified and biochemically characterized a geraniol: acetyl coenzyme A acetyltransferase (GAAT) from Palmarosa aroma grass (Cymbopogon martinii) leaf. MALDI-assisted proteomic study of the 43kDa monomeric enzyme revealed its sequence motif novelties e.g. relaxed conservation at Phe and Trp in DFGWG'. This suggests permissiveness of variations in the conserved motif without loss of catalytic ability. Also, some new conserved/semi-conserved motifs of AATs were recognized. The GAAT k(cat)/K(m) values (300-700M(-1)s(-1)) were low (a generic characteristic for secondary metabolism enzyme) but higher than those of some floral AATs. Wide substrate acceptability for catalyzing acetylation of diverse primary alcohols (chain of ≥C(6)) implied its catalytic description as a 'primary aliphatic alcohol acetyltransferase'. It signifies metabolic ability to deliver diverse aroma esters, should the acceptor alcohols be available in planta. To our knowledge, this is the first report of detailed kinetics of a vegetal monoterpenol acyltransferase.

Efficient genetic transformation of Withania coagulans (Stocks) Dunal mediated by Agrobacterium tumefaciens from leaf explants of in vitro multiple shoot culture.

An efficient and reproducible Agrobacterium-mediated genetic transformation of Withania coagulans was achieved using leaf explants of in vitro multiple shoot culture. The Agrobacterium strain LBA4404 harboring the binary vector pIG121Hm containing ß-glucuronidase gene (gusA) under the control of CaMV35S promoter was used in the development of transformation protocol. The optimal conditions for the Agrobacterium-mediated transformation of W. coagulans were found to be the co-cultivation of leaf explants for 20 min to agrobacterial inoculum (O.D. 0.4) followed by 3 days of co-cultivation on medium supplemented with 100 µM acetosyringone. Shoot bud induction as well as differentiation occurred on Murashige and Skoog medium supplemented with 10.0 µM 6-benzylaminopurine, 8.0 µM indole 3-acetic acid, and 50.0 mgl(-1) kanamycin after three consecutive cycles of selection. Elongated shoots were rooted using a two-step procedure involving root induction in a medium containing 2.5 µM indole 3-butyric acid for 1 week and then transferred to hormone free one-half MS basal for 2 weeks. We were successful in achieving 100 % frequency of transient GUS expression with 5 % stable transformation efficiency using optimized conditions. PCR analysis of T0 transgenic plants showed the presence of gusA and nptII genes confirming the transgenic event. Histochemical GUS expression was observed in the putative transgenic W. coagulans plants. Thin layer chromatography showed the presence of similar type of withanolides in the transgenic and non-transgenic regenerated plants. A. tumefaciens mediated transformation system via leaf explants developed in this study will be useful for pathway manipulation using metabolic engineering for bioactive withanolides in W. coagulans, an important medicinal plant.

Qualitative and quantitative variations in withanolides and expression of some pathway genes during different stages of morphogenesis in Withania somnifera Dunal.

Withania somnifera Dunal is an important and extensively studied medicinal plant; however, there is no report available that relates withanolide content and its profile in relation to the expression of pathway genes during different morphogenic stages. In this study, withanolide A, withaferin A, and withanone, the major withanolides of W. somnifera, were measured in different in vitro stages during organogenesis, viz., shoot to root (direct rhizogenesis)/root to shoot (indirect via callus phase) transition vis-à-vis expression levels of key pathway genes involved in withanolide biosynthetic pathways. The morphogenic transitions were found to be tightly linked to the pattern of accumulation of withanolides. The high expression levels of most of the pathway genes in in vitro shoots in comparison to in vitro root and callus tissues exhibited a direct co-relation with the maximum withanolide content (>2.7 mg/gDW). The biogenesis of withaferin A, a major constituent of the leaves, was however found to be tightly linked to shoots/green tissue. In addition, we were also able to establish an efficient regeneration system from roots for their further utilization in biotechnological applications.

Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf.

Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.

Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice.

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10-100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3-30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses.

Bioconversion of artemisinin to its nonperoxidic derivative deoxyartemisinin through suspension cultures of Withania somnifera Dunal.

Biotransformation of artemisinin was investigated with two different cell lines of suspension cultures of Withania somnifera. Both cell lines exhibited potential to transform artemisinin into its nonperoxidic analogue, deoxyartemisinin, by eliminating the peroxo bridge of artemisinin. The enzyme involved in the reaction is assumed to be artemisinin peroxidase, and its activity in extracts of W. somnifera leaves was detected. Thus, the non-native cell-free extract of W. somnifera and suspension culture-mediated bioconversion can be a promising tool for further manipulation of pharmaceutical compounds.

Metabolic clustering of a core collection of Indian ginseng Withania somnifera Dunal through DNA, isoenzyme, polypeptide and withanolide profile diversity.

Withania somnifera is one of the most important medicinal plants of Ayurveda and finds extensive uses in Indian traditional herbal preparations. In this investigation, selected accessions of the plant were examined for diversity through RAPDs, isoenzymes, polypeptide polymorphism and withanolide profiles. The accessions clustered together with respect to their characteristic profile of major withanolides and represented withaferin A, withanone, withanolide D or withanolide A rich groups. This level of phytochemical diversity as discrete chemotypes is widest and is being first ever documented to occur in Indian population of the plant.

Cellulase production by six Trichoderma spp. fermented on medicinal plant processings.

Capabilities of cellulase production, using delignified bioprocessings of medicinal and aromatic plants, viz. citronella (Cymbopogon winterianus) and Artemisia annua (known as marc of Artemisia) and garden waste (chiefly containing Cynodon dactylon), by the six species of Trichoderma were comparatively evaluated. Among the members of Trichoderma studied, T. citrinoviride was found to be the most efficient producer of cellulases along with a high level of beta-glucosidase (produced 102.4 IU g(-1) on marc of Artemisia; 101.33 IU g(-1) on garden waste; 81.86 IU g(-1) on distillation waste of citronella and 94.77 IU g(-1) on pure cellulose). Although T. virens was noticed to be the minimal enzyme producer fungus, it interestingly could not produce complete cellulase enzyme complex on any test waste or pure cellulose, except on marc of Artemisia, where it produced all three enzymes of the complex. Immediate reduction in pH was also noticed during fermentation in the case of pure polymer (cellulose) by all tested fungi, while it was delayed with delignified agrowastes. The pH profile varied with the substrate used as well as with individual species of Trichoderma. On the other hand, no alteration in pH with any species of Trichoderma was noticed when grown on marc of A. annua, which might be due to the buffering capacity of this marc.

Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade.

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin.

Withanolides from Withania somnifera roots.

Two new and seven known withanolides along with beta-sitosterol, stigmasterol, beta-sitosterol glucoside, stigmasterol glucoside, alpha+beta glucose were isolated from the roots of Withania somnifera. Among the known compounds, Viscosa lactone B, stigmasterol, stigmasterol glucoside and alpha+beta glucose are being reported from the roots of W. somnifera for the first time. One of the new compounds contained the rare 16beta-acetoxy-17(20)-ene the other contained unusual 6alpha-hydroxy-5,7alpha-epoxy functional groups in the withasteroid skeleton. The structures were elucidated by spectroscopic methods and chemical transformations.

Selective reactivity of 2-mercaptoethanol with 5beta,6beta-epoxide in steroids from Withania somnifera.

2-Mercaptoethanol reacts selectively with the 5beta,6beta-epoxy steroids isolated from Withania somnifera substituting the epoxide by a six-membered oxyethylene-2'-thio ring whereas it failed to show such reactivity on 6alpha,7alpha-epoxy withasteroids. The structure of the product has been elucidated by spectroscopic methods, especially applying extensive 2D NMR methods. The anticancer activity of withaferin A was lost in the reaction product indicating that its activity is also linked to the free 5beta,6beta-epoxide functional group.