A critical assessment of the whole plant-based phytotherapeutics from Withania somnifera (L.) Dunal with respect to safety and efficacy vis-a-vis leaf or root extract-based formulation.

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,ß unsaturated ketone which binds to amines, alcohols, and esters and 5ß, 6ß 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.

Assessment of Chemopreventive Potential of the Plant Extracts against Liver Cancer Using HepG2 Cell Line.

The edible parts of the plants Camellia sinensis, Vitis vinifera and Withania somnifera were extensively used in ancient practices such as Ayurveda, owing to their potent biomedical significance. They are very rich in secondary metabolites such as polyphenols, which are very good antioxidants and exhibit anti-carcinogenic properties. This study aims to evaluate the anti-cancerous properties of these plant crude extracts on human liver cancer HepG2 cells. The leaves of Camellia sinensis, Withania somnifera and the seeds of Vitis vinifera were collected and methanolic extracts were prepared. Then, these extracts were subjected to DPPH, α- amylase assays to determine the antioxidant properties. A MTT assay was performed to investigate the viability of the extracts of HepG2 cells, and the mode of cell death was detected by Ao/EtBr staining and flow cytometry with PI Annexin- V FITC dual staining. Then, the protein expression of BAX and BCl2 was studied using fluorescent dye to determine the regulation of the BAX and BCl2 genes. We observed that all the three extracts showed the presence of bioactive compounds such as polyphenols or phytochemicals. The W. somnifera bioactive compounds were found to have the highest anti-proliferative activity on human liver cancer cells.

Withaferin A exerts an anti-obesity effect by increasing energy expenditure through thermogenic gene expression in high-fat diet-fed obese mice.

BACKGROUND: The enhancement of energy expenditure has attracted attention as a therapeutic target for the management of body weight. Withaferin A (WFA), a major constituent of Withania somnifera extract, has been reported to possess anti-obesity properties, however the underlying mechanism remains unknown. PURPOSE: To investigate whether WFA exerts anti-obesity effects via increased energy expenditure, and if so, to characterize the underlying pathway. METHODS: C57BL/6 J mice were fed a high-fat diet (HFD) for 10 weeks, and WFA was orally administered for 7 days. The oxygen consumption rate of mice was measured at 9 weeks using an OxyletPro™ system. Hematoxylin and eosin (H&E), immunohistochemistry, immunoblotting, and real-time PCR methods were used. RESULTS: Treatment with WFA ameliorated HFD-induced obesity by increasing energy expenditure by improving of mitochondrial activity in brown adipose tissue (BAT) and promotion of subcutaneous white adipose tissue (scWAT) browning via increasing uncoupling protein 1 levels. WFA administration also significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the BAT of obese mice. Additionally, WFA activated mitogen-activated protein kinase (MAPK) signaling, including p38/extracellular signal-regulated kinase MAPK, in both BAT and scWAT. CONCLUSION: WFA enhances energy expenditure and ameliorates obesity via the induction of AMPK and activating p38/extracellular signal-regulated kinase MAPK, which triggers mitochondrial biogenesis and browning-related gene expression.

Withania somnifera Extract Enhances Energy Expenditure via Improving Mitochondrial Function in Adipose Tissue and Skeletal Muscle.

 Withania somnifera (WS), commonly known as ashwagandha, possesses diverse biological functions. WS root has mainly been used as an herbal medicine to treat anxiety and was recently reported to have an anti-obesity effect, however, the mechanisms underlying its action remain to be explored. We hypothesized that WS exerts its anti-obesity effect by enhancing energy expenditure through improving the mitochondrial function of brown/beige adipocytes and skeletal muscle. Male C57BL/6J mice were fed a high-fat diet (HFD) containing 0.25% or 0.5% WS 70% ethanol extract (WSE) for 10 weeks. WSE (0.5%) supplementation significantly suppressed the increases in body weight and serum lipids, and lipid accumulation in the liver and adipose tissue induced by HFD. WSE supplementation increased oxygen consumption and enhanced mitochondrial activity in brown fat and skeletal muscle in the HFD-fed mice. In addition, it promoted browning of subcutaneous fat by increasing mitochondrial uncoupling protein 1 (UCP1) expression. Withaferin A (WFA), a major compound of WS, enhanced the differentiation of pre-adipocytes into beige adipocytes and oxygen consumption in C2C12 murine myoblasts. These results suggest that WSE ameliorates diet-induced obesity by enhancing energy expenditure via promoting mitochondrial function in adipose tissue and skeletal muscle, and WFA is a key regulator in this function.

A validated HPTLC method for the simultaneous quantifications of three phenolic acids and three withanolides from Withania somnifera plants and its herbal products.

A simple, rapid and selective high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for simultaneous determination of three withanolides (withaferin A, withanone and withanolide A) and three phenolic acids (caffeic acid, ferulic acid and benzoic acid) from different parts (root, stem and leaf) of Withania somnifera and its two commercially available polyherbal formulations. The extraction efficiency of withanolides and phenolic acids were tested using two solvents, chloroform and methanol, respectively. HPTLC separation was performed on silica coated aluminium plates Si 60F254; using toluene, ethyl acetate and acetic acid (60:40:4). The samples were quantitated at 231 nm. The purity and identity of peaks of all the six analytes were confirmed by matching Rf values and UV-spectrum with authentic standards. The identity of three withanolides was further confirmed by positive ion electrospray ionization mass spectrometry (ESI-MS/MS) analyses. The developed method was validated for sensitivity, linearity, reproducibility, accuracy, the limit of detection (LOD) and limit of quantification (LOQ) following the guidelines of the International Conference on Harmonization (ICH). The method was found to be linear (r > 0.99) in the range of 50-2000 ng/band for benzoic acid and 50-1000 ng/band for the other five studied metabolites. This simple and accurate HPTLC method provided enhanced resolution of studied analytes as compared to other phytoconstituents present in W. somnifera extracts. It has also been successfully applied in the analysis and quantification of two polyherbal formulations containing W. somnifera plant parts.

Green and cost effective synthesis of silver nanoparticles from endangered medicinal plant Withania coagulans and their potential biomedical properties.

Silver nanoparticles (AgNPs) have great potential for their mechanistic role in biomedical researches. Recently, green biosynthetic approaches have been received much attention in plant science for nanoparticles production. Therefore, in the present study AgNPs have been synthesized utilizing in-vitro grown leaf extract of anti-diabetic medicinal plant Withania coagulans Dunal by the reduction of silver nitrate solution. W. coagulans synthesized silver nanoparticles (WcAgNPs) were characterized by UV-visible spectroscopy, scanning electron microscopy, energy dispersive X-ray analysis, transmission electron microscopy, X-ray powder diffraction and Fourier transform Infra-Red spectroscopy. All cumulative results showed that WcAgNPs were ~14 nm in size having spherical shape with face centered cubic structure. High performance liquid chromatography confirmed the involvement of withanolides in AgNPs synthesis as a reducing/capping agent. Synthesized WcAgNPs showed greater antioxidative potential when compared with W. coagulans leaf extract. WcAgNPs have efficient antimicrobial potential and suppresses the growth of both gram positive and gram negative bacteria. In our finding we also observed cytotoxicity of WcAgNPs against SiHa (cervical cancerous, hyper-triploid) cell lines and apoptosis in SiHa cells after 48 hour incubation with 13.74 µg ml-1 (IC50) concentration of WcAgNPs. As results suggested, this is the first report which explain that W. coagulans leaf extract have potential as bio-reducing agent for synthesis of silver nanoparticles, which can be exploited as anti-oxidant, antimicrobial and anti-cancerous agent and depicting an effective way for utilizing bioactive resources in restoration of medicinal properties of this plant with high efficacy.

Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum.

Larval packet test was used for detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids, in the multi-host tick Hyalomma anatolicum collected from district Moga, Punjab (India). Results indicated the presence of level I resistance against deltamethrin (RF = 2.81), whereas the tick isolate was susceptible to cypermethrin (RF = 0.2). The aqueous and ethanolic extracts of leaves of Cymbopogon winterianus, Vitex negundo and Withania somnifera along with roots of Vitex negundo were assessed for their acaricidal activity against the larvae of deltamethrin resistant H. anatolicum. The efficacy was assessed by measuring per cent larval mortality and determination of LC50 values. The various ethanolic extracts produced a concentration dependent increase in larval tick mortality, whereas the aqueous extracts exhibited a much lower mortality. The highest mortality (93.7 ± 0.66 %) was observed at the 5.0 % concentration of ethanolic extract of leaves of C. winterianus and the lowest LC50 value (0.011 %) was recorded for ethanolic extracts of leaves of V. negundo. The results indicated that these plant extracts have potential to be developed as herbal acaricides.

Acute and sub-acute oral toxicity assessment of the hydroalcoholic extract of Withania somnifera roots in Wistar rats.

Withania somnifera is a widely used medicinal plant for several disorders. Toxicity studies on Withania somnifera are not available. Acute and sub-acute oral toxicities of Withania somnifera root extract in Wistar rats were evaluated in the present study. In the acute toxicity study, WSR extract was administered to five rats at 2000 mg/kg, once orally and were observed for 14 days. No toxic signs/mortality were observed. In the sub-acute study, WSR extract was administered once daily for 28 days to rats at 500, 1000 and 2000 mg/kg, orally. No toxic signs/mortality were observed. There were no significant changes (P < 0.05) in the body weights, organ weights and haemato-biochemical parameters in any of the dose levels. No treatment related gross/histopathological lesions were observed. The present investigation demonstrated that the no observed adverse effect level was 2000 mg/kg body weight per day of hydroalcoholic extract of W. somnifera in rats and hence may be considered as non-toxic.

Wastewater treatment by batch adsorption method onto micro-particles of dried Withania frutescens plant as a new adsorbent.

A new adsorbent for removing metallic elements, nitrate and phosphate ions from municipal and industrial wastewaters has been investigated. This new adsorbent consists of micro-particles of dried Withania frutescens plant (<500 µm). Batch experiments were conducted to evaluate the removal of metallic elements and anions from raw wastewaters by W. frutescens particles. The results show that the micro-particles of W. frutescens plant presented a good adsorption of metallic elements, nitrate and phosphate ions from both real wastewaters. This adsorption increased with increasing of contact time. The percentage of metallic elements removal from industrial wastewater by W. frutescens plant was 98 ≈ 99% for Pb(II), 92 ≈ 93% for Cd(II), 91 ≈ 92% for Cu(II) and 92 ≈ 93% for Zn(II). The maximum adsorption capacity was dependent on the type of ions. The results also indicate that the values of chemical oxygen demand (COD) decrease after the contact with W. frutescens particles. Based on the results it can be concluded that the dried W. frutescens plant appears to be an economical and environmentally friendly material for wastewater treatment.