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Breast cancer is the second most common malignancy in females worldwide and poses a great challenge that necessitates the identification of novel therapeutic agents from several sources. This research aimed to study the molecular docking and molecular dynamics simulations of four proteins (such as PDB: 6CBZ, 1FDW, 5GWK and 2WTT) with the selected phytochemicals from Withania somnifera to identify the potential inhibitors for breast cancer. The molecular docking result showed that among 44 compounds, two of them, Ashwagandhanolide and Withanolide sulfoxide have the potential to inhibit estrogen receptor alpha (ERα), 17-beta-hydroxysteroid -dehydrogenase type 1 (17β-HSD1), topoisomerase II alpha (TOP2A) and p73 tetramerization domain that are expressed during breast cancer. The molecular dynamics (MD) simulations results suggested that Ashwagandhanolide remained inside the binding cavity of four targeted proteins and contributed favorably towards forming a stable protein-ligand complex throughout the simulation. Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties confirmed that Ashwagandhanolide is hydrophobic and has moderate intestinal permeability, good intestinal absorption, and poor skin permeability. The compound has a relatively low VDss value (-1.652) and can be transported across ABC transporter and good central nervous system (CNS) permeability but did not easily cross the blood-brain barrier (BBB). This compound does not possess any mutagenicity, hepatotoxicity and skin sensitization. Based on the results obtained, the present study highlights the anticancer potential of Ashwagandhanolide, a compound from W. somnifera. Furthermore, in vitro and in vivo studies are necessary to perform before clinical trials to prove the potentiality of Ashwagandhanolide., Competing Interests: The authors have declared that no competing interests exist.

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.)

A new withanolide named as withacoagulin J (1) along with a known withanolide H (2) from Withania coagulans Dunal are reported in this paper. The isolated compounds were elucidated by using 1D-NMR ( 1 H NMR, 13 C NMR) and 2D-NMR including homonuclear (COSY, NOESY) and heteronuclear (HSQC, HMBC); along with Mass spectrometry, UV-Visible and IR spectroscopic techniques. The molecular formula based on Fast-Atom Bombardment Mass Spectrometry [FAB-MS (M + 1)] for 1 and 2 were deduced as C 28 H 37 O 5 and C 28 H 39 O 6 with m/z values 453.2624 and 471.6041, respectively. The quantum mechanical studies of both compounds are based on DFT calculations. The DFT studies show band gaps of 4.86 and 4.83 eV for 1 and 2, respectively. The band gaps of 1 and 2 reflect high stability and resistivity towards oxidation-reduction reactions. The energies of HOMO and LUMO for compound 1 are -6.11 and -1.25 eV and for compound 2: -6.47 and -1.64 eV respectively. Theoretical and experimental FTIR data closely match for both the compounds which support the high accuracy of the computational protocol selection. Other parameters such as bond lengths, bond angles and dihedral angles for both compounds are also studied., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

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

Background: Nuclear Factor kappa B (NF-κB) is a transcription factor involved in the regulation of cell signaling responses and is a key regulator of cellular processes involved in the immune response, differentiation, cell proliferation, and apoptosis. The constitutive activation of NF-κB contributes to multiple cellular outcomes and pathophysiological conditions such as rheumatoid arthritis, asthma, inflammatory bowel disease, AIDS and cancer. Thus there lies a huge therapeutic potential beneath inhibition of NF-κB signalling pathway for reducing these chronic ailments. Withania somnifera, a reputed herb in ayurvedic medicine, comprises a large number of steroidal lactones known as withanolides which show plethora of pharmacological activities like anti- inflammatory, antitumor, antibacterial, antioxidant, anticonvulsive, and immunosuppressive. Though a few studies have been reported depicting the effect of WA (withaferin A) on suppression of NF-κB activation, the mechanism behind this is still eluding the researchers. The study conducted here is an attempt to explore NF-κB signalling pathway modulating capability of Withania somnifera's major constituent WA and to elucidate its possible mode of action using molecular docking and molecular dynamics simulations studies., Results: Formation of active IKK (IκB kinase) complex comprising NEMO (NF-κB Essential Modulator) and IKKβ subunits is one of the essential steps for NF-κB signalling pathway, non-assembly of which can lead to prevention of the above mentioned vulnerable disorders. As observed from our semi-flexible docking analysis, WA forms strong intermolecular interactions with the NEMO chains thus building steric as well as thermodynamic barriers to the incoming IKKβ subunits, which in turn pave way to naive complex formation capability of NEMO with IKKβ. Docking of WA into active NEMO/IKKβ complex using flexible docking in which key residues of the complex were kept flexible also suggest the disruption of the active complex. Thus the molecular docking analysis of WA into NEMO and active NEMO/IKKβ complex conducted in this study provides significant evidence in support of the proposed mechanism of NF-κB activation suppression by inhibition or disruption of active NEMO/IKKβ complex formation being accounted by non-assembly of the catalytically active NEMO/IKKβ complex. Results from the molecular dynamics simulations in water show that the trajectories of the native protein and the protein complexed with WA are stable over a considerably long time period of 2.6 ns., Conclusions: NF-κB is one of the most attractive topics in current biological, biochemical, and pharmacological research, and in the recent years the number of studies focusing on its inhibition/regulation has increased manifolds. Small ligands (both natural and synthetic) are gaining particular attention in this context. Our computational analysis provided a rationalization of the ability of naturally occurring withaferin A to alter the NF-κB signalling pathway along with its proposed mode of inhibition of the pathway. The absence of active IKK multisubunit complex would prevent degradation of IκB proteins, as the IκB proteins would not get phosphorylated by IKK. This would ultimately lead to non-release of NF-κB and its further translocation to the nucleus thus arresting its nefarious acts. Conclusively our results strongly suggest that withaferin A is a potent anticancer agent as ascertained by its potent NF-κB modulating capability. Moreover the present MD simulations made clear the dynamic structural stability of NEMO/IKKβ in complex with the drug WA, together with the inhibitory mechanism.

Cell suspension cultures of Withania somnifera were established in shake flasks and the effect of different growth regulators (auxins, combination of auxin and cytokinin), inoculum density (2.5-20 g L(-1)), different media (MS, B5, NN and N6), the strength of the MS medium (0.25-2.0 x), carbon source (sucrose, glucose, fructose, maltose), concentration of the sucrose (1-8% (w/v)) and the initial pH (4.0-6.5) of the medium were determined for the production of withanolide A. The optimized conditions for biomass accumulation and withanolide A production were found to be 10 g L(-1) of the inoculum on fresh weight basis, the full strength MS medium, 3% (w/v) sucrose, four weeks culture period and the initial medium pH of 5.8. The results of present study are useful for scale-up process., ((c) 2010 Elsevier Ltd. All rights reserved.)

Stability testing at preformulation stages is a crucial part of drug development. We studied physicochemical stability and biological activity of Withania somnifera (ashwagandha) dried root aqueous extract during six months real-time and under accelerated storage conditions. The characteristic constituents of ashwagandha roots include withanolides such as withaferin A and withanolide A. We modified and validated the HPLC-DAD method for quantitative measurement of withanolides and fingerprint analysis. The results suggest a significant decline in withaferin A and withanolide A content under real and accelerated conditions. The HPLC fingerprint analysis showed significant changes in some peaks during real and accelerated storage (> 20 %). We also observed incidences of clump formation and moisture sensitivity (> 10 %) under real-time and accelerated storage conditions. These changes were concurrent with a significant decline in immunomodulatory activity (p < 0.01) during the third month of the accelerated storage. Thus, adequate control of temperature and humidity is important for WSE containing formulations. This study may help in proposing suitable guidance for storage conditions and shelf life of ashwagandha formulations., ((c) Georg Thieme Verlag KG Stuttgart . New York.)

Ashwagandha (Withania somnifera) is widely used in the Indian traditional system of medicine, Ayurveda. Although it is claimed to have a large variety of health-promoting effects, including therapeutic effects on stress and disease, the mechanisms of action have not yet been determined. In the present study, we aimed to investigate the growth inhibition and differentiation potential of the alcoholic extract of Ashwagandha leaves (i-Extract), its different constituents (Withaferin A, Withanone, Withanolide A) and their combinations on glioma (C6 and YKG1) cell lines. Withaferin A, Withanone, Withanolide A and i-Extract markedly inhibited the proliferation of glioma cells in a dose-dependent manner and changed their morphology toward the astrocytic type. Molecular analysis revealed that the i-Extract and some of its components caused enhanced expression of glial fibrillary acidic protein, change in the immunostaining pattern of mortalin from perinuclear to pancytoplasmic, delay in cell migration, and increased expression of neuronal cell adhesion molecules. The data suggest that the i-Extract and its components have the potential to induce senescence-like growth arrest and differentiation in glioma cells. These assays led us to formulate a unique combination formula of i-Extract components that caused enhanced differentiation of glial cells.

This study was taken up to see the effect of Withanolide A (WS-1), a compound isolated from Withania somnifera root extract on chronic stress-induced alterations on T lymphocyte subset distribution and corresponding cytokine secretion patterns in experimental Swiss albino mice. Stress disturbs the homeostatic state of the organism and brings about behavioral, endocrine and immunological changes. The chronic suppression induced by stress depresses the immune functioning and increases susceptibility to diseases. Oral administration of WS-1 once daily at the graded doses of 0.25, 0.5, 1 and 2 mg/kg p.o. caused significant recovery of stress-induced depleted T cell population causing an increase in the expression of IL-2 and IFN-gamma (a signature cytokine of Th1 helper cells) and a decrease in the concentration of corticosterone in stressed experimental animals. It also reversed the restraint stress-induced increase in plasma alanine aminotransferase (ALT), aspartate aminotransferase(AST) and hepatic lipid peroxidation (LP) levels and improved the restraint stress-induced decrease in hepatic glutathione (GSH), and glycogen levels, thus showing the significant antistress potential of the test drug.

Preparations of the roots of the medicinal plant Withania somnifera (L.) Dunal commonly called ashwagandha have been used for millennia in the Ayurvedic medical tradition of India as a general tonic to relieve stress and enhance health, especially in the elderly. In modern times, ashwagandha has been shown to possess intriguing antiangiogenic and anticancer activity, largely attributable to the presence of the steroidal lactone withaferin A as the major constituent. When cultured using the aeroponic technique, however, this plant was found to produce a new natural product, 2,3-dihydrowithaferin A-3beta-O-sulfate (1), as the predominant constituent of methanolic extracts prepared from aerial tissues. The characteristic bioactivities exhibited by 1 including inhibition of cancer cell proliferation/survival, disruption of cytoskeletal organization and induction of the cellular heat-shock response paralleled those displayed by withaferin A (2). The delayed onset of action and reduced potency of 1 in cell culture along with previous observations demonstrating the requirement of the 2(3)-double bond in withanolides for bioactivity suggested that 1 might be converted to 2 in cell culture media and this was confirmed by HPLC analysis. The abundant yield of 1 from aeroponically cultivated plants, its good aqueous solubility and spontaneous conversion to 2 under cell culture conditions, suggest that 1 could prove useful as a readily formulated prodrug of withaferin A that merits further evaluation in animal models.

For successful conservation and breeding of a medicinal species, it is important to evaluate its genetic diversity as well as its content of phytochemical compounds. The aim of the present study was to investigate the genetic variation of Iranian natural populations of W. somnifera and W. coagulans, using the RAPD (random amplified polymorphic DNA) markers, and their withaferin A content. Using 16 RAPD primers, a total of 282 RAPD bands were achieved. The highest and lowest percentages of polymorphism were observed with primers OPAD-15 (100.0%) and OPC-06 (75.0%), respectively. Cluster analysis of the genotypes was performed based on data from polymorphic RAPD bands, using Dice's similarity coefficient and the UPGMA clustering method. Variations in the RAPD results were found to reflect geographical distribution and genetic factors of the plant populations. The HPTLC analysis of the studied samples revealed the presence of withaferin A in W. coagulans and W. somnifera. Moreover, the concentration of withaferin A had a range from 2.2 to 32.5 microg/g DW and was higher in the aerial part than in the root in all used samples. The results of the present study show that there is a high level of variation in the Iranian natural population of Withania, which is significant for conservation and breeding programs to improve production of withaferin A.

Withaferin A (WA) is an important withanolide holding promise in cancer treatment and as a relatively safe radiosensitive/chemotherapeutic agent, which is present in traces in all parts of Withania somnifera except the leaves, where as it is reported to be present in only two non-Indian chemotypes (South African chemotype/Israel chemotype 1). The present studies have marked its presence in all Indian populations (wild/cultivated), as well as two identified Indian chemotypes (AGB002 and AGB025). The quantitative dynamics of WA production in Indian populations and interchemotypic hybrids developed at our institute have been studied, and the results were compared with five previously reported chemotypes from Israel, South Africa and India. An analysis on inheritance characteristics based on presence/absence of WA in hybrid plants and their respective parents is given for future studies on the chemogenetics of this complex species in greater detail. Further, the production potential of WA in vitro propagated plants of elite varieties developed at our institute is discussed, in view of maintaining chemotypic fidelity and stability from a production point of view. Also, evidence-based clues suggesting the leaves as the site of the synthesis of WA is provided.

High yielding transformed callus culture of W. somnifera was established by infecting hypocotyls with Agrobacterium tumefaciens MTCC-2250. Maximum withaferin A content of 0.0875 mg/g dry cell weight and transformation efficiency of 80% were obtained. Confirmation of transformation was done on the basis of the presence of the ags gene by using polymerase chain reaction. Various abiotic elicitors (arachidonic acid, methyl jasmonate, calcium chloride, and copper sulfate) and biotic elicitors (cell extracts and culture filtrates of Alternia alternata, Fusarium solani, and Verticilium dahaliae) were tested at different concentrations to enhance withaferin A production in suspension culture of transformed cells. Maximum enhancements of 5.4 times and 9.7 times, respectively, were obtained when copper sulfate (100 microM) and the cell extract of V. dahaliae (5% v/v) were added separately to suspension cultures. The dual elicitation strategy by the combined addition of these two elicitors resulted in 13.8-fold enhancement of withaferin A content in comparison to control cultures (2.65 mg/L). The present study indicates the potential of this biotechnology-based methodology for the large-scale production of withaferin A.

Withania sominifera (Indian ginseng) was transformed by Agrobacterium rhizogenes. Explants from seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain R1601. Hairy (transformed) roots were induced from cotyledons and leaf explants. The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptII and rolB specific primers and, subsequently, by Southern analysis for the presence of nptII and rolB genes in the genomes of transformed roots. Four clones of hairy roots were established; these differed in their morphology. The doubling time of faster growing cultures was 8-14 d with a fivefold increase in biomass after 28 d compared with cultured, non-transformed seedling roots. MS-based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated (SH, LS and N6), with MS-based medium supplemented with 40 g/L sucrose being optimal for biomass production. Cultured hairy roots synthesized withanolide A, a steroidal lactone of medicinal and therapeutic value. The concentration of withanolide A in transformed roots (157.4 microg/g dry weight) was 2.7-fold more than in non-transformed cultured roots (57.9 microg/g dry weight).

Ashwagandha (root of Withania somnifera) has been used for many purposes, it is mainly considered a tonic in traditional Ayurvedic medicine. This review focuses on the effects of compounds isolated from Ashwagandha on dementia models and on the spinal cord injury model. Our study demonstrated that the active constituents, withanolide A, withanoside IV, and withanoside VI, restored presynapses and postsynapses, in addition to both axons and dendrites in cortical neurons after Abeta(25-35)-induced injury. In vivo, oral withanolide A, withanoside IV, and withanoside VI (10 micromol/kg/day for 12 days) improved Abeta(25-35)-induced memory impairment, neurite atrophy, and synaptic loss in the cerebral cortex and hippocampus in mice. Since spinal cord injury (SCI) is also difficult to treat, and therefore practical and curable strategies for SCI are desired. Oral treatment with withanoside IV improved locomotor functions in mice with SCI. In mice treated with withanoside IV (10 micromol/kg/day for 21 days), the axonal density and peripheral nervous system myelin level increased. The loss of CNS myelin and increase in reactive gliosis were not affected by withanoside IV. Additionally, sominone, an aglycone of withanoside IV, was identified as the main metabolite after oral administration of withanoside IV in mice. Withanolide A, withanoside IV, and withanoside VI are therefore important candidates for the therapeutic treatment of neurodegenerative diseases. In particular, withanoside IV was shown to control neurons as well as glial cells for reconstruction neuronal networks. To clarify key events in overcoming neurodegeneration, we are now studying the molecular targets and signal cascades of sominone.

Ashwagandha (Withania somnifera Dunal., Solanaceae) is one of the most reputed medicinal plants of Ayurveda, the traditional medical system. Several of its traditionally proclaimed medicinal properties have been corroborated by recent molecular pharmacological investigations and have been shown to be associated with its specific secondary metabolites known as withanolides, the novel group of ergostane skeletal phytosteroids named after the plant. Withanolides are structurally distinct from tropane/nortropane alkaloids (usually found in Solanaceae plants) and are produced only by a few genera within Solanaceae. W. somnifera contains many structurally diverse withanolides in its leaves as well as roots. To date, there has been little biosynthetic or metabolism-related research on withanolides. It is thought that withanolides are synthesized in leaves and transported to roots like the tropane alkaloids, a group of bioactive secondary metabolites in Solanaceae members known to be synthesized in roots and transported to leaves for storage. To examine this, we have studied incorporation of (14)C from [2-(14)C]-acetate and [U-(14)C]-glucose into withanolide A in the in vitro cultured normal roots as well as native/orphan roots of W. somnifera. Analysis of products by thin layer chromatography revealed that these primary metabolites were incorporated into withanolide A, demonstrating that root-contained withanolide A is de novo synthesized within roots from primary isoprenogenic precursors. Therefore, withanolides are synthesized in different parts of the plant (through operation of the complete metabolic pathway) rather than imported.

In vitro multiple shoots, root, callus and cell suspension cultures of Withania somnifera exhibited the potentiality to produce pharmacologically active withanolides. Multiple shoots cultures exhibited an increase in withanolide A accumulation compared to shoots of the mother plant. In vitro generated root cultures as well as callus and suspension cultures also produced withanolides albeit at lower levels.

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

Withania somnifera (WS) is an important herb with known antiinflammatory activity. Its molecular mechanism of action has not been investigated. The effect of a WS crude ethanol extract was studied on peripheral blood mononuclear cells of normal individuals and rheumatoid arthritis (RA) patients and synovial fluid mononuclear cells of RA patients in vitro. The WS extract significantly suppressed lipopolysaccharide (LPS) induced production of proinflammatory cytokines TNF-alpha, IL-1beta and IL-12p40 in normal individuals and RA patients, but had no effect on IL-6 production at the protein and transcript level. WS also suppressed LPS activated nitric oxide production in the mouse macrophage cell line, RAW 264.7. The extract inhibited nuclear translocation of the transcription factors NF-kappaB and AP-1 and phosphorylation of IkappaBalpha in normal and RA patients' mononuclear cells. HPLC analysis of the crude extract showed the presence of withaferin A and pure withaferin A also inhibited NF-kappaB translocation. The study demonstrated that the WS crude ethanol extract suppressed the production of proinflammatory molecules in vitro. This activity is partly through the inhibition of transcription factors NF-kappaB and AP-1 by the constituent withanolide. The role of additional constituents needs to be studied. Studies on the mechanism of action of the extract may yield potentially useful compounds for the treatment of inflammatory diseases.

Sterol glycosyltransferases catalyze the synthesis of diverse glycosterols in plants. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a novel 27beta-hydroxy glucosyltransferase was purified to near homogeneity from cytosolic fraction of W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme showed activity with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols/withanolides with beta-OH group at C-17, C-21 and C-27 positions. It transferred glucose to the alkanol at C-25 position of the lactone ring, provided an alpha-OH was present at C-17 in the sterol skeleton. A comparable enzyme has not been reported earlier from plants. The enzyme is distinct from the previously purified W. somnifera 3beta-hydroxy specific sterol glucosyltransferase and does not glucosylate the sterols at C-3 position; though it also follows an ordered sequential bisubstrate reaction mechanism, in which UDP-glucose and sterol are the first and second binding substrates. The enzyme activity with withanolides suggests its role in secondary metabolism in W. somnifera. Results on peptide mass fingerprinting showed its resemblance with glycuronosyltransferase like protein. The enzyme activity in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses.

Multiple shoot cultures of two experimental lines of Withania somnifera plants (RS-Selection-1 and RS-Selection-2) were established using nodal segments as explants. The hormonal combinations of benzyl adenine and kinetin not only influenced their morphogenetic response but also differentially modulated the level of biogeneration of withanolide A in the in vitro shoots of the two lines. Interestingly, withanolide-A, that was hardly detectable in the aerial parts of field-grown Withania somnifera (explant source), accumulated considerably in the in vitro shoot cultures of the plant. The productivity of withanolide A in the cultures varied considerably (ca. 10-fold, 0.014 to 0.14 mg per gram fresh weight) with the change in the hormone composition of the culture media as well as genotype used as source of the explant. The shoot culture of RS-Selection-1 raised at 1.00 ppm of BAP and 0.50 ppm of kinetin displayed the highest concentration of withanolide A in the green shoots of 0.238 g per 100 g dry weight tissue. This was a more analytical concentration keeping in view the isolation yields so far reported from the dried roots of the field-grown plant (ca. 0.015 g per 100 g dry weight), even if isolation losses are considered during purification. The enhanced de novo biogenesis of withanolide A in shoot cultures was corroborated with radiolabel incorporation studies using [2-(14)C] acetate as a precursor. Production of withaferin A was also found in the in vitro shoot cultures. As this compound is a predominant withanolide of native shoots as well and has been already reported to be accumulated in in vitro shoot cultures, its biogeneration observed in these shoot cultures is not discussed in detail.

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.

The present study is on the growth inhibitory effect of Withania somnifera methanolic leaf extract and its active component, withanolide on HL-60 promyelocytic leukemia cells. The decrease in survival rate of HL-60 cells was noted to be associated with a time dependent decrease in the Bcl-2/Bax ratio, leading to up regulation of Bax. Both the crude leaf extract and the active component activated the apoptotic cascade through the cytochrome c release from mitochondria. The activation of caspase 9, caspase 8 and caspase 3 revealed that caspase was a key mediator in the apoptotic pathway. DNA fragmentation analysis revealed typical ladders as early as 12h indicative of caspase 3 role in the apoptotic pathway. Flow cytometry data demonstrated an increase of sub-G1 peak upon treatment by 51% at 24h, suggesting the induction of apoptotic cell death in HL-60 cells.

A new dimeric withanolide, ashwagandhanolide (1), was isolated from the roots of an Ayurvedic medicinal herb, Withania somnifera. A detailed spectroscopic evaluation revealed its identity as a dimer with an unusual thioether linkage. Compound 1 displayed growth inhibition against human gastric (AGS), breast (MCF-7), central nervous system (SF-268), colon (HCT-116), and lung (NCI H460) cancer cell lines, with IC50 values in the range 0.43-1.48 microg/mL. In addition, it inhibited lipid peroxidation and the activity of the enzyme cyclooxygenase-2 in vitro.

The plant Withania somnifera Dunal (Ashwagandha), also known as Indian ginseng, is widely used in the Ayurvedic system of medicine to treat tumors, inflammation, arthritis, asthma, and hypertension. Chemical investigation of the roots and leaves of this plant has yielded bioactive withanolides. Earlier studies showed that withanolides inhibit cyclooxygenase enzymes, lipid peroxidation, and proliferation of tumor cells. Because several genes that regulate cellular proliferation, carcinogenesis, metastasis, and inflammation are regulated by activation of nuclear factor-kappaB (NF-kappaB), we hypothesized that the activity of withanolides is mediated through modulation of NF-kappaB activation. For this report, we investigated the effect of the withanolide on NF-kappaB and NF-kappaB-regulated gene expression activated by various carcinogens. We found that withanolides suppressed NF-kappaB activation induced by a variety of inflammatory and carcinogenic agents, including tumor necrosis factor (TNF), interleukin-1beta, doxorubicin, and cigarette smoke condensate. Suppression was not cell type specific, as both inducible and constitutive NF-kappaB activation was blocked by withanolides. The suppression occurred through the inhibition of inhibitory subunit of IkappaB alpha kinase activation, IkappaB alpha phosphorylation, IkappaB alpha degradation, p65 phosphorylation, and subsequent p65 nuclear translocation. NF-kappaB-dependent reporter gene expression activated by TNF, TNF receptor (TNFR) 1, TNFR-associated death domain, TNFR-associated factor 2, and IkappaB alpha kinase was also suppressed. Consequently, withanolide suppressed the expression of TNF-induced NF-kappaB-regulated antiapoptotic (inhibitor of apoptosis protein 1, Bfl-1/A1, and FADD-like interleukin-1beta-converting enzyme-inhibitory protein) and metastatic (cyclooxygenase-2 and intercellular adhesion molecule-1) gene products, enhanced the apoptosis induced by TNF and chemotherapeutic agents, and suppressed cellular TNF-induced invasion and receptor activator of NF-kappaB ligand-induced osteoclastogenesis. Overall, our results indicate that withanolides inhibit activation of NF-kappaB and NF-kappaB-regulated gene expression, which may explain the ability of withanolides to enhance apoptosis and inhibit invasion and osteoclastogenesis.

At the present, medication of dementia is limited to symptomatic treatments such as the use of cholinesterase inhibitors. To cure dementia completely, that is regaining neuronal function, reconstruction of neuronal networks is necessary. Therefore, we have been exploring antidementia drugs based on reconstructing neuronal networks in the damaged brain and found that withanoside IV (a constituent of Ashwagandha; the root of Withania somnifera) induced neurite outgrowth in cultured rat cortical neurons. Oral administration of withanoside IV (10 micromol/kg/day) significantly improved memory deficits in Abeta(25-35)-injected (25 nmol, i.c.v.) mice and prevented loss of axons, dendrites, and synapses. Sominone, an aglycone of withanoside IV, was identified as the main metabolite after oral administration of withanoside IV. Sominone (1 microM) induced axonal and dendritic regeneration and synaptic reconstruction significantly in cultured rat cortical neurons damaged by 10 microM Abeta(25-35). These data suggest that orally administrated withanoside IV may ameliorate neuronal dysfunction in Alzheimer's disease and that the active principle after metabolism is sominone.

Histopathological studies of the cutaneous tissues of Wistar rats exposed to UV B radiation (294 nm) for 20 days and rats exposed to UV B radiation for 20 days, followed by topical treatment with benzoyl peroxide, a tumor promoter (20 mg/animal/0.2 ml acetone) twice a week for 1 month, and kept under observation for 12 weeks, demonstrate the development of malignancy. Pretreatment of the animals with 1-oxo-5beta, 6beta-epoxy-witha-2-enolide (20 mg/kg bwt.), isolated from the roots of Withania somnifera, prior to exposing the animals to UV B radiation, prevents the incidence of skin carcinoma. The administration of 1-oxo-5beta, 6beta-epoxy-witha-2-enolide, to the animals after exposing them to UV B radiation/UV B radiation and benzoyl peroxide also prevents the occurrence of malignancy in the cutaneous tissue. Immunohistochemical staining of the cutaneous tissues of rats exposed to UV B radiation show the presence of p53 + foci (clusters of cells containing the mutated p53 protein), whereas an absence of p53 + foci is observed in animals pretreated with 1-oxo-5beta, 6beta-epoxy-witha-2-enolide. These results prove that 1-oxo-5beta, 6beta-epoxy-witha-2-enolide has the potential for acting as an effective agent to prevent the incidence of skin carcinoma induced by UV B radiation.

This paper describes a method for separation, identification, and quantification of selected withanolides in Withania somnifera plant extracts by HPLC-UV(DAD)-Mass Spectrometry (HPLC-MS). Withaferin-A (WS-3), 12-deoxywithastramonolide (WS-12DS), Withanolide A (WS-1), and Withanone (WS-2) were used as external standards. The compounds were isolated from Withania somnifera by repeated column chromatography of the root extract and their identity was established by 1H- and 13C-NMR and mass spectral data. The compounds were chromatographed on a Merck (250 x 4.6 mm ID, 5 microm) column and analyzed by Electrospray Ionization on a mass spectrometer in Selected Ion Mode (SIM). For quantification, [M + Na]+ ions were monitored. Linear calibration curves were obtained in the concentration range of 1.50 microg/mL to 6.5 microg/mL. The method was applied successfully to the detection and quantification of the said withanolides in a number of samples.

Objective: Isolation of biologically active fractions and compounds from the roots of Withania somnifera, a plant used extensively as a constituent of rasayana, in Ayurveda and to test their adaptogenic activity on stress indices using the cold-hypoxia-restraint (C-H-R) model., Design: Bioactivity-guided fractionation of an aqueous extract of the roots of Withania somnifera led to the isolation of a new species of withanolide 1-oxo-5beta, 6beta-epoxy-witha-2-ene-27-ethoxy-olide. Structure elucidation, was carried out using proton nuclear magnetic resonance, infrared (IR), ultraviolet (UV), and mass spectroscopic analysis. Stress-related indices were evaluated, namely serum creatine phosphokinase (CPK) activity, serum lactate dehydrogenase (LDH) activity, serum corticosterone levels, and serum lipid peroxidation (LPO) levels., Results: There was a significant decrease in a serum CPK, LDH, and LPO levels in animals pretreated with (1) fraction-I (20 mg/kg body weight), (2) 1-oxo-5beta, 6beta-epoxy-witha-2-ene-27-ethoxy-olide (2.5 mg/kg body weight) in comparison to control when subjected to C-H-R stress., Conclusions: The results show that the a new species of withanolide, 1-oxo-5beta, 6beta-epoxy-witha-2-ene-27-ethoxy-olide (compound-1) could prove to be an effective agent to counteract C-H-R stress.

Ayurvedic medicines prepared in India consist of Withania somnifera roots as one of the main ingredients. It is consumed as a dietary supplement around the world. The leaves of W. somnifera were used in the treatment of tumors and inflammation in several Asian countries. We have isolated twelve withanolides such as withaferin A (1), sitoindoside IX (2), 4-(1-hydroxy-2, 2-dimethylcyclpropanone)-2, 3-dihydrowithaferin A (3), 2, 3-dihydrowithaferin A (4), 24, 25-dihydro-27-desoxywithaferin A (5), physagulin D (1-->6)-beta-D-glucopyranosyl- (1-->4)-beta-D-glucopyranoside (6), 27-O-beta-D-glucopyranosylphysagulin D (7), physagulin D (8), withanoside IV (9), and 27-O-beta-D-glucopyranosylviscosalactone B (10), 4, 16-dihydroxy-5beta, 6beta-epoxyphysagulin D (11), viscosalactone B (12) from the leaves of this species. Compounds 1-12 and diacetylwithaferin A (13) were tested for their antiproliferative activity on NCI-H460 (Lung), HCT-116 (Colon), SF-268 (Central Nervous System; CNS and MCF-7 (Breast) human tumor cell lines. The inhibitory concentration to afford 50% cell viability (IC50) for these compounds was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Withaferin A and its derivatives exhibited inhibitory concentrations (50%) ranging from 0.24 +/- 0.01 to 11.6 +/- 1.9 microg/mL. Viscosalactone B (12) showed the 50% inhibition at concentrations ranging from 0.32 +/- 0.05 to 0.47 +/- 0.15 microg/mL whereas its 27-O-glucoside derivative (10) exhibited IC50 between 7.9 +/- 2.9 and 17.3 +/- 3.9 microg/ml. However, Physagulin D type withanolides showed either weak or no activity at 30 microg/mL. Therefore, incorporation of withanolides in the diet may prevent or decrease the growth of tumors in human.

Two withanolides, 20beta-hydroxy-1-oxo-(22R)-witha-2,5,24-trienolide (1) and withacoagulin (2), along with a known withanolide, 17beta-hydroxy-14alpha, 20alpha-epoxy-1-oxo-(22R)-witha-3,5,24-trienolide (3) were isolated from Withania coagulans. Their structures were elucidated with the help of different spectroscopic techniques.

One of the most widely used herbs in Ayurvedic medicine is Ashwaghanda, Withania somnifera, a shrub commonly found on the Indian subcontinent. As this plant is increasingly becoming a popular adaptogenic in the western world, analytical methods for its identification and quality control are in demand. Thus, a HPLC method for the determination of withaferin A and withanolide D was developed. The system was successfully used to investigate the presence of the markers in different W. somnifera plant parts as well as to analyze their content in market products.

The roots of Withania somnifera (WS) are used extensively in Ayurveda, the classical Indian system of medicine, and WS is categorized as a rasayana, which are used to promote physical and mental health, to provide defence against disease and adverse environmental factors and to arrest the aging process. WS has been used to stabilize mood in patients with behavioural disturbances. The present study investigated the anxiolytic and antidepressant actions of the bioactive glycowithanolides (WSG), isolated from WS roots, in rats. WSG (20 and 50 mg/kg) was administered orally once daily for 5 days and the results were compared by those elicited by the benzodiazepine lorazepam (0.5 mg/kg, i.p.) for anxiolytic studies, and by the tricyclic anti-depressant, imipramine (10 mg/kg, i.p.), for the antidepressant investigations. Both these standard drugs were administered once, 30 min prior to the tests. WSG induced an anxiolytic effect, comparable to that produced by lorazepam, in the elevated plus-maze, social interaction and feeding latency in an unfamiliar environment, tests. Further, both WSG and lorazepam, reduced rat brain levels of tribulin, an endocoid marker of clinical anxiety, when the levels were increased following administration of the anxiogenic agent, pentylenetetrazole. WSG also exhibited an antidepressant effect, comparable with that induced by imipramine, in the forced swim-induced 'behavioural despair' and 'learned helplessness' tests. The investigations support the use of WS as a mood stabilizer in clinical conditions of anxiety and depression in Ayurveda.