New withanolides from leaves along with alkyl glucoside from roots and stem as well as variations in withanolide contents in leaves of accessions of Withania somnifera, Ashwagandha- the Indian ginseng$.

Based on the major components in the leaves, the ashwagandha has been found to exist in several chemotypic forms in India. From the leaves of various accessions of Withania somnifera, which were maintained in our institute, three new steroids namely, 4-acetoxy-20ß-hydroxy-1-oxo-witha-2,5,24-trienolide (7), 24,25-dihydro-14α-hydroxy withanolide D (9), 5α,6ß,17α,27-tetrahydroxy-1-oxo-witha-2,24-dienolide (12) together with thirteen known withanolides were identified by spectroscopic methods. From the roots and stem of one accession and leaves of another, a new alkyl ester glucoside (4) has also been isolated. The new withanolides 7, 9 and 12 have been tentatively named as withanolide 135 A, withanolide 135B and withanolide 108, respectively.

Enzymatic processing of Citrus reticulata (Kinnow) pomace using naringinase and its valorization through preparation of nutritionally enriched pasta.

Citrus fruits are consumed either as whole fruits or as juice after processing. Processing of fruits yields a significant number of by-products in the form of pulp, peel and seeds, which are often discarded and major cause of environmental concern. Bitterness in the waste residue of citrus products is one of the leading hindrance in its valorization and supplementation in other food products. Aim of this study was to reduce the bitterness of Citrus reticulata (kinnow) pomace using enzymatic method and its supplementation in production of nutritionally rich pasta. Under optimized conditions (1U/mg enzyme naringinase concentration, temperature 50 °C, at pH 4.5 and treatment time 4 h), the maximum reduction (65.95%) of naringin (bitterness causing compound) was observed coupled with increase (60.13%) in naringenin (non-bitter compound). The debittered kinnow pomace has been further characterized for physio-chemical changes and morphological changes before and after treatment. The debittered kinnow pomace was then supplemented for the preparation of antioxidant and nutrient enriched pasta.

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.

Value additon of kinnow industry byproducts for the preparation of fiber enriched extruded products.

In the present study dietary fiber enriched vermicelli from wheat flour supplemented with debittered kinnow industry by-products (pulp residue and pomace) has been developed. Functional, cooking and textural properties of both supplemented and unsupplemented vermicelli were evaluated. Vermicelli containing 15% debittered kinnow pulp residue and pomace showed minimum cooking loss (18.5, 20.0%) but higher swelling index (2.06, 1.87), water absorption capacity (153, 202 g/100 g) and optimal cooking time (9.34, 9.02 min). Firmness and fracturability of vermicelli supplemented debittered pulp residue (10.0 and 21.5) and pomace (16.7 and 16.1) was higher as compared to control sample (6.1 and 2.1) respectively. Further, redness, firmness, TPC, DPPH activity and water absorption capacity of vermicelli got increased with addition of debittered kinnow pulp and pomace. The utilization of debittered kinnow pulp and pomace in vermicelli can provide dual benefit like production of healthy food products along with solving the problem of solid waste disposal of kinnow industry byproducts.

De novo sequencing, assembly and characterisation of Aloe vera transcriptome and analysis of expression profiles of genes related to saponin and anthraquinone metabolism.

BACKGROUND: Aloe vera is a perennial, succulent, drought-resistant plant that exhibits many pharmacological characteristics such as wound healing ability against skin burns, anti-ulcer, anti-inflammatory, anti-tumor, anti-viral, anti-hypercholesterolemic, anti-hyperglycemic, anti-asthmatic and much more. Despite great medicinal worth, little genomic information is available on Aloe vera. This study is an initiative to explore the full-scale functional genomics of Aloe vera by generating whole transcriptome sequence database, using Illumina HiSeq technology and its progressive annotation specifically with respect to the metabolic specificity of the plant. RESULTS: Transcriptome sequencing of root and leaf tissue of Aloe vera was performed using Illumina paired-end sequencing technology. De novo assembly of high quality paired-end reads, resulted into 1,61,733 and 2,21,792 transcripts with mean length of 709 and 714 nucleotides for root and leaf respectively. The non-redundant transcripts were clustered using CD-HIT-EST, yielding a total of 1,13,063 and 1,41,310 unigenes for root and leaf respectively. A total of 6114 and 6527 CDS for root and leaf tissue were enriched into 24 different biological pathway categories using KEGG pathway database. DGE profile prepared by calculating FPKM values was analyzed for differential expression of specific gene encoding enzymes involved in secondary metabolite biosynthesis. Sixteen putative genes related to saponin, lignin, anthraquinone, and carotenoid biosynthesis were selected for quantitative expression by real-time PCR. DGE as well as qRT PCR expression analysis represented up-regulation of secondary metabolic genes in root as compared to leaf. Furthermore maximum number of genes was found to be up-regulated after the induction of methyl jasmonate, which stipulates the association of secondary metabolite synthesis with the plant's defense mechanism during stress. Various transcription factors including bHLH, NAC, MYB were identified by searching predicted CDS against PlantTFdb. CONCLUSIONS: This is the first transcriptome database of Aloe vera and can be potentially utilized to characterize the genes involved in the biosynthesis of important secondary metabolites, metabolic regulation, signal transduction mechanism, understanding function of a particular gene in the biology and physiology of plant of this species as well as other species of Aloe genus.

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.

An integrated bio-process for production of functional biomolecules utilizing raw and by-products from dairy and sugarcane industries.

The study investigated an integrated bioprocessing of raw and by-products from sugarcane and dairy industries for production of non-digestible prebiotic and functional ingredients. The low-priced feedstock, whey, molasses, table sugar, jaggery, etc., were subjected to transglucosylation reactions catalyzed by dextransucrase from Leuconostoc mesenteroides MTCC 10508. HPLC analysis approximated production of about 11-14 g L-1 trisaccharide i.e. 2-α-D-glucopyranosyl-lactose (4-galactosyl-kojibiose) from the feedstock prepared from table sugar, jaggery, cane molasses and liquid whey, containing about 30 g L-1 sucrose and lactose each. The trisaccharide was hydrolysed into the prebiotic disaccharide, kojibiose, by employing recombinant ß-galactosidase from Escherichia coli. The enzyme ß-galactosidase achieved about 90% conversion of 2-α-D-glucopyranosyl-lactose into kojibiose. The D-fructose generated by catalytic reactions of dextransucrase was targeted for catalytic transformation into rare sugar, D-allulose (or D-psicose), by treating the samples with Smt3-D-psicose 3-epimerase. The catalytic reactions resulted in the conversion of ~ 25% D-fructose to D-allulose. These bioactive compounds are known to exert a plethora of benefits to human health, and therefore, are preferred ingredients for making functional foods.

Tomato processing byproduct valorization in bread and muffin: improvement in physicochemical properties and shelf life stability.

Tomato processing industry generates huge waste like tomato skin, seed, and pulp which creates environmental issues. Since tomato pomace contains bioactive compounds and pigments, present study was conducted to investigate the effect of tomato pomace addition on physicochemical characteristics and shelf-life stability of the developed bread and muffin. Refined flour was partially substituted with 35 and 40% tomato pomace in bread and muffin, respectively. Tomato pomace addition in bread and muffin was observed to significantly (p < 0.05) increase the dietary fiber, vitamin C, antioxidant activity and minerals (Na, K, Mg, Ca, Fe). The color parameters for bread and muffins were quantified in terms of L* (lightness), a* (redness/greenness) and b* (yellowness/blueness). There was an increase in a* and b*, while L* values decreased. Tomato based bread and muffin were found to possess softer texture as compared to control products. Microbial study has depicted the enhanced shelf-life of tomato based bread and muffin. Shelf life of preservative added tomato based bread was 8 days and muffins were 12 days. Tomato pomace could be a very useful commodity for incorporation into bread and muffin to have a complete nutritive food product.

De novo transcriptome analysis of rose-scented geranium provides insights into the metabolic specificity of terpene and tartaric acid biosynthesis.

BACKGROUND: Rose-scented geranium (Pelargonium sp.) is a perennial herb that produces a high value essential oil of fragrant significance due to the characteristic compositional blend of rose-oxide and acyclic monoterpenoids in foliage. Recently, the plant has also been shown to produce tartaric acid in leaf tissues. Rose-scented geranium represents top-tier cash crop in terms of economic returns and significance of the plant and plant products. However, there has hardly been any study on its metabolism and functional genomics, nor any genomic expression dataset resource is available in public domain. Therefore, to begin the gains in molecular understanding of specialized metabolic pathways of the plant, de novo sequencing of rose-scented geranium leaf transcriptome, transcript assembly, annotation, expression profiling as well as their validation were carried out. RESULTS: De novo transcriptome analysis resulted a total of 78,943 unique contigs (average length: 623 bp, and N50 length: 752 bp) from 15.44 million high quality raw reads. In silico functional annotation led to the identification of several putative genes representing terpene, ascorbic acid and tartaric acid biosynthetic pathways, hormone metabolism, and transcription factors. Additionally, a total of 6,040 simple sequence repeat (SSR) motifs were identified in 6.8% of the expressed transcripts. The highest frequency of SSR was of tri-nucleotides (50%). Further, transcriptome assembly was validated for randomly selected putative genes by standard PCR-based approach. In silico expression profile of assembled contigs were validated by real-time PCR analysis of selected transcripts. CONCLUSION: Being the first report on transcriptome analysis of rose-scented geranium the data sets and the leads and directions reflected in this investigation will serve as a foundation for pursuing and understanding molecular aspects of its biology, and specialized metabolic pathways, metabolic engineering, genetic diversity as well as molecular breeding.

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.

Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal).

Withanolides are pharmaceutically important C(28)-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of (13)C label from (13)C(1)-D-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of (13)C abundance at each carbon of withaferin A from (13)C(1)-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific (13)C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids. Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.

1,4-Dioxane and ergosterol derivatives from Withania somnifera roots.

The chemical investigation on the n-hexane extract of Withania somnifera roots has yielded octacosane, oleic and stearic fatty acids, stigmasterone, stigmasterol, sitostanone, oleanolic acid along with the ergosterol and 1,4-dioxane derivatives as new compounds. The isolation of alkenyl-1,4-dioxane compound is rare, whereas the ergosterol derivative may have biogenetic significance in the lactone formation in the E ring of withanolides. The presence of a 1,4-dioxane derivative in the nonpolar extract of roots assumes importance as this type of compound has not been reported earlier from W. somnifera. The structures of new compounds were elucidated by spectroscopic methods and chemical transformations.

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.

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.

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.

Efficient and economic process for the production of bacterial cellulose from isolated strain of Acetobacter pasteurianus of RSV-4 bacterium.

In the present investigation, several residues from agro-forestry industries such as rice straw acid hydrolysate, corn cob acid hydrolysate, tomato juice, cane molasses and orange pulp were evaluated as the economical source for the production of bacterial cellulose. The bacterial cellulose attained the significant yield of 7.8 g/L using tomato juice, followed by 3.6 g/L using cane molasses and 2.8 g/L using orange pulp after 7 days of incubation. Furthermore, the optimum pH and temperature of fermentation for maximum production of bacterial cellulose was 4.5 and 30 ±â€¯1 °C. The identified bacterium Acetobacter pasteurianus RSV-4 has been deposited at repository under the accession number MTCC 25117. The produced bacterial cellulose was characterized through FTIR, SEM, TGA and DSC and found to be of very good quality. The bacterial cellulose produced by identified strain on these various agro-waste residues could be a cost effective technology for commercial its production.

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.

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.