Showing papers on "Withania somnifera published in 2015"

Pharmacologic overview of Withania somnifera, the Indian Ginseng

Abstract: Withania somnifera, also called 'Indian ginseng', is an important medicinal plant of the Indian subcontinent. It is widely used, singly or in combination, with other herbs against many ailments in Indian Systems of Medicine since time immemorial. Withania somnifera contains a spectrum of diverse phytochemicals enabling it to have a broad range of biological implications. In preclinical studies, it has shown anti-microbial, anti-inflammatory, anti-tumor, anti-stress, neuroprotective, cardioprotective, and anti-diabetic properties. Additionally, it has demonstrated the ability to reduce reactive oxygen species, modulate mitochondrial function, regulate apoptosis, and reduce inflammation and enhance endothelial function. In view of these pharmacologic properties, W. somnifera is a potential drug candidate to treat various clinical conditions, particularly related to the nervous system. In this review, we summarize the pharmacologic characteristics and discuss the mechanisms of action and potential therapeutic applications of the plant and its active constituents.

Antimicrobial activity of cream incorporated with silver nanoparticles biosynthesized from Withania somnifera

Abstract: We report on the antimicrobial activity of a cream formulation of silver nanoparticles (AgNPs), biosynthesized using Withania somnifera extract. Aqueous extracts of leaves promoted efficient green synthesis of AgNPs compared to fruits and root extracts of W. somnifera. Biosynthesized AgNPs were characterized for their size and shape by physical-chemical techniques such as UV-visible spectroscopy, laser Doppler anemometry, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, X-ray diffraction, and X-ray energy dispersive spectroscopy. After confirming the antimicrobial potential of AgNPs, they were incorporated into a cream. Cream formulations of AgNPs and AgNO3 were prepared and compared for their antimicrobial activity against human pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Candida albicans) and a plant pathogen (Agrobacterium tumefaciens). Our results show that AgNP creams possess significantly higher antimicrobial activity against the tested organisms.

Withania somnifera (Ashwagandha) in neurobehavioural disorders induced by brain oxidative stress in rodents: a systematic review and meta-analysis.

Abstract: Objectives Withania somnifera has been in use for several thousand years in Ayurveda to treat various neurological disorders. There is, however, not much scientific data on its protective role in neuronal pathology specifically against brain oxidative stress. Hence, an attempt is made in this work for systematic review and meta-analysis of W. somnifera on neurobehavioural disorders induced by brain oxidative stress in rodents. Methods A systematic search of the effect of W. somnifera on brain oxidative stress-induced neuronal pathology was performed using electronic databases. The systematic review was performed on neurobehavioural parameters, whereas meta-analysis of W. somnifera effect was done on oxidative stress markers (superoxide dismutase, catalase, glutathione peroxidase, glutathione and lipid peroxidation), nitrite, protein carbonyl, AchE, ChAT and Ach of rodent brain. Data were analysed using Review Manager Software. Key findings Twenty-eight studies were selected based upon the inclusion and exclusion criteria. W. somnifera appreciably inhibited the neurological abnormalities due to oxidative stress in rodent brain produced by different physical and chemical stimuli. W. somnifera also significantly restored the altered oxidative and other stress markers in different parts of rodent brain. Summary The systematic review provides scientific evidence for the traditional claim of W. somnifera use in different neurological aliments. However, future clinical trials are mandated to establish the therapeutic efficacy and safety in human beings.

Virus‐induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence‐related genes resulting in reduced withanolides and biotic stress tolerance

Abstract: Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides formation in W. somnifera leaves.

Chemistry and pharmacology of withania somnifera: An update

Abstract: Withania somnifera (Ashwagandha) is an important Rasayana herb and widely considered as Indian ginseng in Ayurveda. In traditional system of Indian medicine, it is used as tonic to rejuvenate the body and increase longevity. In Ayurvedic preparations, various parts of the plant have been used to treat variety of ailments that affect the human health. However, dried roots of the plant are widely used for the treatment of nervous and sexual disorders. The major active chemical constituents of this plant are withanolides, which is responsible for its wide range of biological activities. Since the beginning of the 20 th century, a significant amount of research has been done and efforts are ongoing to further explore other bioactive constituents, and many pharmacological studies have been carried out to describe their disease preventing mechanisms. In this chapter, we have reviewed the chemistry and pharmacological basis of W. somnifera in various human ailments.

Withaferin-A Inhibits Colon Cancer Cell Growth by Blocking STAT3 Transcriptional Activity.

Abstract: BACKGROUND: Withania somnifera (known as Ashwagandha) is a medicinal plant used in the ayurvedic medicines in India. Withaferin-A, a withanolide derived from the leaf extract of W. somnifera, has been reported to exhibit anti-tumor activity against various cancer cells, such as leukemia, breast cancer and colon cancer cells. METHODS: We investigated the anti-cancer effects of withaferin-A on the proliferation and migration of human colorectal cancer (HCT116) cells. And we evaluated the effects of withaferin-A on the transcriptional activity of STAT3 and the growth of HCT116 cells in xenograft mouse tumor model. RESULTS: In the present study, we found that withaferin-A inhibited the proliferation and migration of HCT116 cells in a concentration-dependent manner. Treatment of HCT116 cells with withaferin-A attenuated interleukin-6-induced activation of STAT3, which has been implicated in the development and progression of colon cancer. To examine the effect of withaferin-A on HCT116 cells proliferation in vivo, we generated HCT116 cells xenograft tumors in Balb/c nude mice and treated the tumor bearing mice with or without withaferin-A intraperitoneally. Treatment with withaferin-A exhibited significant decrease in the volume and weight of tumors as compared to untreated controls. CONCLUSIONS: The present study suggests that withaferin-A holds the potential to be developed as a small molecule inhibitor of STAT3 for the treatment of HCT116.

Anti-cancer activity of withaferin A in B-cell lymphoma

Abstract: Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90.

A Decade of Molecular Understanding of Withanolide Biosynthesis and In vitro Studies in Withania somnifera (L.) Dunal: Prospects and Perspectives for Pathway Engineering.

Abstract: Withania somnifera, a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids that are steroidal lactones known as withanolides. Though the plant has been well-characterized in terms of phytochemical profiles as well as pharmaceutical activities, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. This scenario limits biotechnological interventions for enhanced production of bioactive compounds. Nevertheless, recent emergent trends vis-a-vis, the exploration of genomic, transcriptomic, proteomic, metabolomics, and in vitro studies have opened new vistas regarding pathway engineering of withanolide production. During recent years, various strategic pathway genes have been characterized with significant amount of regulatory studies which allude toward development of molecular circuitries for production of key intermediates or end products in heterologous hosts. Another pivotal aspect covering redirection of metabolic flux for channelizing the precursor pool toward enhanced withanolide production has also been attained by deciphering decisive branch point(s) as robust targets for pathway modulation. With these perspectives, the current review provides a detailed overview of various studies undertaken by the authors and collated literature related to molecular and in vitro approaches employed in W. somnifera for understanding various molecular network interactions in entirety.

Promising Anti-influenza Properties of Active Constituent of Withania somnifera Ayurvedic Herb in Targeting Neuraminidase of H1N1 Influenza: Computational Study

Abstract: Neuraminidase (NA) is a membrane surface antigen which helps in the release of influenza viruses from the host cells after replication. Anti-influenza drugs such as zanamivir bind with eight highly conserved functional residues (R118, D151, R152, R224, E276, R292, R371, and Y406) in the active site of NA, thus restricting the viral release the from host cells. Binding of the drug in active site inhibits the ability of enzyme to cleave sialic acid residues on the cell membrane. Reports on the emergence of zanamivir-resistant strains of H1N1 Influenza virus necessitated a search for alternative drug candidates, preferably from plant source due to their known benefits such as less or no side effects, availability, and low cost. Withaferin A (WA), an active constituent of Withania somnifera ayurvedic herb, has been shown to have a broad range of medicinal properties including its anti-viral activity. The present study demonstrated that WA has the potential to attenuate the neuraminidase of H1N1 influenza. Our docking and simulation results predicted high binding affinity of the WA toward NA and revealed several interesting molecular interactions with the residues which are catalytically important during molecular dynamic simulations. The results presented in the article could be of high importance for further designing of target-specific anti-influenza drug candidates.

Standardized extract of Withania somnifera (Ashwagandha) markedly offsets rotenone-induced locomotor deficits, oxidative impairments and neurotoxicity in Drosophila melanogaster.

Abstract: Withania somnifera (Ashwagandha, WS) or Indian ginseng possesses multiple pharmacological properties which are mainly attributed to the active constituents, withanolides. Despite its extensive usage as a memory enhancer and a nerve tonic, few attempts have been made to ascertain its usage in the management of Parkinson’s disease. In the present study, we investigated the neuroameliorative effects of WS in a rotenone (ROT) model of Drosophila melanogaster (Oregon-K). Initially, we ascertained the ability of WS-enriched diet (0–0.05 %) to protect against ROT induced lethality and locomotor phenotype in adult male flies. Further, employing a co-exposure paradigm, we investigated the propensity of WS to offset ROT-induced oxidative stress, mitochondrial dysfunctions and neurotoxicity. WS conferred significant protection against ROT-induced lethality, while the survivor flies exhibited improved locomotor phenotype. Biochemical investigations revealed that ROT-induced oxidative stress was significantly diminished by WS enrichment. WS caused significant elevation in the levels of reduced GSH/non-protein thiols. Furthermore, the altered activity levels of succinate dehydrogenase, MTT, membrane bound enzymes viz., NADH-cytochrome-c reductase and succinate-cytochrome-c reductase were markedly restored to normalcy. Interestingly, ROT-induced perturbations in cholinergic function and depletion in dopamine levels were normalized by WS. Taken together these data suggests that the neuromodulatory effect of WS against ROT- induced neurotoxicity is probably mediated via suppression of oxidative stress and its potential to attenuate mitochondrial dysfunctions. Our further studies aim to understand the underlying neuroprotective mechanisms of WS and withanolides employing neuronal cell models.

Transcriptome analysis reveals in vitro cultured Withania somnifera leaf and root tissues as a promising source for targeted withanolide biosynthesis

Abstract: The production of metabolites via in vitro culture is promoted by the availability of fully defined metabolic pathways. Withanolides, the major bioactive phytochemicals of Withania somnifera, have been well studied for their pharmacological activities. However, only a few attempts have been made to identify key candidate genes involved in withanolide biosynthesis. Understanding the steps involved in withanolide biosynthesis is essential for metabolic engineering of this plant to increase withanolide production. Transcriptome sequencing was performed on in vitro adventitious root and leaf tissues using the Illumina platform. We obtained a total of 177,156 assembled transcripts with an average unigene length of 1,033 bp. About 13% of the transcripts were unique to in vitro adventitious roots but no unique transcripts were observed in in vitro-grown leaves. A putative withanolide biosynthetic pathway was deduced by mapping the assembled transcripts to the KEGG database, and the expression of candidate withanolide biosynthesis genes -were validated by qRT PCR. The accumulation pattern of withaferin A and withanolide A varied according to the type of tissue and the culture period. Further, we demonstrated that in vitro leaf extracts exhibit anticancer activity against human gastric adenocarcinoma cell lines at sub G1 phase. We report here a validated large-scale transcriptome data set and the potential biological activity of in vitro cultures of W. somnifera. This study provides important information to enhance tissue-specific expression and accumulation of secondary metabolites, paving the way for industrialization of in vitro cultures of W. somnifera.

Biotechnological interventions in Withania somnifera (L.) Dunal

Abstract: Withania somnifera is one of the most valued plants and is extensively used in Indian, Unani, and African systems of traditional medicine. It possess a wide array of therapeutic properties including anti-arthritic, anti-aging, anti-cancer, anti-inflammatory, immunoregulatory, chemoprotective, cardioprotective, and recovery from neurodegenerative disorders. With the growing realization of benefits and associated challenges in the improvement of W. somnifera, studies on exploration of genetic and chemotypic variations, identification and characterization of important genes, and understanding the secondary metabolites production and their modulation has gained significant momentum. In recent years, several in vitro and in vivo preclinical studies have facilitated the validation of therapeutic potential of the phytochemicals derived from W. somnifera and have provided necessary impetus for gaining deeper insight into the mechanistic aspects involved in the mode of action of these important pharmaceutically active constituents. The present review highlights some of the current developments and future prospects of biotechnological intervention in this important medicinal plant.

Comparative transcriptome analysis of different chemotypes elucidates withanolide biosynthesis pathway from medicinal plant Withania somnifera

Abstract: Withania somnifera is one of the most valuable medicinal plants synthesizing secondary metabolites known as withanolides. Despite pharmaceutical importance, limited information is available about the biosynthesis of withanolides. Chemo-profiling of leaf and root tissues of Withania suggest differences in the content and/or nature of withanolides in different chemotypes. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis, we established transcriptomes of leaf and root tissues of distinct chemotypes. Genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis with their alternatively spliced forms and paralogous have been identified. Analysis suggests differential expression of large number genes among leaf and root tissues of different chemotypes. Study also identified differentially expressing transcripts encoding cytochrome P450s, glycosyltransferases, methyltransferases and transcription factors which might be involved in chemodiversity in Withania. Virus induced gene silencing of the sterol ∆7-reductase (WsDWF5) involved in the synthesis of 24-methylene cholesterol, withanolide backbone, suggests role of this enzyme in biosynthesis of withanolides. Information generated, in this study, provides a rich resource for functional analysis of withanolide-specific genes to elucidate chemotype- as well as tissue-specific withanolide biosynthesis. This genomic resource will also help in development of new tools for functional genomics and breeding in Withania.

Effect of Withania somnifera (Ashwagandha) root extract on amelioration of oxidative stress and autoantibodies production in collagen-induced arthritic rats.

Abstract: BACKGROUND Rheumatoid arthritis is an inflammatory autoimmune disorder Withania somnifera Dunal (Solanaceae) (WS), is a common medicinal plant used in traditional systems of medicine for the treatment of arthritis, and is an ingredient of anti-arthritic polyherbal formulations such as Habb-e-Asgand® and Arthritin™ In the present study, we evaluated the antioxidant and anti-arthritic activity of aqueous extract of WS root (WSAq) in collagen-induced arthritic (CIA) rats METHODS CIA rats were treated by using three doses of WSAq (100, 200, 300 mg/kg b wt, orally) and methotrexate (MTX, 025 mg/kg b wt ip) as a standard reference drug for 20 days The anti-arthritic effect was assayed by measuring the arthritic index, autoantibodies such as rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (a-CCP), anti-nuclear antibody (ANA), anti-collagen type II antibody (a-CII) and inflammatory marker like C-reactive protein (CRP) The oxidative stress parameters were also measured RESULTS Treatment with WSAq resulted in a dose-dependent reduction in arthritic index, autoantibodies and CRP (p < 005) with maximum effect at dose of 300 mg/kg b wt and the results were comparable to that of MTX-treated rats Similarly, oxidative stress in CIA rats was ameliorated by treatment with different doses of WSAq, as evidenced by a decrease in lipid peroxidation and glutathione-S-transferase activity and an increase in the glutathione content and ferric-reducing ability of plasma (p < 005) CONCLUSIONS The results showed that WSAq exhibited antioxidant and anti-arthritic activity and reduced inflammation in CIA rats and suggests the potential use of this plant in the treatment of arthritis

Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera (L.) Dunal using sonication and heat treatment

Abstract: An improved Agrobacterium rhizogenes-mediated genetic transformation of Withania somnifera (L.) Dunal was developed using the bacterial strain R1000 with leaf segment explants of in vitro raised plantlets. Out of the three strains used (R1000, MTCC 2364 and MTCC 532), the strain R1000 proved to be more efficient than others. Among the different conditions tested, the highest (93.3 %) transformation rate was observed after 3 weeks when the explants were subjected to sonication (15 s) and heat treatment (41 °C for 5 min). Transgenic status of the hairy roots was confirmed by PCR using rol B-specific primers. HPLC analysis showed the ability of hairy roots to synthesize withaferin A and withanolide A, both steroidal lactones of medicinal value. This protocol offers new avenue in A. rhizogenes-mediated hairy root induction and is useful for large-scale production of these bioactive compounds from W. somnifera.

Alterations in metabolite profile and free radical scavenging activities of Withania somnifera leaf and root extracts under supplemental ultraviolet-B radiation

Abstract: The effects of supplemental ultraviolet-B (s-UV-B; 3.6 kJ m−2 day−1 above ambient) radiation on plant metabolite profile and free radical scavenging activities of Withania somnifera (an indigenous medicinal plant) under field conditions were investigated. The metabolite profiles of both leaves and roots were analyzed via GC–MS. The methanolic extracts were examined for their DPPH radical-, superoxide radical-, hydrogen peroxide-, hydroxyl radical-, and nitric oxide radical scavenging activities, ferrous ion chelating activity, and reducing power. GC–MS profile of leaves revealed increment in compounds such as isophytol (138.1 %), β-stigmasterol (183.05 %), trans-squalene (233.3 %), and Withaferin A (155.0 %), while compounds such as eugenol, β-carotene, lycopene, and vitamin E were detected in s-UV-B-treated leaves only. In roots, compounds such as ledol, neophytadiene, palmitic acid, retinol, sitosteryl oleate, and campesterol registered their presence only under s-UV-B. Methanolic extracts of treated plant organs were found to be more potent as free radical scavengers (their EC50 values being lower than those of control extracts). Anomalies were observed for nitric oxide radical scavenging in both leaves and roots. The present study indicates that s-UV-B alters the composition and contents of plant metabolites leading to an increase in their free radical scavenging activities. Hence, s-UV-B-treated plant organs might be more effective in combating oxidative stress as well as from a nutritional and health perspective.

Evaluation of the bioavailability of major withanolides of Withania somnifera using an in vitro absorption model system.

Abstract: Withania somnifera (L.) Dunal, shows several pharmacological properties which are attributed mainly to the withanolides present in the root. The efficacy of medicinally active withanolides constituents depends on the absorption and transportation through the intestinal epithelium. We examined these characteristics by employing the Sino-Veda Madin-Darby canine kidney cells culture system, which under in vitro condition shows the absorption characteristics similar to the human intestinal epithelium. Thus, the aim of the present investigation was to assess the bioavailability of individual withanolides. Withanolides were diluted in Hank's buffered saline at a concentration of 2 μg/ml were tested for permeability studies carried out for 1 h duration. Permeability was measured in terms of efflux pump (P eff) in cm/s. P eff values of withanolide A (WN A), withanone (WNN), 1,2-deoxywithastramonolide (1,2 DWM), withanolide B (WN B), withanoside IV-V (WS IV-V), and withaferin A were 4.05 × 10(-5), 2.06 × 10(-5), 1.97 × 10(-5), 1.80 × 10(-5), 3.19 × 10(-6), 3.03 × 10(-6) and 3.30 × 10(-7) respectively. In conclusion, the nonpolar and low molecular weight compounds (WN A, WNN, 1,2 DWM, and WN B) were highly permeable. As against this, the glycosylated and polar WS IV and WS V showed low permeability. Surprisingly and paradoxically, the highly biologically active withaferin A was completely impermeable, suggesting that further studies possibly using human epithelial colorectal adenocarcinoma (Caco-2) cells may be needed to delineate the absorption characteristics of withanolides, especially withaferin A.

Withania somnifera Improves Ischemic Stroke Outcomes by Attenuating PARP1-AIF-Mediated Caspase-Independent Apoptosis

Abstract: Withania somnifera (WS), popularly known as “Ashwagandha” has been used for centuries as a nerve tonic. Its protective effect has been elucidated in many neurodegenerative pathologies, although there is a paucity of data regarding its effects in ischemic stroke. We examined the neuroprotective properties of an aqueous extract of WS in both pre- and poststroke treatment regimens in a mouse model of permanent distal middle cerebral artery occlusion (pMCAO). WS (200 mg/kg) improved functional recovery and significantly reduced the infarct volume in mice, when compared to those treated with vehicle, in both paradigms. We investigated the protective mechanism/s induced by WS using brain cortices by testing its ability to modulate the expression of key proteins in the ischemic-apoptotic cascade. The Western blots and immunofluorescence analyses of mice cortices revealed that WS upregulated the expression of hemeoxygenase 1 (HO1) and attenuated the expression of the proapoptotic protein poly (ADP-ribose) polymerase-1 (PARP1) via the PARP1-AIF pathway, thus preventing the nuclear translocation of apoptosis-inducing factor (AIF), and subsequent apoptosis. Semaphorin-3A (Sema3A) expression was reduced in WS-treated group, whereas Wnt, pGSK3β, and pCRMP2 expression levels were virtually unaltered. These results indicate the interplay of antioxidant-antiapoptic pathways and the possible involvement of angiogenesis in the protective mechanism of WS while emphasizing the noninvolvement of one of the prime pathways of neurogenesis. Our results suggest that WS could be a potential prophylactic as well as a therapeutic agent aiding stroke repair, and that part of its mechanism could be attributed to its antiapoptotic and antioxidant properties.

Withania somnifera Root Extract Has Potent Cytotoxic Effect against Human Malignant Melanoma Cells.

Abstract: In Ayurveda, Withania somnifera is commonly known as Ashwagandha, its roots are specifically used in medicinal and clinical applications. It possesses numerous therapeutic actions which include anti-inflammatory, sedative, hypnotic and narcotic. Extracts from this plant have been reported for its anticancer properties. In this study we evaluated for the first time, the cytotoxic effect of Withania root extract on human malignant melanoma A375 cells. The crude extract of Withania was tested for cytotoxicity against A375 cells by MTT assay. Cell morphology of treated A375 cells was visualized through phase contrast as well as fluorescence microscopy. Agarose gel electrophoresis was used to check DNA fragmentation of the crude extract treated cells. Crude extract of Withania root has the potency to reduce viable cell count in dose as well as time dependent manner. Morphological change of the A375 cells was also observed in treated groups in comparison to untreated or vehicle treated control. Apoptotic body and nuclear blebbing were observed in DAPI stained treated cells under fluorescence microscope. A ladder of fragmented DNA was noticed in treated cells. Thus it might be said that the crude water extract of Withania somnifera has potent cytotoxic effect on human malignant melanoma A375 cells.

Analysis of arsenic induced physiological and biochemical responses in a medicinal plant, Withania somnifera

Abstract: Withania somnifera has been an important herb in the Ayurvedic and indigenous medical systems for centuries in India However, these grow as weeds mostly in the wastelands, which receive contaminated water from municipal and industrial sources In the present investigation, plants of Withania somnifera were exposed to various concentrations of arsenate (AsV) and arsenite (AsIII) (0, 10, 25, 50, 100 μM) for 10 days and analysed for accumulation of arsenic (As) and physiological and biochemical changes Plants showed more As accumulation upon exposure to AsIII (320 μg g−1 DW in roots and 161 μg g−1 DW in leaves) than to AsV (173 μg g−1 DW in roots and 100 μg g−1 DW in leaves) after 10 days of treatment Consequently, AsIII exposure caused more toxicity to plants as compared to that AsV, as evaluated in terms of the level of photosynthetic pigments and oxidative stress parameters (superoxide, hydrogen peroxide and lipid peroxidation), particularly at higher concentrations and on longer durations Plants could tolerate low concentrations (variable for AsIII and AsV) until longer durations (10 days) and high concentrations for shorter durations (1–5 days) through increase in antioxidant enzymes and by augmented synthesis of thiols In conclusion, As tolerance potential of Withania plants on one hand advocates its prospective use for remediation under proper supervision and on the other demonstrates possible threat of As entry into humans due to medicinal uses

Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.

Abstract: In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants.

Micromorphology, histochemistry and ultrastructure of the foliar trichomes of Withania somnifera (L.) Dunal (Solanaceae)

Abstract: The leaves of Withania somnifera contained four morphologically distinct trichome types: glandular capitate, non-glandular dendritic (branched), non-glandular bicellular and non-glandular multicellular trichomes. Major phytochemical compounds present within glandular and non-glandular trichomes were alkaloids and phenolic compounds. The aim of this study was to characterize the micromorphology of the foliar trichomes of Withania somnifera as well as to elucidate the location and composition of the secretory products. Trichome density and length was also determined in three developmental stages of the leaves. Light microscopy and scanning electron microscopy showed the presence of four morphologically distinct trichome types: glandular capitate, non-glandular dendritic, non-glandular bicellular and non-glandular multicellular. The dendritic trichomes exhibited cuticular warts which are involved in the “Lotus-Effect”. Glandular capitate and non-glandular dendritic trichomes were aggregated on the mid-vein of young and mature leaves, possibly to protect underlying vasculature. Histochemical staining also revealed the presence of two major classes of phytochemical compounds that are of medicinal importance, i.e. alkaloids and phenolic compounds. These compounds are used to treat a wide variety of ailments and also act as chemical deterrents in plants. The results of this study explain possible roles of four morphologically distinct trichome types based on their morphology, foliar distribution and content.

Physiological performance, secondary metabolite and expression profiling of genes associated with drought tolerance in Withania somnifera

Abstract: Physiological, biochemical, and gene expression responses under drought stress were studied in Withania somnifera Photosynthesis rate, stomatal conductance, transpiration rate, relative water content, chlorophyll content, and quantum yield of photosystems I and II (PSI and PSII) decreased in response to drought stress Comparative expression of genes involved in osmoregulation, detoxification, signal transduction, metabolism, and transcription factor was analyzed through quantitative RT-PCR The genes encoding 1-pyrroline-5-carboxylate synthetase (P5CS), glutathione S-transferase (GST), superoxide dismutase (SOD), serine threonine-protein kinase (STK), serine threonine protein phosphatase (PSP), aldehyde dehydrogenase (AD), leucoanthocyanidin dioxygenase/anthocyanin synthase (LD/AS), HSP, MYB, and WRKY have shown upregulation in response to drought stress condition in leaf tissues Enhanced detoxification and osmoregulation along with increased withanolides production were also observed under drought stress The results of this study will be helpful in developing stress-tolerant and high secondary metabolite yielding genotypes

Free radical scavenging activity and comparative metabolic profiling of in vitro cultured and field grown Withania somnifera roots.

Abstract: Free radical scavenging activity (FRSA), total phenolic content (TPC), and total flavonoid content (TFC) of in vitro cultured and field grown Withania somnifera (Ashwagandha) roots were investigated. Withanolides analysis and comprehensive metabolic profiling between 100% methanol extracts of in vitro and field grown root tissues was performed using high performance thin layer chromatography (HPTLC) and gas chromatography-mass spectrometry (GC-MS), respectively. Significantly higher levels of FRSA, TPC, and TFC were observed in in-vitro cultured roots compared with field grown samples. In addition, 30 day-cultured in vitro root samples (1MIR) exhibited a significantly higher FRSA (IC50 81.01 μg/mL), TPC (118.91 mg GAE/g), and TFC (32.68 mg CE/g) compared with those in 45 day-cultured samples (1.5MIR). Total of 29 metabolites were identified in in vitro cultured and field grown roots by GC-MS analysis. The metabolites included alcohols, organic acids, purine, pyrimidine, sugars, and putrescine. Vanillic acid was only observed in the in vitro cultured root samples, and higher level of the vanillic acid was observed in 1MIR when compared to 1.5MIR. Therefore, it is suggested that 1MIR might serve as an alternative to field grown roots for the development of medicinal and functional food products.

Overexpression of squalene synthase in Withania somnifera leads to enhanced withanolide biosynthesis

Abstract: Genetic engineering of secondary metabolic pathways is an emerging area of research for production and improvement of natural products in plant biotechnology. Here, we describe a systematic approach to manipulate a key regulatory step of isoprenoid biosynthetic pathway in Withania somnifera to study its effect on withanolide production. We generated T0 W. somnifera plants overexpressing squalene synthase (WsSQS) by Agrobacterium tumefaciens mediated transformation, which were analyzed by Gus biochemical assay and PCR of hygromycin phosphotransferase (hptII) and WsSQS. qRT-PCR analyses of various transformed tissues indicated 2–5 fold increase in WsSQS transcripts in both T0 and T1 generations. The tissue specific protein expression studies revealed 2–3 fold increase in WsSQS, which was further confirmed by enzyme activity. These observations were corroborated with the 1.5–2 fold increase in total withanolide content of the transformed tissues. However, in leaf tissue, the levels of Withaferin A and Withanolide A increased significantly up to 4–4.5 fold. These findings demonstrate genetic engineering of isoprenoid pathway in W. somnifera resulting in enhanced production of withanolides, and also provide insights into such metabolic pathways for their manipulation to improve the pharmacological content of different medicinally important plants.

Light and auxin responsive cytochrome P450s from Withania somnifera Dunal: cloning, expression and molecular modelling of two pairs of homologue genes with differential regulation

Abstract: Cytochrome P450s (CYPs) catalyse a wide variety of oxygenation/hydroxylation reactions that facilitate diverse metabolic functions in plants. Specific CYP families are essential for the biosynthesis of species-specialized metabolites. Therefore, we investigated the role of different CYPs related to secondary metabolism in Withania somnifera, a medicinally important plant of the Indian subcontinent. In this study, complete complementary DNAs (cDNAs) of four different CYP genes were isolated and christened as WSCYP93Id, WSCYP93Sm, WSCYP734B and WSCYP734R. These cDNAs encoded polypeptides comprising of 498, 496, 522 and 550 amino acid residues with their deduced molecular mass of 56.7, 56.9, 59.4 and 62.2 kDa, respectively. Phylogenetic study and molecular modelling analysis of the four cloned WSCYPs revealed their categorization into two CYP families (CYP83B1 and CYP734A1) belonging to CYP71 and CYP72 clans, respectively. BLASTp searches showed similarity of 75 and 56 %, respectively, between the two CYP members of CYP83B1 and CYP734A1 with major variances exhibited in their N-terminal regions. The two pairs of homologues exhibited differential expression profiles in the leaf tissues of selected chemotypes of W. somnifera as well as in response to treatments such as methyl jasmonate, wounding, light and auxin. Light and auxin regulated two pairs of WSCYP homologues in a developing seedling in an interesting differential manner. Their lesser resemblance and homology with other CYP sequences suggested these genes to be more specialized and distinct ones. The results on chemotype-specific expression patterns of the four genes strongly suggested their key/specialized involvement of the CYPs in the biosynthesis of chemotype-specific metabolites, though their further biochemical characterization would reveal the specificity in more detail. It is revealed that WSCYP93Id and WSCYP93Sm may be broadly involved in the oxygenation reactions in the plant and, thereby, control various pathways involving such metabolic reactions in the plant. As a representative experimental validation of this notion, WSCYP93Id was heterologouly expressed in Escherichia coli and catalytic capabilities of the recombinant WSCYP93Id protein were evaluated using withanolides as substrates. Optimized assays with some major withanolides (withanone, withaferin A and withanolide A) involving spectrophotometric as well as high-pressure liquid chromatography (HPLC)-based evaluation (product detection) of the reactions showed conversion of withaferin A to a hydroxylated product. The genes belonging to other CYP group are possibly involved in some specialised synthesis such as that of brassinosteroids.

Withania somnifera shows a protective effect in monocrotaline-induced pulmonary hypertension.

Abstract: Context: Withania somnifera (Linn.) Dunal (Solanaceae), a clinically used herbal drug in Ayurveda, shows potent antioxidant, anti-inflammatory, pro-apoptotic, and cardioprotective effects. However, the efficacy of W. somnifera in pulmonary hypertension (PH), a cardiopulmonary disorder, remains unexplored.Objective: The present study investigates the effect of W. somnifera root powder on monocrotaline (MCT)-induced PH in rats.Materials and methods: In preventive studies, W. somnifera root powder (50 and 100 mg/kg/d, p.o.) was administered from day 1 following single administration of MCT (60 mg/kg, s.c.) in Sprague–Dawley (SD) rats. After 35 d, right ventricular pressure (RVP) was measured in anesthetized rats. Various physical markers of right ventricular hypertrophy (RVH) were measured in isolated hearts. Markers of endothelial function, inflammation, and oxidative stress were estimated in lung homogenate. Vasoreactivity of pulmonary arteries was also studied. In therapeutic treatment, W. somnife...