Biotransformation of withanolides by cell suspension cultures of Withania somnifera (Dunal)

The biotransformation potential of cell suspension cultures generated from Withania somnifera leaf was investigated, using withanolides, i.e. withanolide A, withaferin A, and withanone as precursor substrates. Interestingly, the cell suspension cultures showed inter-conversion of withanolides, as well converted to some unknown compounds, released to the culture media. The bio-catalyzed withanolide was detected and quantified by TLC and HPLC, respectively. There is noticeable conversion of withanolide A to withanone, and vice versa though at a lower level. The type of reaction of this biotransformation appears to be substitution of 20-OH group to 17-OH in withanolide A. In this paper, we present for the first time the possibility of biotransformation by inter-conversion of withanolides of pharmacological importance through cell suspension culture of W. somnifera. The possible role of putative cytochrome P₄₅₀ hydroxylases is implicated in the conversion.     

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.     

Withanolide production by <Emphasis Type="Italic">in vitro</Emphasis> cultures of <Emphasis Type="Italic">Withania somnifera</Emphasis> and its association with differentiation

Withanolides-steroidal lactones, isolated from various Solanaceous plants have received considerable attention due to their potential biological activities. Five selected withanolides (withanone, withaferin A, withanolide A, withanolide B, withanolide E) were identified by HPLC-UV (DAD) — positive ion electrospray ionization mass spectroscopy in Withania somnifera (L.) Dunal cv. WSR plants and tissues cultured in vitro at different developmental phases. Cultures were established from five explants on Murashige and Skoog’s medium supplemented with different plant growth regulators. Results suggest that production of withanolides is closely associated with morphological differentiation.     

Arbuscular mycorrhizal fungal diversity in high-altitude hypersaline Andean wetlands studied by 454-sequencing and morphological approaches

Withania somnifera, also known as Indian ginseng is known to contain valuable bioactive compounds, called withanolides that structurally resemble ginsenosides of Panax ginseng. These compounds provide the basis of pharmacological relevance in traditional systems of medicine. In the present study, 150 hairy root lines of W. somnifera were induced of which nine fast growing lines were analysed for their growth and withanolide content. Hairy root line W9 was selected due to its high specific growth rate (0.196 ± 0.005 d^?1) and high withanolide content. The response to different concentrations of elicitors (methyl jasmonate and P. indica cell homogenate) and various exposure durations was assessed in the W9 hairy root line. The withanolide content as well as the pattern of gene expression from MVA, MEP and sterol pathway, was evaluated using qPCR. Though gene expression and withanolide content were found to be elevated in almost all MeJ and CHP treatments, the exposure of hairy roots to 15 μM MeJ for 4 h gave the maximum withanolide yield. The results suggest that the elicitation potential of methyl jasmonate was higher than that of P. indica cell homogenate for increasing withanolide levels in hairy roots of W. somnifera.     

Differential expression of farnesyl diphosphate synthase gene from <Emphasis Type="Italic">Withania somnifera</Emphasis> in different chemotypes and in response to elicitors

Withania somnifera (L.) Dunal (Family, Solanaceae), commonly known as Ashwagandha is one of the most valuable medicinal plants synthesizing large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized a gene encoding farnesyl diphosphate synthase (FPPS; EC 2.5.1.10), a key enzyme in the pathway of biosynthesis of isoprenoids, from W. somnifera. The full-length cDNA of Withania somnifera FPPS (WsFPPS) of 1,253 bps encodes a polypeptide of 343 amino acids. The amino acid sequence homology and phylogenetic analysis suggest that WsFPPS has close similarity to its counterparts from tomato (SlFPPS) and capsicum (CaaFPPS). Using semi quantitative RT–PCR, the expression pattern of the WsFPPS gene was analyzed in different tissues of Withania chemotypes (NMITLI-101, NMITLI-108, NMITLI-118 and NMITLI-135) as well as in response to elicitors (salicylic acid and methyl jasmonate) and mechanical wounding. The expression analysis suggests that WsFPPS expression varies in different tissues (with maximal expression in flower and young leaf) and chemotypes (with highest level in NMITLI-101) and was significantly elevated in response to salicylic acid, methyl jasmonate and mechanical injury. This is the first report on characterization of an isoprenoid pathway gene involved in withanolide biosynthesis.     

Comprehensive assessment of the genes involved in withanolide biosynthesis from <Emphasis Type="Italic">Withania somnifera</Emphasis>: chemotype-specific and elicitor-responsive expression

Withania somnifera (L.) Dunal (Family, Solanaceae), is among the most valuable medicinal plants used in Ayurveda owing to its rich reservoir of pharmaceutically active secondary metabolites known as withanolides. Withanolides are C₂₈-steroidal lactones having a triterpenoidal metabolic origin synthesised via mevalonate (MVA) pathway and methyl-D-erythritol-4-phosphate (MEP) pathway involving metabolic intermediacy of 24-methylene (C₃₀-terpenoid) cholesterol. Phytochemical studies suggest differences in the content and/or nature of withanolides in different tissues of different chemotypes. Though development of genomic resources has provided information about putative genes encoding enzymes for biosynthesis of intermediate steps of terpenoid backbone, not much is known about their regulation and response to elicitation. In this study, we generated detailed molecular information about genes catalysing key regulatory steps of withanolide biosynthetic pathway. The full-length sequences of genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis and their paralogs have been characterized for their functional and structural properties as well as phylogeny using bioinformatics approach. The expression analysis suggests that these genes are differentially expressed in different tissues (with maximal expression in young leaf), chemotypes and in response to salicylic acid (SA) and methyl jasmonate (MJ) treatments. Sub-cellular localization studies suggest that both paralogs of sterol ?-7 reductase (WsDWF5-1 and WsDWF5-2) are localized in the endoplasmic reticulum (ER) thus supporting their indispensible role in withanolide biosynthesis. Comprehensive information developed, in this study, will lead to elucidation of chemotype- as well as tissue-specific withanolide biosynthesis and development of new tools for functional genomics in this important medicinal plant.     

Metabolite Profiling in Withania somnifera Roots Hydroalcoholic Extract Using LC/MS,GC/MS and NMR Spectroscopy

Ashwagandha (Withania somnifera) is a very well‐known herbal medicine and it was well studied for its active metabolites throughout the World. Although, nearly 40 withanolides were isolated from W. somnifera root extract, still there is remaining unidentified metabolites due to very low abundance and geographical variation. Advanced separation technology with online identification by mass and nuclear magnetic resonance (NMR) are nowadays used to find out the new compounds in the crude herbal extract. This article described the metabolite profiling of ashwagandha root hydroalcoholic extract using ultra‐performance liquid chromatography coupled with a positive ion electrospray ionization tandem mass spectrometry through gas chromatography mass spectrometry (GC/MS) and NMR spectroscopy. A total of 43 possible withanolides was identified and proposed their structures based on the mass of molecular and fragment ions. GC/MS and NMR analysis indicated the presence of several known withanolides including withaferin A, withanolide D, withanoside IV or VI, withanolide sulfoxide, etc. To the best of our knowledge, dihydrowithanolide D at m/z 473 (t_R 7.86 min) and ixocarpalactone A at m/z 505 (t_R 8.43 min) were first time identified in the ashwagandha root hydroalcoholic extract. The current study that described the identification of withanolides with summarized literature review might be helpful for designing the experiment to identify of the new chemical constituents in Withania species.     

Indirect regeneration of Withania somnifera and comparative analysis of withanolides in in vitro and greenhouse grown plants

The present study reports an efficient protocol for indirect shoot organogenesis and plantlets regeneration of Withania somnifera (L.) Dunal. Leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA). The highest callus induction rate (89.5 %) and shoot regeneration rate (92 %) were obtained when 2 mg dm⁻³ BAP was combined with 0.5 mg dm⁻³ IAA. Three major withanolides (withaferine A, 12-deoxywithastramonolide and withanolide A) were investigated in different plant organs from in vitro and greenhouse grown plants. Leaves contained higher contents of withanolides and phenolics than roots or stems, whereas roots contained the highest contents of flavonoids and polysacharides. In vitro grown plants contained greater contents of phenolics, flavonoids and polysaccharides while lower contents of withanolides than greenhouse grown plants.     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of Withania somnifera (L.) Dunal in Shake-Flask Culture and Bioreactor

The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28^th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.     

Growth kinetics and withanolide production in novel transformed roots of <Emphasis Type="Italic">Withania somnifera</Emphasis> and measurement of their antioxidant potential using chemiluminescence

Markedly increased withanolide content was found in transformed roots (TR) of Withania somnifera germplasm grown in low mineral minimal media and withanolides showed high antioxidant potential when analysed using acidic potassium permanganate chemiluminescence. Transformation frequency of explants infected with Agrobacterium rhizogenes strain A4 varied between the three germplasms tested with the highest observed as 75?±?0.9. Transformed root production was explant specific with leaves being the most productive among the different explants used. Withanolides, namely withaferin A, withanolide A, withanolide B and 12-deoxywithastramonolide were detected in TR cultures and differences in their content were found between germplasms. The highest concentrations of secondary metabolites were found in 4-week-old cultures and concentrations declined by the 8th and 12th week of culture. In 4-week-old cultures, the biomass of TR cultures was 4.5 fold higher than their respective non-transformed roots (NTR). Withaferin A was found in TR at levels that were 28–34 times higher than that found in NTR. A rapid method for the determination of the antioxidant potential of W. somnifera TR extracts was developed using post-column acidic potassium permanganate chemiluminescence (APPC) detection. The APPC chromatographic peaks for extract constituents showed strong alignment with those found for ultraviolet absorbance detection. The methods developed in this study for TR culture establishment and the use of a fast and sensitive way for the qualitative and quantitative determination of the antioxidant activity of their metabolites provides a new platform that will have use for similar studies in other species.     

Studies on leaf spot disease of <Emphasis Type="Italic">Withania somnifera</Emphasis> and its impact on secondary metabolites

During an investigation of the disease profile of Withania somnifera, it was observed that leaf spot is the most prevalent disease. Repeated isolations from infected leaf tissues and pathogenicity tests showed the association of fungal pathogen identified as Alternaria alternata (Fr.) Keissler. Scanning electron microscopy showed various histological changes in the leaf tissues of infected plants. A decrease in total content of reducing sugars (20%) and chlorophyll (26.5%) was observed in diseased leaves whereas an increase was noticed in proline (25%), free amino acids (3%) and proteins (74.3%). High performance thin layer chromatography (HPTLC) analysis of secondary metabolites viz. withanolides, withaferin-A and total alkaloids of the diseased leaves vis-à-vis control revealed reduction in withaferin-A and withanolides contents by 15.4% and 76.3% respectively, in contrast to an increase in total alkaloids by 49.3%, information hitherto unreported in W. somnifera.     

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of <Emphasis Type="Italic">Withania somnifera</Emphasis>

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the T_L T-DNA, and 90% of them carried the T_R T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the T_L and the T_R-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the T_R T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the T_L T-DNA genes, while all the callusing lines induced by strain A4 contained the T_L DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.     

A Δ16-withanolide in Withania somnifera as a possible precursor for α-side-chains

The structure of a new naturally occurring steroidal lactone of the withanolide group isolated from Withania somnifera chemotype III has been elucidated as (20R,22R)-14α,2Oα_F-dihydroxy-1-oxowitha-2,5,16,24-tetraenolid. This compound is considered to be an intermediate in the biosynthesis of withanolide E, and is at the origin of the unusual α-oriented side-chain in this compound. The comparative composition of withanolides in different sub-chemotypes of III is provided.