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Ashwagandha (Withania somnifera L. Dunal) is a versatile medicinal plant of Solanaceae family, renowned for its potent therapeutic properties, due to which it is extensively used in Indian traditional systems of medicine such as Ayurveda. The medicinal properties are attributed to specialized metabolites known as withanolides, which are chemically triterpenoid steroidal lactones. Despite their significance, the biosynthetic pathway of withanolides remains poorly understood. It is hypothesized that withanolides are synthesized through the universal sterol pathway, wherein sterol precursors undergo various biochemical modifications such as hydroxylation, oxidation, cyclization, and glycosylation, yielding a diverse array of downstream withanolides and withanosides. Consequently, comprehending the biosynthetic pathway of withanolides is crucial to facilitate advancements in withanolides productivity through metabolic engineering or synthetic biology approaches. This article aims to provide an update on the efforts made toward understanding withanolides formation and regulation and highlights gaps and approaches to elucidate the withanolides biosynthesis in W. somnifera., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Silver (Ag) is a non-essential heavy metal with substantial environmental toxicity but an excellent promotor for plant organogenesis. It is used as an elicitor for secondary metabolite production and for in planta synthesis of metal nanoparticles (MNPs). In the present study, the Ag accumulation and reduction capability of in vitro shoots of Withania somnifera and the toxicity and elicitation effect of Ag on in vitro shoots were explored. In vitro shoot cultures of W. somnifera were treated with different concentrations of silver nitrate for a specific treatment period. Growth index, withaferin A, elemental and electron microscopy analyses were done on silver-treated in vitro shoots of W. somnifera. 1 mM silver nitrate treatment for 12 days period was found to give increased growth index (1.425 ± 0.05c) and withaferin A (2.568 ± 0.08e mg g -1 ) content. The concentration of bioaccumulated Ag in 1 mM silver nitrate treated in vitro shoot was found to be 50.8 ppm. The presence of nano-Ag was also found in the leaves of 1 mM silver nitrate-treated in vitro shoots. In summary, this is the first report portraying the bioaccumulation and in planta reduction capability of the in vitro shoot system of W. somnifera, which makes it a potential medicinal plant of commercial value for silver contaminated soils., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Withanolides represent an important category of natural products with a steroidal lactone core. Many of them contain an α,β-unsaturated carbonyl moiety with a high reactivity toward sulfhydryl groups, including protein cysteine thiols. Different withanolides endowed with marked antitumor and anti-inflammatory have been shown to form stable covalent complexes with exposed cysteines present in the active site of oncogenic kinases (BTK, IKKβ, Zap70), metabolism enzymes (Prdx-1/6, Pin1, PHGDH), transcription factors (Nrf2, NFκB, C/EBPβ) and other structural and signaling molecules (GFAP, β-tubulin, p97, Hsp90, vimentin, M pro , IPO5, NEMO, …). The present review analyzed the covalent complexes formed through Michael addition alkylation reactions between six major withanolides (withaferin A, physalin A, withangulatin A, 4β-hydroxywithanolide E, withanone and tubocapsanolide A) and key cysteine residues of about 20 proteins and the resulting biological effects. The covalent conjugation of the α,β-unsaturated carbonyl system of withanolides with reactive protein thiols can occur with a large set of soluble and membrane proteins. It points to a general mechanism, well described with the leading natural product withaferin A, but likely valid for most withanolides harboring a reactive (electrophilic) enone moiety susceptible to react covalently with cysteinyl residues of proteins. The multiplicity of reactive proteins should be taken into account when studying the mechanism of action of new withanolides. Proteomic and network analyses shall be implemented to capture and compare the cysteine covalent-binding map for the major withanolides, so as to identify the protein targets at the origin of their activity and/or unwanted effects. Screening of the cysteinome will help understanding the mechanism of action and designing cysteine-reactive electrophilic drug candidates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14 mg l -1 ) induced 11.49 -fold increase in withanolides (89.07 ± 2.75 mg g -1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11 mg g - 1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63 mg g -1 DW of withanolides and 15.66 ± 0.92 mg g -1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids., Competing Interests: Declaration of Competing Interest Authors declare that the investigation reported in this paper was not influenced by any competing financial interests or relationships., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Withanolides are steroidal lactones with diverse bioactive potential and their production from plant sources varies with genotype, age, culture conditions, and geographical region. Endophytic fungi serve as an alternative source to produce withanolides, like their host plant, Withania somnifera (L.) Dunal. The present study aimed to isolate endophytic fungi capable of producing withanolides, characterization and investigation of biological activities of these molecules. The methanolic fungal crude extract of one of the fungal isolates WSE16 showed maximum withanolide production (219 mg/L). The fungal isolate WSE16 was identified as Penicillium oxalicum based on its morphological and internal transcribed spacer (ITS) sequence analysis and submitted in NCBI (accession number OR888725). The methanolic crude extract of P. oxalicum was further purified by column chromatography, and collected fractions were assessed for the presence of withanolides. Fractions F3 and F4 showed a higher content of withanolides (51.8 and 59.1 mg/L, respectively) than other fractions. Fractions F3 and F4 exhibited antibacterial activity against Staphylococcus aureus with an IC 50 of 23.52 and 17.39 µg/ml, respectively. These fractions also showed antioxidant activity (DPPH assay with IC 50 of 39.42 and 38.71 µg/ml, superoxide anion scavenging assay with IC 50 of 41.10 and 38.84 µg/ml, and reducing power assay with IC 50 of 42.61 and 41.40 µg/ml, respectively) and acetylcholinesterase inhibitory activity (IC 50 of 30.34 and 22.05 µg/ml, respectively). The withanolides present in fraction 3 and fraction 4 were identified as (20S, 22R)-1a-Acetoxy-27-hydroxywitha-5, 24-dienolide-3b-(O-b-D-glucopyranoside) and withanamide A, respectively, using UV, FTIR, HRMS, and NMR analysis. These results suggest that P. oxalicum, an endophytic fungus isolated from W. somnifera, is a potential source for producing bioactive withanolides., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Ethnopharmacological Relevance: Withania somnifera (L.) Dunal, known as Ashwagandha, has long been used in traditional medicine in Ayurveda, India, a representative adaptogen. The main active constituents of W. somnifera are withanolides, and the root is often used as a medicine with a wide range of pharmacological activities, which can be used to treat insomnia, neurasthenia, diabetes mellitus and skin cancer., Aim of the Study: Whole-component qualitative and quantitative analyses were performed on W. somnifera. We explored the ameliorative effect of the adaptogen representative plant W. somnifera on the senescence events of MGO-injured fibroblasts and its action mechanism and verified the hypotheses that WS can inhibit the accumulation of AGEs and regulate the dynamic balance among the components of the ECM by modulating the expression of integrin β1 receptor; as a result, WS maintains cellular behavioural and biological functions in a normal range and retards the aging of skin from the cellular level., Materials and Methods: In this study, the components of WS were first qualitatively and quantitatively analysed by HPLC fingerprinting and LC-MS detection. Second, a model of MGO-induced injury of CML-overexpressing fibroblasts was established. ELISA was used to detect CML expression and the synthesis of key extracellular matrix ECM protein components COL1, FN1, LM5 and TNC synthesis; CCK-8 was used to detect cell viability; EDU was used to detect cell proliferation capacity; fluorescence was used to detect cell adhesion capacity; and migration assay were used to detect cell migration capacity; qRT-PCR was used to detect the regulatory pathway TGF-β1 and MMP-2, MMP-9 in ECMs; immunofluorescence was used to detect the expression of ITGB1; and WB was used to detect the expression of COL1, FN1, LM5, Tnc, TGF-β1, MMP-2, MMP-9 and ITGB1., Results: In total, 27 active ingredients were analysed from WS, which mainly consisted of withanolide components, such as withaferin A and withanolide A. Based on the model of MGO-induced fibroblast senescence injury, WS significantly inhibited CML synthesis. By up-regulating the expression of integrin β1, it upregulated the expression of the TGF-β1 gene, which is closely related to the generation of ECMs, downregulated the expression of the MMP-2 and MMP-9 genes, which are closely related to the degradation of ECMs, maintained the dynamic balance of the four types of ECMs, and improved cell viability as well as proliferation, migration and adhesion abilities., Conclusions: WS can prevent cellular behavioural dysfunction and delay skin ageing by reducing the accumulation of CML, upregulating the expression of the ITGB1 receptor, maintaining the normal function of ECM-integrin receptor interaction and preventing an imbalance between the production and degradation of protein components of ECMs. The findings reported in this study suggest that WS as a CML inhibitor can modulate ECM-integrin homeostasis and has great potential in the field of aging retardation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Withania somnifera (Ashwagandha), is one of the most reputed Indian medicinal plants, having immense pharmacological activities due to the occurrence of withanolides. The withanolides are biosynthesized through triterpenoid biosynthetic pathway with the involvement of WsCAS leading to cyclization of 2, 3 oxidosqualene, which is a key metabolite to further diversify to a myriad of phytochemicals. In contrast to the available reports on the studies of WsCAS in withanolide biosynthesis, its involvement in phytosterol biosynthesis needs investigation. Present work deals with the understanding of role of WsCAS triterpenoid synthase gene in the regulation of biosynthesis of phytosterols & withanolides. Docking studies of WsCAS protein revealed Conserved amino acids, DCATE motif, and QW motif which are involved in efficient substrate binding, structure stabilization, and catalytic activity. Overexpression/silencing of WsCAS leading to increment/decline of phytosterols confers its stringent regulation in phytosterols biosynthesis. Differential regulation of WsCAS on the metabolic flux towards phytosterols and withanolide biosynthesis was observed under abiotic stress conditions. The preferential channelization of 2, 3 oxidosqualene towards withanolides and/or phytosterols occurred under heat/salt stress and cold/water stress, respectively. Stigmasterol and β-sitosterol showed major contribution in high/low temperature and salt stress, and campesterol in water stress management. Overexpression of WsCAS in Arabidopsis thaliana led to the increment in phytosterols in general. Thus, the WsCAS plays important regulatory role in the biosynthetic pathway of phytosterols and withanolides under abiotic stress conditions., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (μ = 0.036 day -1 ) and growth index (GI = 9.18), and the shortest doubling time (T d  = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM β-CD + 100 μM MeJA (9.57 mg/g DW) and 5.0 mM β-CD + 100 μM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined β-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and β-CD on withaferin A production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Ashwagandha ( Withania somnifera ) has gained worldwide popularity for a multitude of health benefits inclusive of cancer-preventive and curative effects. Despite numerous research data supporting the benefits of this wonder herb, the actual use of ashwagandha for cancer treatment in clinics is limited. The primary reason for this is the inconsistent therapeutic outcome due to highly variable composition and constitution of active ingredients in the plant extract impacting ashwagandha's pharmacology. We investigate here an engineered yield: an ashwagandha extract (Oncowithanib) that has a unique and fixed portion of active ingredients to achieve consistent and effective therapeutic activity. Using the MCF7 cell line, Oncowithanib was studied for its anti-neoplastic efficacy and drug targets associated with cell cycle regulation, translation machinery, and cell survival and apoptosis. Results demonstrate a dose-dependent decline in Oncowithanib-treated MCF7 cell viability and reduced colony-forming ability. Treated cells showed increased cell death as evidenced by enhancement of Caspase 3 enzyme activity and decreased expressions of cell proliferation markers such as Ki67 and Aurora Kinase A. Oncowithanib treatment was also found to be associated with expressional suppression of key cellular kinases such as RSK1, Akt1, and mTOR in MCF7 cells. Our findings indicate that Oncowithanib decreases MCF7 cell survival and propagation, and sheds light on common drug targets that might be good candidates for the development of cancer therapeutics. Further in-depth investigations are required to fully explore the potency and pharmacology of this novel extract. This study also highlights the importance of the standardization of herbal extracts to get consistent therapeutic activity for the disease indication., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: All the authors have approved the manuscript and all but Franco Cavaleri have unanimously declared that there is no potential conflict of interest for them. Franco Cavaleri is the owner and CEO of Biologic Pharmamedical Research, the manufacturer of one of the subject treatment protocols, Oncowithanib.

Withania somnifera (L.) Dunal, popularly known as Ashwagandha or Indian ginseng, is well acclaimed for its health-enhancing effects, including its potent immunomodulatory, anti-inflammatory, neuroprotective, and anti-tumorigenic properties. The prime biological effectors of these attributes are a diverse group of ergostane-based steroidal lactones termed withanolides. Withanones and withanosides are distributed differentially across the plant body, whereas withanolides and withanones are known to be more abundant in leaves, while withanosides are found exclusively in the roots of the plants. Standardized W. somnifera extract is Generally Recognized as Safe (GRAS)-affirmed, however, moderate to severe toxic manifestations may occur at high dosages. Withaferin A, which also happens to be the primary bioactive ingredient for the effectiveness of this plant. There have been contrasting reports regarding the distribution of withaferin A in W. somnifera . While most reports state that the roots of the plant have the highest concentrations of this phytochemical, several others have indicated that leaves can accumulate withaferin A in proportionately higher amounts. A comprehensive survey of the available reports suggests that the biological effects of Ashwagandha are grossly synergistic in nature, with many withanolides together mediating the desired physiological effect. In addition, an assorted formulation of withanolides can also neutralize the toxic effects (if any) associated with withaferin A. This mini-review presents a fresh take on the recent developments regarding the safety and toxicity of the plant, along with a critical assessment of the use of roots against leaves as well as whole plants to develop therapeutic formulations. Going by the currently available scientific evidence, it is safe to infer that the use of whole plant formulations instead of exclusively root or leaf recipes may present the best possible option for further exploration of therapeutic benefits from this novel medicinal plant.HighlightsTherapeutic potential of withanolides owes to the presence of α,β unsaturated ketone which binds to amines, alcohols, and esters and 5β, 6β epoxy group which react with side chain thiols of proteins.At concentrations above NOAEL (no observed adverse effect level), the same mechanisms contribute towards toxicity of the molecule.Although withanosides are found exclusively in roots, whole plants have higher contents of withanones and withanolides.Whole plant-based formulations have other metabolites which can nullify the toxicity associated with roots.Extracts made from whole plants, therefore can holistically impart all therapeutic benefits as well as mitigate toxicity.

Cancer is characterized by the abnormal development of cells that divide in an uncontrolled manner and further take over the body and destroy the normal cells of the body. Although several therapies are practiced, the demand and need for new therapeutic agents are ever-increasing because of issues with the safety, efficacy and efficiency of old drugs. Several plant-based therapeutics are being used for treatment, either as conjugates with existing drugs or as standalone formulations. Withania somnifera (L.) Dunal is a highly studied medicinal plant which is known to possess immunomodulatory activity as well as anticancer properties. The pivotal role of KAT6A in major cellular pathways and its oncogenic nature make it an important target in cancer treatment. Based on the literature and curated datasets, twenty-six compounds from the root of W . somnifera and a standard inhibitor were docked with the target KAT6A using Autodock vina. The compounds and the inhibitor complexes were subjected to molecular dynamics simulation (50 ns) using Desmond to understand the stability and interactions. The top compounds (based on the docking score of less than -8.5 kcal/mol) were evaluated in comparison to the inhibitor. Based on interactions at ARG655, LEU686, GLN760, ARG660, LEU689 and LYS763 amino acids with the inhibitor WM-8014, the compounds from W . somnifera were evaluated. Withanolide D, Withasomniferol C, Withanolide E, 27-Hydroxywithanone, Withanolide G, Withasomniferol B and Sitoindoside IX showed high stability with the residues of interest. The cell viability of human breast cancer MCF-7 cells was evaluated by treating them with W. Somnifera root extract using an MTT assay, which showed inhibitory activity with an IC50 value of 45 µg/mL. The data from the study support the traditional practice of W . somnifera as an anticancer herb.

SARS-CoV-2 outbreak in China in December 2019 and its spread as worldwide pandemic has been a major global health crisis. Extremely high infection and mortality rate has severely affected all sectors of life and derailed the global economy. While drug and vaccine development have been prioritized and have made significant progression, use of phytochemicals and herbal constituents is deemed as a low-cost, safer and readily available alternative. We investigated therapeutic efficacy of eight withanolides (derived from Ashwagandha) against the angiotensin-converting enzyme 2 (ACE2) proteins, a target cell surface receptor for SARS-CoV-2 and report results on the (i) computational analyses including binding affinity and stable interactions with ACE2, occupancy of ACE2 residues in making polar and nonpolar interactions with different withanolides/ligands and (2) in vitro mRNA and protein analyses using human cancer (A549, MCF7 and HSC3) cells. We found that among all withanolides, Withaferin-A, Withanone, Withanoside-IV and Withanoside-V significantly inhibited the ACE2 expression. Analysis of withanolides-rich aqueous extracts derived from Ashwagandha leaves and stem showed a higher ACE2 inhibitory potency of stem-derived extracts. Taken together, we demonstrated the inhibitory potency of Ashwagandha withanolides and its aqueous extracts against ACE2.Communicated by Ramaswamy H. Sarma.

Traditionally, Withania somnifera is widely used as an immune booster, anti-viral, and for multiple medicinal purposes. The present study investigated the withanolides as an immune booster and anti-viral agents against the coronavirus-19. Withanolides from Withania somnifera were retrieved from the open-source database, their targets were predicted using DIGEP-Pred, and the protein-protein interaction was evaluated. The drug-likeness score and intestinal absorptivity of each compound were also predicted. The network of compounds, proteins, and modulated pathways was constructed using Cytoscape, and docking was performed using autodock4.0, and selected protein-ligand complexes were subjected to 100 ns Molecular Dynamics simulations. The molecular dynamics trajectories were subjected to free energy calculation by the MM-GBSA method. Withanolide_Q was predicted to modulate the highest number of proteins, showed human intestinal absorption, and was predicted for the highest drug-likeness score. Similarly, combined network interaction identified Withanolide_Q to target the highest number of proteins; RAC1 was majorly targeted, and fluid shear stress and atherosclerosis associated pathway were chiefly regulated. Similarly, Withanolide_D and Withanolide_G were predicted to have a better binding affinity with PLpro, Withanolide_M with 3CLpro, and Withanolide_M with spike protein based on binding energy and number of hydrogen bond interactions. MD studies suggested Withanoside_I with the highest binding free energy (ΔG bind -31.56 kcal/mol) as the most promising inhibitor. Among multiple withanolides from W. somnifera, Withanolide_D, Withanolide_G, Withanolide_M, and Withanolide_Q were predicted as the lead hits based on drug-likeness score, modulated proteins, and docking score to boost the immune system and inhibit the COVID-19 infection, which could primarily act against COVID-19. HighlightsWithanolides are immunity boosters.Withanolides are a group of bio-actives with potential anti-viral properties.Withanolide_G, Withanolide_I, and Withanolide_M from Withania somnifera showed the highest binding affinity with PLpro, 3CLpro, and spike protein, respectively.Withanolides from Withania somnifera holds promising anti-viral efficacy against COVID-19.Communicated by Vsevolod Makeev.

Main Conclusion: Overexpression of a novel geranylgeranyl pyrophosphate synthase gene (WsGGPPS) in planta resulted in increased levels of gibberellic acid and decrease in withanolide content. Withania somnifera (L.) Dunal, the herb from family Solanaceae is one of the most treasured medicinal plant used in traditional medicinal systems owing to its unique stockpile of pharmaceutically active secondary metabolites. Phytochemical and pharmacological studies in this plant were well established, but the genes affecting the regulation of biosynthesis of major metabolites were not well elucidated. In this study cloning and functional characterization of a key enzyme in terpenoid biosynthetic pathway viz. geranylgeranyl pyrophosphate synthase (EC 2.5.1.29) gene from Withania somnifera was performed. The full length WsGGPPS gene contained 1,104 base pairs that encode a polypeptide of 365 amino acids. The quantitative expression analysis suggested that WsGGPPS transcripts were expressed maximally in flower tissues followed by berry tissues. The expression levels of WsGGPPS were found to be regulated by methyl jasmonate (MeJA) and salicylic acid (SA). Amino acid sequence alignment and phylogenetic studies suggested that WsGGPPS had close similarities with GGPPS of Solanum tuberosum and Solanum pennellii. The structural analysis provided basic information about three dimensional features and physicochemical parameters of WsGGPPS protein. Overexpression of WsGGPPS in planta for its functional characterization suggested that the WsGGPPS was involved in gibberellic acid biosynthesis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Identification of novel anti-inflammatory strategies are needed to avoid the side effects associated with the currently available therapies. Use of anti-inflammatory herbal remedies is gaining attention. The purpose of the present investigation was to evaluate the pharmacological potential of the withanolide-rich root extracts of the medical plant Withania somnifera (L.) Dunal using in vivo and in vitro models of endotoxin-induced inflammation and oxidative stress. The pharmacological effects of W. somnifera root extracts were evaluated using a mouse model of endotoxin (lipopolysaccharide)-induced peritonitis and various relevant human cell lines. HPLC analysis of the W. somnifera root extracts identified the presence of various bioactive withanolides. In vivo challenge of mice with endotoxin resulted in the infiltration of various leukocytes, specifically neutrophils, along with monocytes and lymphocytes into the peritoneal cavity. Importantly, prophylactic treatment with W. somnifera inhibited the migration of neutrophils, lymphocytes, and monocytes and decreased the release of interleukin-1 β , TNF- α , and interleukin-6 cytokines into the peritoneal cavity as identified by ELISA. Liver (glutathione peroxidase, glutathione, glutathione disulfide, superoxide dismutase , m alondialdehyde , myeloperoxidase) and peritoneal fluid (nitrite) biochemical analysis revealed the antioxidant profile of W. somnifera . Similarly, in human HepG2 cells, W. somnifera significantly modulated the antioxidant levels. In THP-1 cells, W. somnifera decreased the secretion of interleukin-6 and TNF- α . In HEK-Blue reporter cells, W. somnifera inhibited TNF- α -induced nuclear factor- κ B/activator protein 1 transcriptional activity. Our findings suggest the pharmacological effects of root extracts of W. somnifera rich in withanolides inhibit neutrophil infiltration, oxidative hepatic damage, and cytokine secretion via modulating the nuclear factor- κ B/activator protein 1 pathway., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Oxidative stress and the AKT serine/threonine kinase (AKT) signaling pathway are essential regulators in cellular migration, metastasis, and angiogenesis. More than 300 withanolides were discovered from the plant family Solanaceae, exhibiting diverse functions. Notably, the relationship between oxidative stress, AKT signaling, and angiogenesis in withanolide treatments lacks comprehensive understanding. Here, we summarize connecting evidence related to oxidative stress, AKT signaling, and angiogenesis in the zebrafish model. A convenient vertebrate model monitored the in vivo effects of developmental and tumor xenograft angiogenesis using zebrafish embryos. The oxidative stress and AKT-signaling-modulating abilities of withanolides were highlighted in cancer treatments, which indicated that further assessments of their angiogenesis-modulating potential are necessary in the future. Moreover, targeting AKT for inhibiting AKT and its AKT signaling shows the potential for anti-migration and anti-angiogenesis purposes for future application to withanolides. This particularly holds for investigating the anti-angiogenetic effects mediated by the oxidative stress and AKT signaling pathways in withanolide-based cancer therapy in the future.

The medicinal properties of Ashwagandha (Withania somnifera) are attributed to triterpenoid steroidal lactones, withanolides, which are proposed to be derived from phytosterol pathway, through the action of cytochrome P450 (CYP450) enzymes. Here, we report the characterization of three transcriptome-mined CYP450 genes (WsCYP749B1, WsCYP76 and WsCYP71B10), which exhibited induced expression in response to methyl jasmonate treatment indicating their role in secondary metabolism. All three WsCYP450s had the highest expression in leaf compared to other tissues. In planta characterization of WsCYP450s through virus induced gene silencing (VIGS) and transient overexpression approaches and subsequent metabolite analysis indicated differential modulation in the accumulation of certain withanolides in W. somnifera leaves. While WsCYP749B1-vigs significantly enhanced withaferin A (~ 450%) and reduced withanolide A (~ 50%), its overexpression drastically led to enhanced withanolide A (> 250%) and withanolide B (> 200%) levels and reduced 12-deoxywithastramonolide (~ 60%). Whereas WsCYP76-vigs led to reduced withanolide A (~ 60%) and its overexpression increased withanolide A (~ 150%) and reduced 12-deoxywithastramonolide (~ 60%). Silencing and overexpression of WsCYP71B10 resulted in significant reduction of withanolide B (~ 50%) and withanolide A (~ 60%), respectively. Further, while VIGS of WsCYP450s negatively affected the expression of pathogenesis-related (PR) genes and compromised tolerance to bacteria P. syringae DC3000, their overexpression in W. somnifera and transgenic tobacco led to improved tolerance to the bacteria. Overall, these results showed that the identified WsCYP450s have a role in one or several steps of withanolides biosynthetic pathway and are involved in conferring tolerance to biotic stress., (© 2022. The Author(s).)

Withanolides constitute one of the most interesting classes of natural products due to their diversity of structures and biological activities. Our recent studies on withanolides obtained from plants of Solanaceae including Withania somnifera and a number of Physalis species grown under environmentally controlled aeroponic conditions suggested that this technique is a convenient, reproducible, and superior method for their production and structural diversification. Investigation of aeroponically grown Physalis coztomatl afforded 29 withanolides compared to a total of 13 obtained previously from the wild-crafted plant and included 12 new withanolides, physacoztolides I-M ( 9 - 13 ), 15 α -acetoxy-28-hydroxyphysachenolide C ( 14 ), 28-oxophysachenolide C ( 15 ), and 28-hydroxyphysachenolide C ( 16 ), 5 α -chloro-6 β -hydroxy-5,6-dihydrophysachenolide D ( 17 ), 15 α -acetoxy-5 α -chloro-6β-hydroxy-5,6-dihydrophysachenolide D ( 18 ), 28-hydroxy-5 α -chloro-6 β -hydroxy-5,6-dihydrophysachenolide D ( 19 ), physachenolide A-5-methyl ether ( 20 ), and 17 known withanolides 3 - 5 , 8 , and 21 - 33 . The structures of 9 - 20 were elucidated by the analysis of their spectroscopic data and the known withanolides 3 - 5 , 8 , and 21 - 33 were identified by comparison of their spectroscopic data with those reported. Evaluation against a panel of prostate cancer (LNCaP, VCaP, DU-145, and PC-3) and renal carcinoma (ACHN) cell lines, and normal human foreskin fibroblast (WI-38) cells revealed that 8, 13 , 15 , and 17 - 19 had potent and selective activity for prostate cancer cell lines. Facile conversion of the 5,6-chlorohydrin 17 to its 5,6-epoxide 8 in cell culture medium used for the bioassay suggested that the cytotoxic activities observed for 17 - 19 may be due to in situ formation of their corresponding 5β,6β-epoxides, 8 , 27 , and 28 .

Medicinal plants have served humans as medicine for centuries. Withania somnifera (L.) (Ashwagandha) leaf extract is traditionally used in managing and treating bacterial infections. A combination of experimental and computational methods was used to investigate the related antibacterial mechanism. Leaf extract showed strong antibacterial activity against S. aureus . Moreover, molecular docking established that withanolide C, a compound obtained from methanolic leaf extract binded strongly to DNA gyrase enzyme. Molecular dynamics simulation and molecular mechanics Poisson-Boltzmann surface area binding free energy suggested withanolide C to be stable at the active site of DNA gyrase B. The compound binded in a different fashion as compared to chlorobiocin a known DNA gyrase inhibitor. Present finding suggests that the antibacterial activity of W. somnifera is due to inhibition of DNA gyrase by withanolide C. This finding serves as the basis for development of novel antimicrobial agents.Communicated by Ramaswamy H. Sarma.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A 3 (4093 μg mL -1 ), Jujubogenin isomer of Bacopasaponin C (65,339 μg mL -1 ), and Bacopasaponin C (1325 μg mL -1 ) while Bacopaside II (13,030 μg mL -1 ) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 μg mL -1 ), and Withanolide B (1024 μg mL -1 ) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions., (© 2021. Sociedade Brasileira de Microbiologia.)

Globally, Alzheimer's disease (AD) is one of the most prevalent age-related neurodegenerative disorders associated with cognitive decline and memory deficits due to beta-amyloid deposition (Aβ) and tau protein hyperphosphorylation. To date, approximately 47 million people worldwide have AD. This figure will rise to an estimated 75.6 million by 2030 and 135.5 million by 2050. According to the literature, the efficacy of conventional medications for AD is statistically substantial, but clinical relevance is restricted to disease slowing rather than reversal. Withaferin A (WA) is a steroidal lactone glycowithanolides, a secondary metabolite with comprehensive biological effects. Biosynthetically, it is derived from Withania somnifera (Ashwagandha) and Acnistus breviflorus (Gallinero) through the mevalonate and non-mevalonate pathways. Mounting evidence shows that WA possesses inhibitory activities against developing a pathological marker of Alzheimer's diseases. Several cellular and animal models' particulates to AD have been conducted to assess the underlying protective effect of WA. In AD, the neuroprotective potential of WA is mediated by reduction of beta-amyloid plaque aggregation, tau protein accumulation, regulation of heat shock proteins, and inhibition of oxidative and inflammatory constituents. Despite the various preclinical studies on WA's therapeutic potentiality, less is known regarding its definite efficacy in humans for AD. Accordingly, the present study focuses on the biosynthesis of WA, the epidemiology and pathophysiology of AD, and finally the therapeutic potential of WA for the treatment and prevention of AD, highlighting the research and augmentation of new therapeutic approaches. Further clinical trials are necessary for evaluating the safety profile and confirming WA's neuroprotective potency against AD.

Ethnopharmacological Relevance: Withania somnifera is one of the most extensively delved Ayurvedic medicine. Apart from rejuvenation and increasing longevity, it has several other properties such as immunomodulation, anti-cancer, anti-stress and neuroprotection. Because of its prevailing use and increasing demand, it becomes prudent to scientifically evaluate and document both its propagation and production of desired phytoconstituents., Aim of the Study: This review aims to highlight the research progress achieved on various biotechnological and tissue culture aspects of Withania somnifera and to cover up-to-date information regarding in-vitro propagation and production of withanolides., Materials and Methods: Significant published studies were identified for the years 2000-2018 using Elsevier-Science Direct, Pubmed and Google scholar and several research studies in our laboratory. Following keywords such as "plant extracts", "in vitro cultures", "callus and suspension culture", "micropropagation", "hairy root cultures" were used. Further, "Withania somnifera", "secondary metabolites specially withanolides", "molecular techniques" and "in vitro conservation" were used to cross-reference the keywords., Results: Ashwagandha comprises a broad spectrum of phytochemicals with a wide range of pharmacological properties. W. somnifera seeds have reduced viability and germination rates; thus, its regular cultivation method fails to achieve commercial demands mainly for the production of desired phytoconstituents. Cultivation of plant cells/tissues under in vitro conditions and development of various biotechnological strategies will help to build an attractive alternative to provide adequate quality and quantity raw materials. Recently, a large number of in vitro protocols has developed for W. somnifera not only for its propagation but for the production of secondary metabolites as well. Present work highlights a variety of biotechnological strategies both for prompt propagation and production of different bioactive secondary metabolites., Conclusion: The present review focuses on the development and opportunities in various biotechnological approaches to accomplish the global demand of W. somnifera and its secondary metabolites. This review underlines the advances in plant biotechnological approaches for the propagation of W. somnifera and production of its bioactive compounds., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Withanone (WN), an active constituent of Withania somnifera commonly called Ashwagandha has remarkable pharmacological responses along with neurological activities. However, for a better understanding of the pharmacokinetic and pharmacodynamic behavior of WN, a comprehensive in-vitro ADME (absorption, distribution, metabolism, and excretion) studies are necessary., Aim of the Study: A precise, accurate, and sensitive reverse-phase ultra-performance liquid chromatographic method of WN was developed and validated in rat plasma for the first time. The developed method was successfully applied to the in-vitro ADME investigation of WN., Material and Methods: The passive permeability of WN was assayed using PAMPA plates and the plasma protein binding (PPB) was performed using the equilibrium dialysis method. Pooled liver microsomes of rat (RLM) and human (HLM) were used for the microsomal stability, CYP phenotyping, and inhibition studies. CYP phenotyping was evaluated using the specific inhibitors. CYP inhibition study was performed using specific probe substrates along with WN or specific inhibitors., Results: WN was found to be stable in the simulated gastric and intestinal environment and has a high passive permeability at pH 4.0 and 7.0 in PAMPA assay. The PPB of WN at 5 and 20 μg/mL concentrations were found to be high i.e. 82.01 ± 1.44 and 88.02 ± 1.15%, respectively. The in vitro half-life of WN in RLM and HLM was found to be 59.63 ± 2.50 and 68.42 ± 2.19 min, respectively. CYP phenotyping results showed that WN was extensively metabolized by CYP 3A4 and1A2 enzymes in RLM and HLM. However, the results of CYP Inhibition studies showed that none of the CYP isoenzymes were potentially inhibited by WN in RLM and HLM., Conclusion: The in vitro results of pH-dependent stability, plasma stability, permeability, PPB, blood partitioning, microsomal stability, CYP phenotyping, and CYP inhibition studies demonstrated that WN could be a better phytochemical for neurological disorders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Here, we have studied the ameliorative effects of Withania somnifera derivatives (Withanolide A, Withanolide B, Withanoside IV, and Withanoside V) on the fibril formation of amyloid-β 42 for Alzheimer's disease. We analyzed reduction in the aggregation of β amyloid protein with these Ashwagandha derivatives by Thioflavin T assay in the oligomeric and fibrillar state. We have tested the cytotoxic activity of these compounds against human SK-N-SH cell line for 48 h, and the IC 50 value found to be 28.61 ± 2.91, 14.84 ± 1.45, 18.76 ± 0.76 and 30.14 ± 2.59 μM, respectively. After the treatment of the cells with half the concentration of IC 50 value, there was a remarkable decrease in the number of apoptotic cells stained by TUNEL assay indicating the DNA damage and also observed significant decrease of reactive oxygen species. Also, the binding and molecular stability of these derivatives with amyloid β was also studied using bioinformatics tools where these molecules were interacted at LVFFA region which is inhibition site of amyloid-β1 42 . These studies revealed that the Withanolides and Withanosides interact with the hydrophobic core of amyloid-β 1- 42 in the oligomeric stage, preventing further interaction with the monomers and diminishing aggregation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Withania somnifera (L.) Dunal, generally well-known as Ashwagandha, is part of Indian traditional medicinal systems like Ayurveda, Siddha, and Unani for over 3000 years for treating an array of disorders. The chief bioactive component of this plant is the withanolides, a group of C 28 -steroidal lactone triterpenoids. These compounds are present in very low concentrations and hence cell culture methods have been used to enhance their production. Low-level laser irradiation has been reported to have elicited the seed germination, agronomical characters, biosynthesis of bioactive compounds in some plants. Therefore, the objective of the study was to investigate the effect of red (He-Ne) laser irradiation on seed germination, growth characters, pigment contents and withanolide content in W. somnifera. The seeds were inoculated onto two different combinations of Murashige and Skoog (MS) media and incubated for germination. The highest germination percentage was observed in ½ MS with pH 6.5 and GA 3 presoaking followed by ½ MS with different pH. Four different doses of Helium-Neon (He-Ne) laser (10, 15, 20 and 25 J/cm 2 ) were used to irradiate the seeds at 632.8 nm and germinated in vitro on ½ MS with pH 6.5. The maximum germination percentage, 63.88% was noted from seeds irradiated with 25 J/cm 2 (P = 0.04). The highest total length of 13.33 cm was observed in the seedlings irradiated with 25 J/cm 2 groups (P = 0.008). The highest total chlorophyll content of 329.5 μg/g fresh weight (FW) was observed for seedlings irradiated with 15 J/cm 2 (P = 0.02) and the highest carotenoid content of 49.6 μg/g FW was observed for 25 J/cm 2 treated seedlings. Further, primary root length was measured and found to be highest (11.14 cm) in seedlings irradiated with 10 J/cm 2 and the highest number of lateral roots were observed for 15 and 25 J/cm 2 groups. The significant amount of Withanolide A (WA) 0.52 μg/g dry weight (DW) and 0.60 μg/g DW was noted in 15 (P = 0.01) and 20 J/cm 2 (P = 0.002) groups, respectively than control. The present investigation thus reveals the positive impact of red laser on the germination of seeds, growth characters and withanolide contents under in vitro environment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Key Message: Withania coagulans (L.) Dunal bio-synthesized silver nanoparticles (WcAgNPs) worked as an abiotic elicitor or auto-catalyst that enhanced root regeneration and withanolides production in in-vitro regenerated W. coagulans. Rapid development in the production / consumption of silver nanoparticles (AgNPs) raised serious concern over its effects on the growth of natural plant community. The knowledge related to impact of AgNPs on plant growth and biocompatibility is increasing day by day, but comprehensive mechanism and gaps regarding their impacts on plant health have yet to be addressed. In the present study, we investigated the impact of Withania coagulans biosynthesized AgNPs (WcAgNPs) on in-vitro plant growth and withanolides production. Obtained results showed that the low concentrations of WcAgNPs significantly induced the plant growth by regulating oxidative stress via anti-oxidative defense system. Physiological, morphology and anatomical features also reflected healthy plant growth under low WcAgNPs exposure. While higher concentrations of WcAgNPs have a negative impact on W. coagulans plant growth due to induced lipid peroxidation, ROS accumulation, and root cell death. At lower concentrations, WcAgNPs have shown a positive effect on in-planta withanolides biosynthesis stimulating withanolide A and withaferin A up to 11.15-22.8-fold, respectively. Furthermore, the expression of withanolides biosynthetic genes were also quantified upon WcAgNPs exposure and terpenes biosynthetic genes showed over-expression. Thus, the present study concludes that the lower concentrations of WcAgNPs positively induced plant growth via improved root organogenesis and also have potential to act as an elicitor for withanolides production.

Obesity is a risk factor for many diseases, including type 2 diabetes and cardiovascular disease, and is related to the rising morbidity and mortality. Discovery of agents targeting adipogenesis, especially from natural sources, is important for the treatment of obesity. Here, we aimed to identify anti-adipogenic substances in methanol extracts of Physalis peruviana and to investigate their effect, along with underlying mechanisms. Activity-guided fractionation of the extract revealed 4β-hydroxywithanolide E (HWE) and withanolide E (WE) as the adipogenesis inhibitors. Both compounds suppressed mRNA expression of central adipogenic transcription factors, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer-binding protein α in the early stage of adipocyte differentiation. The inhibitory action of these two withanolides on adipogenesis was largely limited to this stage. The proliferation of preadipocytes was markedly suppressed by treatment with HWE and WE for 24 and 48 h in the differentiation medium, and cell-cycle arrest in the G 0 /G 1 phase was observed. Therefore, our results suggested that withanolides from P. peruviana to be novel anti-adipogenic compounds that modulate mitotic clonal expansion.

Withanolides constitute an extensive and vital class of metabolites displaying wide array of structural and therapeutic properties with unique side-chain modifications. These show diversified scaffolds and are promising pharmaceutical molecules with well documented anti-inflammatory and anti-cancer properties. Sterols are dynamic class of compounds and essential molecules having structural and functional significance. These contribute to the synthesis of withanolides by providing structural precursors. In this context, we have characterized sterol Δ 22 -desaturase from Withania somnifera and also functionally validating it by confirming its desaturase nature in conjunction with quantitative real-time expression profiling and metabolite evaluation. Further, transgenic hairy roots of W. somnifera displayed a higher accumulation of stigmasterol and withanolides. The increase in chemical constituents was concomitant with an increased gene copy number predicted via Southern blotting. Additionally, transgenic lines of tobacco over-expressing WsCYP710A11 displayed a substantial increase in its expression, corroborating well with enhanced stigmasterol content. Characterization of CYP710A11 from W. somnifera and its homologous transgenic expression has demonstrated its role in the regulation of withanolides biosynthesis. It also exhibited a differential transcriptional profile in response to exogenous elicitations. These empirical findings suggest the crucial role of CYP710A11 in stigmasterol biosynthesis. This in turn has implications for the overproduction of withanolides via pathway channelling., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Among various in vitro plant culture systems, hairy root systems seem to be one of the most appealing methods of recombinant protein production due to their advantages in combining both whole-plant cultivation and suspension cell culture platform. This is a report on production and secretion of a recombinant pharmaceutically active protein from hairy roots cultures of Withania somnifera to improve the economic potential of this plant for the production pharmaceutical compounds. In this study, we selected and synthesized a codon-optimized globular adiponectin (gAd) gene with a calreticulin signal peptide and cloned the sequence into a plant expression binary vector containing a nptII gene as a selectable marker gene. The transgenic hairy roots were produced by Agrobacterium rhizogenes-mediated transformation protocol developed by our group. Among ten established nptII positive hairy roots lines, six colons significantly accumulated gAd protein in the biomass and extracellular medium. The presence of gAd was confirmed by western blot analysis of root extracts. The maximum level of hairy root biomass, growth rate (GR), intra- and extracellular gAd expressions were obtained after 25-26 days of culture on MS medium. The maximum level of intra- and extracellular gAd proteins were found to be 15.19 μg/gFW and 215.7 μg/L, respectively, which resulted in a significant decrease in the amount of intra- and extracellular withanolide A and withaferin A production. The addition of PVP, KNO3 and NaCl significantly increased the level of extracellular gAd by approximately 13 folds. This improvement could significantly increase the amount of intra- and extracellular withanolide A and withaferin A production, too. The recombinant gAd produced from W. somnifera is functional as proved by induction the phosphorylation of ACC in C2C12 muscle cells, as its functional amount was 5.1-fold more than gAd produced from E. coli and 45 % lower than CHO cells., (Copyright © 2020. Published by Elsevier B.V.)

Status epilepticus (SE) is a medical emergency with high mortality and a risk factor for the development of chronic epilepsy. Given that effective treatments for the pathophysiology following SE are still lacking, suppressing pathophysiological mechanisms of SE may be important to inhibit epileptogenesis. Withanolide-A (WA), a major bioactive component of Withania somnifera, is a potential medicinal natural compound showing improvement of some neurological diseases, such as cerebral ischemia. In the present study, we examined whether administration of WA can exert the beneficial effects involved in neuroprotection and anti-inflammatory effects in a mouse model of pilocarpine-induced SE. Our results showed that WA treatment ameliorated SE-induced apoptotic neuronal cell death in the hippocampus. Moreover, WA treatment reduced immunoreactivity of both ionized calcium binding adapter molecule 1-positive microglia/macrophage and glial fibrillary acidic protein-positive reactive astrocytes, and the SE-induced increase in both interleukin-1 β and tumor necrosis factor in the hippocampus, suggesting that inhibiting pro-inflammatory factors by WA treatment might induce neuroprotection after SE. These results suggest that WA may be useful in improving the treatment efficacy for pathophysiology following SE., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Withaferin A (WFA), a C 5 ,C 6 -epoxy steroidal lactone isolated from the medicinal plant Withania somnifera (L.) Dunal, inhibits growth of tumor cells in different cancer types. However, the mechanisms underlying the effect of WFA on tumor cells are not fully understood. In the present study, we evaluated the blockade of TASK-3 channels by WFA in TASK-3-expressing HEK-293 cells. Explore if the WFA-mediated TASK-3 blockade can be used as a pharmacological tool to decrease the cell viability in cancer cells. A combination of functional experiments (patch-clamp, gene downregulation, overexpression and pharmacological inhibition) and molecular docking analysis were used to get insights into the mechanism by which the inhibition of TASK-3 by WFA affects the growth and viability of cancer cells. Withaferin A was found to inhibit the activity of TASK-3 channels. The inhibitory effect of Withaferin A on TASK-3 potassium currents was dose-dependent and independent of voltage. Molecular modeling studies identified putative WFA-binding sites in TASK-3 channel involved the channel blockade. In agreements with the molecular modeling predictions, mutation of residues F125 to A (F125A), L197 to V (L197 V) and the double mutant F125A-L197 V markedly decreased the WFA-induced inhibition of TASK-3. Finally, the cytotoxic effect of WFA was tested in MDA-MB-231 human breast cancer cells transfected with TASK-3 or shRNA that decreases TASK-3 expression. Together, our results show that the cytotoxic effect of WFA on fully transformed MDA-MB-231 cells depends on the expression of TASK-3. Herein, we also provide insights into the mechanism of TASK-3 inhibition by WFA., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Virus-induced gene silencing (VIGS) has emerged as a fast and efficient reverse and forward genetics tool to study gene function in model plants as well as in agriculturally important plants. In addition, VIGS approach has been successfully used to provide insights into the role of several genes and regulators involved in plant secondary metabolism. Ashwagandha (Withania somnifera) is an important Indian medicinal plant that accumulates pharmacologically important triterpenoid steroidal lactones, which are collectively termed as withanolides. W. somnifera being a highly recalcitrant plant for genetic transformation, Tobacco rattle virus (TRV)-mediated VIGS was established by our group to facilitate understanding of withanolides' pathway. Here, we describe a detailed procedure to carry out VIGS for gene function studies in W. somnifera.

Withania somnifera (Ashwagandha) is considered as Rasayana in Indian systems of medicine. This study reports a novel transcriptome of W. somnifera berries, with high depth, quality and coverage. Assembled and annotated transcripts for nearly all genes related with the withanolide biosynthetic pathway were obtained. Tissue-wide gene expression analysis reflected almost similar definitions for the terpenoid pathway in leaf, root and berry tissues with relatively higher abundance of transcripts linked to steroid, phenylpropanoid metabolism as well as flavonoid metabolism in berries. The metabolome map generated from the data embodied transcripts from 143 metabolic pathways connected together and mediated collectively by about 1792 unique enzyme functions specific to berry, leaf and root tissues, respectively. Transcripts specific to cytochrome p450 (CYP450), methyltransferases and glycosyltransferases were distinctively located in a tissue specific manner and exhibited a complex network. Significant distribution of transcription factor genes such as MYB, early light inducible protein (ELI), minichromosome maintenance1, agamous, deficiens and serum response factor (MADS) and WRKY etc. was observed, as the major transcriptional regulators of secondary metabolism. Validation of the assembly was ascertained by cloning WsELI, which has a light dependent regulatory role in development. Quantitative expression of WsELI was observed to be considerably modulated upon exposure to abiotic stress and elicitors. Co-relation of over-expression of WsELI, may provide new aspects for the functional role of ELI proteins in plants linked to secondary metabolism. The study offers the first comprehensive and comparative evaluation of W. somnifera transcriptome data between the three tissues and across other members of Solanaceae (Nicotiana, Solanum and Capsicum) with respect to major pathways and their metabolome regulation., (© 2019 Scandinavian Plant Physiology Society.)

Withaferin A (WIT) is a natural product possessing a wide range of pharmacologic activities. Previous studies have reported covalent binding of WIT to tubulin and down-of tubulin protein levels although the underlying mechanisms remain to be established. In the current investigation, we showed that WIT induces down-regulation of tubulin in a post-transcriptional manner, suggestive of direct and potent activity in tubulin degradation. The N , N '-ethylene bis(iodoacetamide) assay and competitive binding experiments with four colchicine site-targeted tubulin inhibitors further revealed that WIT interacts with the colchicine site of tubulin to promote degradation. WIT irreversibly inhibited tubulin polymerization, and mass spectrometry results disclosed binding to cysteine at position 239 (Cys239) and Cys303 sites of β -tubulin. Interestingly, WIT promoted degradation of the β -tubulin isoforms containing Cys239 [ β 2, β 4, and β 5( β )] but had no effect on those containing Ser239 ( β 3 and β 6). Moreover, a C239S but not C303S mutation in β -tubulin completely abolished the degradation effect of WIT, suggesting that the Cys239-WIT covalent bond accounts for this activity. Our collective results clearly demonstrate that covalent interactions between WIT and Cys239 of β -tubulin promote tubulin degradation, supporting its potential utility as a therapeutic compound. SIGNIFICANCE STATEMENT: Withaferin A, a natural product possessing a wide range of pharmacologic activities, covalently binds to Cys239 of β -tubulin near the colchicine site, and the WIT-Cys239 covalent bond accounts for WIT-induced tubulin degradation, fully clarifying the underlying mechanisms and supporting its potential utility a therapeutic compound., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Key Message: Functional characterization of WsMYC2 via artificial microRNA mediated silencing and transient over-expression displayed significant regulatory role vis-à-vis withanolides and stigmasterol biosyntheses in Withania somnifera. Further, metabolic intensification corroborated well with higher expression levels of putative pathway genes. Additionally, copious expression of WsMYC2 in response to exogenous elicitors resulted in enhanced withanolides production. Withania somnifera, a high value multipurpose medicinal plant, is a rich reservoir of structurally diverse and biologically active triterpenoids known as withanolides. W. somnifera has been extensively pursued vis-à-vis pharmacological and chemical studies. Nonetheless, there exists fragmentary knowledge regarding the metabolic pathway and the regulatory aspects of withanolides biosynthesis. Against this backdrop, a jasmonate-responsive MYC2 transcription factor was identified and functionally characterized from W. somnifera. In planta transient over-expression of WsMYC2 showed significant enhancement of mRNA transcript levels which corroborated well with the enhanced content of withanolides and stigmasterol. Further, a comparative analysis of expression levels of some of the genes of triterpenoid pathway viz. WsCAS, WsCYP85A, WsCYP90B and WsCYP710A in corroboration with the over-expression and silencing of WsMYC2 suggested its positive influence on their regulation. These corroboratory approaches suggest that WsMYC2 has cascading effect on over-expression of multiple pathway genes leading to the increased triterpenoid biosynthesis in infiltered plants. Further, the functional validation of WsMYC2 was carried out by artificial micro-RNA mediated silencing. It resulted in significant reduction of withanolides and stigmasterol levels, indicative of crucial role of WsMYC2 in the regulation of their biosyntheses. Taken together, these non-complementary approaches provided unambiguous understanding of the regulatory role of WsMYC2 in context to withanolides and stigmasterol biosyntheses. Furthermore, the upstream promoter of WsMYC2 presented several cis-regulatory elements primarily related to phytohormone responsiveness. WsMYC2 displayed inducible nature in response to MeJA. It had substantial influence on the higher expression of WsMYC2 which was in consonance with enhanced accumulation of withanolides.

To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the present study, root cell suspension cultures of W. somnifera were elicited with mycelial extract (1% w/v) and culture filtrate (5% v/v) of their native endophytic fungus Aspergillus terreus 2aWF in shake flask. Culture filtrate of A. terreus 2aWF significantly elicits withanolide A at 6H (12.20 ± 0.52 µg/g FCB). However, with A. terreus 2aWF mycelial extract, withanolide A content was higher at 24H (10.29 µg/g FCB). Withanolide A content was maximum with salicylic acid (0.1 mM) treatment at 24H (8.3 ± 0.20 µg/g FCB). Further, expression analysis of withanolide pathway genes, hydrogen peroxide production, and lipid peroxidation was carried out after 48H of elicitation with 2aWF mycelial extract and culture filtrate. The expression levels of withanolides biosynthetic pathway genes, viz. HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were quantified by real time PCR at 48H of elicitation. In all the treatments, the expression levels of key genes were significantly upregulated as compared to untreated suspension cells. Hydrogen peroxide was noticeably enhanced in SA, mycelia extract and culture filtrate, at 20% (115 ± 4.40 nM/g FCB), 42% (137.5 ± 3.62 nM/g FCB), and 27% (122.8 ± 1.25 nM/g FCB) respectively; however, lipid peroxidation was 0.288 ± 0.014, 0.305 ± 0.041 and 0.253 ± 0.007 (µM/gm FCB) respectively, higher than the control (0.201 ± 0.007 µM/gm FCB).

The medicinal properties of Ashwagandha (Withania somnifera) are accredited to a group of compounds called withanolides. 24-Methylene cholesterol is the intermediate for sterol biosynthesis and a proposed precursor of withanolide biogenesis. However, conversion of 24-methylene cholesterol to withaferin A and other withanolides has not yet been biochemically dissected. Hence, in an effort to fill this gap, an important gene, encoding S-adenosyl l-methionine-dependent sterol-C24-methyltransferase type 1 (SMT1), involved in the first committed step of sterol biosynthesis, from W. somnifera was targeted in the present study. Though SMT1 has been characterized in model plants such as Nicotiana tabacum and Arabidopsis thaliana, its functional role in phytosterol and withanolide biosynthesis was demonstrated for the first time in W. somnifera. Since SMT1 acts at many steps preceding the withanolide precursor, the impact of this gene in channeling of metabolites for withanolide biosynthesis and its regulatory nature was illustrated by suppressing the gene in W. somnifera via the RNA interference (RNAi) approach. Interestingly, down-regulation of SMT1 in W. somnifera led to reduced levels of campesterol, sitosterol and stigmasterol, with an increase of cholesterol content in the transgenic RNAi lines. In contrast, SMT1 overexpression in transgenic N. tabacum enhanced the level of all phytosterols except cholesterol, which was not affected. The results established that SMT1 plays a crucial role in W. somnifera withanolide biosynthesis predominantly through the campesterol and stigmasterol routes., (� The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Withania somnifera (Ashwagandha) is an efficient medicinal plant known in Ayurveda and Chinese medicine since ancient times, whose extracts are consumed orally as food supplement or as a health tonic owing to its several restorative properties for various CNS disorders, inflammation, tumour, stress, rheumatism etc. In this study, we have analyzed the binding interaction of four derivatives of Withania somnifera (Withanolide A, Withanolide B, Withanoside IV and Withanoside V) with HSA because of their important pharmacological properties. To unravel the binding between derivatives of Withania somnifera and HSA, fluorescence spectroscopy was used. Binding studies were further studied by molecular docking and dynamics and results confirmed greater stability upon binding of derivatives with HSA. Circular dichroism data illustrated change in the secondary structure of protein upon interaction with these derivatives, particularly the helical structure was increased and β-sheets and random coils were decreased. Furthermore, morphological and topological changes were observed using AFM and TEM upon binding of ligands with HSA indicating that HSA-withnoside/withanolide complexes were formed. All the results cumulatively demonstrate strong binding of withanosides and withanolides derivatives with serum albumin, which should further be explored to study the pharmacokinetics and pharmacodynamics of these derivatives., Competing Interests: The authors have declared that no competing interests exist.

A large part of chemodiversity of plant triterpenes is due to the modification of their side chains. Reduction or isomerization of double bonds in the side chains is often an important step for the diversification of triterpenes, although the enzymes involved are not fully understood. Withanolides are a large group of structurally diverse C 28 steroidal lactones derived from 24-methylenecholesterol. These compounds are found in the Indian medicinal plant Withania somnifera , also known as ashwagandha, and other members of the Solanaceae. The pathway for withanolide biosynthesis is unknown, preventing sustainable production via white biotechnology and downstream pharmaceutical usages. In the present study, based on genome and transcriptome data we have identified a key enzyme in the biosynthesis of withanolides: a DWF1 paralog encoding a sterol Δ 24 -isomerase (24ISO). 24ISO originated from DWF1 after two subsequent duplication events in Solanoideae plants. Withanolides and 24ISO appear only in the medicinal plants in the Solanoideae, not in crop plants such as potato and tomato, indicating negative selection during domestication. 24ISO is a unique isomerase enzyme evolved from a reductase and as such has maintained the FAD-binding oxidoreductase structure and requirement for NADPH. Using phylogenetic, metabolomic, and gene expression analysis in combination with heterologous expression and virus-induced gene silencing, we showed that 24ISO catalyzes the conversion of 24-methylenecholesterol to 24-methyldesmosterol. We propose that this catalytic step is the committing step in withanolide biosynthesis, opening up elucidation of the whole pathway and future larger-scale sustainable production of withanolides and related compounds with pharmacological properties., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Withania somnifera, popularly known as Indian ginseng, is one of the most important medicinal plants. The plant is well studied in terms of its pharmaceutical activities and genes involved in biosynthetic pathways. However, not much is known about the regulatory mechanism of genes responsible for the production of secondary metabolites. The idea was to identify miRNA transcriptome responsible for the regulation of withanolide biosynthesis, specifically of root and leaf tissues individually. The transcriptome data of in vitro culture of root and leaf tissues of the plant was considered for miRNA identification. A total of 24 and 39 miRNA families were identified in root and leaf tissues, respectively. Out of these, 15 and 27 miRNA families have shown their involvement in different biological functions in root and leaf tissues, respectively. We report here, specific miRNAs and their corresponding target genes for corresponding root and leaf tissues. The target genes have also been analyzed for their role in withanolide metabolism. Endogenous root-miR5140, root-miR159, leaf-miR477, and leaf-miR530 were reported for regulation of withanolide biosynthesis.

Withanolides, the secondary metabolite from Withania species is used in Ayurvedic medicine and now proved to have potential use in treating cardiovascular, Alzheimer's disease, etc. Its production in plants varies between genotypes with very low yield. For improved industrial commercialisation, there is a need to increase its production. Endophytic fungi are symbiotically associated with plants and can synthesise the same bioactive compounds and natural products as their host plant. There are no reports available on withanolide-producing (endophytic) fungi. The present study identified an endophytic fungus (Taleromyces pinophilus) from leaves of Withania somnifera which produces withanolides in the medium. The structure of withanolide was confirmed by 1 H NMR, LC-MS analyses and quantified by HPLC analysis. The fungus produces high amount of withanolide when compared to leaf and root of W. somnifera. The fungus can be exploited to produce the withanolide to meet its demand.

Despite the vast exploration of endophytic microbes for growth enhancement in various crops, knowledge about their impact on the production of therapeutically important secondary metabolites is scarce. In the current investigation, chitinolytic bacterial endophytes were isolated from selected medicinal plants and assessed for their mycolytic as well as plant growth promoting potentials. Among them the two most efficient bacterial endophytes namely Bacillus amyloliquefaciens (MPE20) and Pseudomonas fluorescens (MPE115) individually as well as in combination were able to modulate withanolide biosynthetic pathway and tolerance against Alternaria alternata in Withania somnifera. Interestingly, the expression level of withanolide biosynthetic pathway genes (3-hydroxy-3-methylglutaryl co-enzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductase, farnesyl di-phosphate synthase, squalene synthase, cytochrome p450, sterol desaturase, sterol Δ-7 reductase and sterol glycosyl transferases) were upregulated in plants treated with the microbial consortium under A. alternata stress. In addition, application of microbes not only augmented withaferin A, withanolide A and withanolide B content (1.52-1.96, 3.32-5.96 and 12.49-21.47 fold, respectively) during A. alternata pathogenicity but also strengthened host resistance via improvement in the photochemical efficiency, normalizing the oxidized and non-oxidized fraction, accelerating photochemical and non-photochemical quantum yield, and electron transport rate. Moreover, reduction in the passively dissipated energy of PSI and PSII in microbial combination treated plants corroborate well with the above findings. Altogether, the above finding highlights novel insights into the underlying mechanisms in application of endophytes and emphasizes their capability to accelerate biosynthesis of withanolides in W. somnifera under biotic stress caused by A. alternata., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Withania somnifera (Ashwagandha) has recently been studied extensively for its health-supplementing and therapeutic activities against a variety of ailments. Several independent studies have experimentally demonstrated pharmaceutical potential of its active Withanolides, Withaferin A (Wi-A), Withanone (Wi-N) and Withanolide A (Wil-A). However, to promote its use in herbal industry, an environmentally sustainable cultivation and high yield are warranted. In modern agriculture strategies, there has been indiscriminate use of chemical fertilizers to boost the crop-yield, however the practice largely ignored its adverse effect on the quality of soil and the environment. In view of these, we attempted to recruit Vermicompost (Vcom, 20-100%) as an organic fertilizer of choice during the sowing and growing phases of Ashwagandha plants. We report that (i) pre-soaking of seeds for 12 h in Vermicompost leachate (Vcom-L) and Vermicompost tea (Vcom-T) led to higher germination, (ii) binary combination of pre-soaking of seeds and cultivation in Vcom (up to 80%) resulted in further improvement both in germination and seedling growth, (iii) cultivated plants in the presence of Vcom+Vcom-L showed higher leaf and root mass, earlier onset of flowering and fruiting and (iv) leaves from the Vcom+Vcom-L cultivated plants showed higher level of active Withanolides, Withanone (Wi-N), Withanolide A (Wil-A) and Withaferin A (Wi-A) and showed anticancer activities in cell culture assays. Taken together, we report a simple and inexpensive method for improving the yield and pharmaceutical components of Ashwagandha leaves.

Tissue specific biosynthesis of secondary metabolites is a distinguished feature of medicinal plants. Withania somnifera, source of pharmaceutically important withanolides biosynthesizes withaferin-A in leaves and withanolide-A in roots. To increase the in planta withanolides production, a sustainable approach needs to be explored. Here, we isolated endophytes from different parts of W. somnifera plants and their promising role in in planta withanolide biosynthesis was established in both in-vivo grown as well in in-vitro raised composite W. somnifera plants. Overall, the fungal endophytes improved photosynthesis, plant growth and biomass, and the root-associated bacterial endophytes enhanced the withanolide content in both in-vivo and in-vitro grown plants by modulating the expression of withanolide biosynthesis genes in leaves and roots. Surprisingly, a few indole-3-acetic acid (IAA)-producing and nitrogen-fixing root-associated endophytes could induce the biosynthesis of withaferin-A in roots by inducing in planta IAA-production and upregulating the expression of withanolide biosynthesis genes especially MEP-pathway genes (DXS and DXR) in roots as well. Results indicate the role of endophytes in modulating the synthesis and site of withanolides production and the selected endophytes can be used for enhancing the in planta withanolide production and enriching roots with pharmaceutically important withaferin-A which is generally absent in roots.

Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.

1. Physalin A is a bioactive withanolide isolated from the natural plant Physalis alkekengi var. franchetii (Solanaceae), a common traditional Chinese herbal medicine. This study aims to investigate the metabolites of physalin A in vivo. 2. Two metabolites (M1 and M2) were characterized as sulfonate metabolites in the feces obtained from rats treated with physalin A orally at a dose of 15 mg/kg/day for 3 days, by application of a UPLC-Q/TOF-MS method. Furthermore, formation of the two sulfonate metabolites was verified by chemical synthesis and NMR, including 1 H NMR, 13 C NMR and two-dimensional NMR. The structures of M1 and M2 were identified to be 3α-sulfo-2,25β,27-trihydrophysalin A and 3α,27-disulfo-2,25α-dihydrophysalin A, respectively. 3. In summary, this study indicated that physalin A could be biotransformed to sulfonate metabolites with strong polarity, which contributed to the elimination of physalin A. A rare metabolic pathway has been revealed in this study.

Transcription factors (TFs) are important regulators of cellular and metabolic functions including secondary metabolism. Deep and intensive RNA-seq analysis of Withania somnifera using transcriptomic databases provided 3532 annotated transcripts of transcription factors in leaf and root tissues, belonging to 90 different families with major abundance for WD-repeat (174 and 165 transcripts) and WRKY (93 and 80 transcripts) in root and leaf tissues respectively, followed by that of MYB, BHLH and AP2-ERF. Their detailed comparative analysis with Arabidopsis thaliana, Capsicum annum, Nicotiana tabacum and Solanum lycopersicum counterparts together gave interesting patterns. However, no homologs for WsWDR representatives, LWD1 and WUSCHEL, were observed in other Solanaceae species. The data extracted from the sequence read archives (SRA) in public domain databases were subjected to re-annotation, re-mining, re-analysis and validation for dominant occurrence of WRKY and WD-repeat (WDR) gene families. Expression of recombinant LWD1 and WUSCHEL proteins in homologous system led to enhancements in withanolide content indicating their regulatory role in planta in the biosynthesis. Contrasting expression profiles of WsLWD1 and WsWUSCHEL provided tissue-specific insights for their participation in the regulation of developmental processes. The in-depth analysis provided first full-spectrum and comparative characteristics of TF-transcripts across plant species, in the perspective of integrated tissue-specific regulation of metabolic processes including specialized metabolism.

Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated. A jasmonate- and salicin-inducible WRKY transcription factor from W. somnifera (WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay. WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W. somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W. somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, WsWRKY1 binds to W-box sequences in promoters of W. somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while WsWRKY1 silencing in W. somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance. Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

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 C 28 -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 (C 30 -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.