Standardized root extract of Withania somnifera and Withanolide A exert moderate vasorelaxant effect in the rat aortic rings by enhancing nitric oxide generation.

ETHNO-PHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal, commonly known as Ashwagandha, belongs to the family Solanaceae. In Ayurveda, Ashwagandha has been defined as one of the most important herb and is considered to be the best adaptogen. It is also an excellent rejuvenator, a general health tonic and cure for various disorders such as cerebrovascular, insomnia, asthma, ulcers, etc. Steroidal lactones (Withanolides: Withanolide A, Withaferin A, Withanolide D, Withanone, etc) isolated from this plant, possess promising medicinal properties such as anti-inflammatory, immune-stimulatory etc. Standardized root extract of the plant NMITLI-118R (NM) was prepared at CSIR-CIMAP, and was investigated for various biological activities at CSIR-CDRI. Among the notable medicinal properties, NM exhibited excellent neuroprotective activity in the middle cerebral artery occlusion (MCAO) rat model. AIM OF THE STUDY: Endothelial dysfunction is the primary event in the cerebrovascular or cardiovascular disorders, present study was thus undertaken to evaluate vasoprotective potential of NM and its biomarker compound Withanolide A (WA) using rat aortic rings and EA.hy926 endothelial cells. MATERIAL AND METHODS: Transverse aortic rings of 10 weeks old Wistar rats were used to evaluate effect of NM and WA on the vasoreactivity. While, mechanism of NM and WA mediated vasorelaxant was investigated in Ea.hy926 cell line by measuring NO generation, nitrite content, Serine 1177 phosphorylation of eNOS, reduced/oxidized biopterin levels and expression of endothelial nitric oxide synthase (eNOS) mRNA and protein. RESULTS: Fingerprinting of NM using HPLC identified presence of WA in the extract. NM as well as WA exerted moderate vasorelaxant effect in the endothelium intact rat aortic rings which was lesser than acetylcholine (ACh). NM and WA augmented ACh induced relaxation in the rat aortic rings. NM and WA dependent vasorelaxation was blocked by N-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ), indicating role of NO/cGMP. Further Ea.hy926 cells treated with NM and WA showed accumulation of nitrite content, enhanced NO levels, eNOS expression and eNOS phosphorylation (Serine 1177). CONCLUSION: Altogether NM and WA dependent improvement in the NO availability seems to be mediated by the enhanced eNOS phosphorylation. WA, seems to be one of the active constituent of NM, and presence of other vasoactive substances cannot be ruled out. The data obtained imply that the vasorelaxant property of NM is beneficial for its neuroprotective potential.

Neurodegenerative diseases and Withania somnifera (L.): An update.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal also known as 'Ashwaghanda' in Sanskrit and as 'Indian Winter Cherry' in english. is an important medicinal herb in India. It is widely used in Indian systems of medicine as an adaptogen, nerve tonic, anti-stress, memory enhancer and against cognitive deficits, insomnia, anxiety, infectious diseases, infertility, rheumatoid arthritis and gout over thousands of years. Its formulations are mainly used in Unani and Ayurvedic system of medicine. It is a remarkable centuries old herbal Rasayana used to treat neuronal ailments and is known as ''Sattvic Kapha Rasayana. AIM OF THE STUDY: To review neuroprotective properties of Withania somnifera (L.)extract as well as its active constituents in neurodegenerative diseases and other neurological ailments. MATERIALS AND METHODS: The sources of information used in present article include Indian system of Medicine reports on the use of natural products, Medicinal books, research articles and scientific databases like PubMed, Google Scholar, Web of Science, Science-Direct, SciFinder, ACS Publications and Wiley Online Library. RESULTS: Research reports based largely on preclinical studies as well as few clinical trials have highlighted the neuroprotective role of Ashwagandha against many neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's disease. The protective effects of Ashwagandha were accomplished by restoring mitochondrial and endothelial function, mitigation of apoptosis, inflammation and oxidative stress mechanisms. CONCLUSION: In this review, we recapitulated neuroprotective properties of Ashwagandha extracts and/or its major constituents and discussed their mechanisms of action and potential therapeutic applications. The pre-clinical as well as clinical studies suggest the use of Withania somnifera (L.) against neurodegenerative disease. However, extensive studies are warranted to validate the use of extract or its single constituents for its clinical use.

Quantification of withaferin-A and withanolide-A in diploid (n = 12) and tetraploid cytotypes (n = 24) of "Rassbhary", Physalis angulata L.

During the present study an analytical method based on reverse-phase high performance liquid chromatography photodiode array detection method was developed for simultaneous determination of withaferin-A and withanolide-A in plant parts of two cytotypes (diploid n = 12 & tetraploid n = 24) of Physalis angulata. All the plant parts were extracted in different solvent solutions i.e., acidic [HCl] methanol (i.e., methanol containing 0.3% of HCl), methanol, n-hexane, chloroform. Both the compounds were comparatively analysed. The results revealed that tetraploid cytotype (n = 24) showed the higher composition of both the reference compounds. The method is simple, rapid and provides better resolution can be easily applied to the quantitative analyses of withanolides in plant matrices.

Withanolide A extends the lifespan in human EGFR-driven cancerous Caenorhabditis elegans.

The conserved EGFR pathway is linked with multiple cancers in humans including breast, ovarian, and lung carcinoma. Withanolide A, one of the major withanolidal active compounds isolated from the Withania somnifera, extends lifespan and ameliorates stress resistance in wild-type C. elegans by targeting the Insulin/IGF-1 signaling pathway. Up-regulation of IGF1 can transactivate EGFR which inturn reduces longevity and promotes tumor development in an organism. We examined the effects of Withanolide A on the lifespan of a human EGFR-driven C. elegans transgenic model exhibiting the multivulva (Muv) phenotype. The results showed that WA extends the lifespan of both wild human EGFR-driven C. elegans model (human wild-type tyrosine kinase) as well as models bearing single (L858R), and double mutations (T790M-L858R). The lifespan extension observed in these transgenic strains was 20.35, 24.21 and 21.27%, respectively. Moreover, the reduced fat levels were noticed in both wild-type N2 worms and transgenic strains. These observations support the heathspan promoting effect of WA as lipid-rich diet has been reported to promote tumor development. In view of the fact that most of the well known FDA approved drugs such as gefitinib fail to inhibit the EGFR-associated cancers because of these mutations, the present findings show the potential of Withanolide A as a foreseen future nutraceutical to improve the average survival of cancer patients.

Features and outcomes of drugs for combination therapy as multi-targets strategy to combat Alzheimer's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Alzheimer's disease (AD), a deleterious neurodegenerative disorder that impairs memory, cognitive functions and may lead to dementia in late stage of life. The pathogenic cause of AD remains incompletely understood and FDA approved drugs are partial inhibitors rather than curative. Most of drugs are synthetic or natural products as galanthamine is an alkaloid obtained from Galanthus spp. Huperzine A, an alkaloid found in Huperzia spp., gingkolides a diterpenoids from Gingko biloba and many ethnobotanicals like Withania somnifera (L.) Dunal., Physostigma venenosum Balf., Bacopa monnieri (L.) Wettst., Centella asiatica (L.) Urb. have been used by traditional Indian, Chinese, and European system of medicines in AD. Clinical significance opioid alkaloid in Papaver somniferum has shown another dimension to this study. Over exploitation of medicinal plants with limited bioactive principles has provided templates to design synthetic drugs in AD e.g. rivastigmine, phenserine, eptastigmine based on chemical structure of physostigmine of Physostigma venenosum Balf. Even ZT-1 a prodrug of Hup A and memogain a prodrug of galantamine has achieved new direction in drug development in AD. All these first-line cholinesterase-inhibitors are used as symptomatic treatments in AD. Single modality of "One-molecule-one-target" strategy for treating AD has failed and so future therapies on "Combination-drugs-multi-targets" strategy (CDMT) will need to address multiple aspects to block the progression of pathogenesis of AD. Besides, cholinergic and amyloid drugs, in this article we summarize proteinopathy-based drugs as AD therapeutics from a variety of biological sources. In this review, an attempt has been made to elucidate the molecular mode of action of various plant products, and synthetic drugs investigated in various preclinical and clinical tests in AD. It also discusses current attempts to formulate a comprehensive CDMT strategy to counter complex pathogenesis in AD. MATERIALS AND METHODS: Information were collected from classical books on medicinal plants, pharmacopoeias and scientific databases like PubMed, Scopus, GoogleScholar, Web of Science and electronic searches were performed using Cochrane Library, Medline and EMBASE. Also published scientific literatures from Elsevier, Taylor and Francis, Springer, ACS, Wiley publishers and reports by government bodies and documentations were assessed. RESULTS: 60 no. of natural and synthetic drugs have been studied with their significant bioactivities. A decision matrix designed for evaluation of drugs for considering to the hypothetic "CDMT" strategy in AD. We have introduced the scoring pattern of individual drugs and based on scoring pattern, drugs that fall within the scoring range of 18-25 are considered in the proposed CDMT. It also highlights the importance of available natural products and in future those drugs may be considered in CDMT along with the qualified synthetic drugs. CONCLUSION: A successful validation of the CDMT strategy may open up a debate on health care reform to explore other possibilities of combination therapy. In doing so, it should focus on clinical and molecular relationships between AD and CDMT. A better understanding of these relationships could inform and impact future development of AD-directed treatment strategies. This strategy also involves in reducing costs in treatment phases which will be affordable to a common man suffering from AD.

Phospholipid complexation of NMITLI118RT+: way to a prudent therapeutic approach for beneficial outcomes in ischemic stroke in rats.

Withania somnifera Dunal (Solanaceae) known as Ashwagandha, a popular plant of Indian origin is known to possess tremedous medicinal potential, often used as anti-inflammatory, anti-platelet, antihypertensive, hypoglycemic, hypolipidemic and adaptogenic candidate. Some of its chemotypes developed by CSIR, India includes NMITLI-101, NMITLI-118, NMITLI-128. In this study the investigators have attempted development of a phytosomal complex of NMITLI118RT + (standardized ethanolic extract of a new chemotype of W. somnifera Dunal.), its pharmaceutical characterization and evaluation of its neuro-protective potential against experimenal stroke in rats in continuation with their previous work in this area. The phytosomal complex (NIMPLC) was prepared by following a cohesive optimization design and was characterized on the basis of solubility, dissolution profile, FT-IR, DSC-TGA analysis, zeta potential, physical stability, forced degradation and photolytic degradation. Results were suggestive of a pharmaceutically acceptable formulation. NIMPLC was taken up further for biological evaluation using the middle cerebral artery occlusion (MCAO) model in rats. It could be demonstrated that the beneficial effects of NMITLI118RT + could be augmented by NIMPLC in 1 h pre and 6 h post treatment as was evident from reduction in MDA levels, increment in GSH levels, reduction in neurological deficit (ND) scores and reduction in infarct size. The study could successfully demonstrate the beneficial effects of NIMPLC in brain function restoration following stroke.

Withania somnifera and Its Withanolides Attenuate Oxidative and Inflammatory Responses and Up-Regulate Antioxidant Responses in BV-2 Microglial Cells.

Withania somnifera (L.) Dunal, commonly known as Ashwagandha, has been used in Ayurvedic medicine for promoting health and quality of life. Recent clinical trials together with experimental studies indicated significant neuroprotective effects of Ashwagandha and its constituents. This study is aimed to investigate anti-inflammatory and anti-oxidative properties of this botanical and its two withanolide constituents, namely, Withaferin A and Withanolide A, using the murine immortalized BV-2 microglial cells. Ashwagandha extracts not only effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and reactive oxygen species (ROS) production in BV-2 cells, but also stimulates the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, leading to induction of heme oxygenase-1 (HO-1), both in the presence and absence of LPS. Although the withanolides were also capable of inhibiting LPS-induced NO production and stimulating Nrf2/HO-1 pathway, Withaferin A was tenfold more effective than Withanolide A. In serum-free culture, LPS can also induce production of long thin processes (filopodia) between 4 and 8 h in BV-2 cells. This morphological change was significantly suppressed by Ashwagandha and both withanolides at concentrations for suppressing LPS-induced NO production. Taken together, these results suggest an immunomodulatory role for Ashwagandha and its withanolides, and their ability to suppress oxidative and inflammatory responses in microglial cells by simultaneously down-regulating the NF-kB and upregulating the Nrf2 pathways.

Withanolide A offers neuroprotection, ameliorates stress resistance and prolongs the life expectancy of Caenorhabditis elegans.

Withanolide A (steroidal lactone) forms the major constituent of the most popular herbal drug in Ayurvedic medicine, Ashwagandha. It has been used since ancient times as an alternative medicine for the treatment of a variety of age related disorders. Here we provide multiple lines of evidence indicating that Withanolide A improves healthspan, delays age-associated physiological changes and also extends the lifespan of Caenorhabditis elegans. We also report several neuroprotective benefits of this natural product, including its anti-amyloidogenic effects, alleviation of α-synuclein aggregation and neuroprotection through modulation of neural mediators like acetylcholine. We observed that Withanolide A mediates lifespan extension and promotes stress resistance via insulin/insulin-like growth factor signaling pathway. Such findings could be helpful to develop a therapeutic medicine from this natural product for the prevention or reversal of age-related ailments and to improve the survival of patients suffering from Alzheimer's or Parkinson's disease.

Neuro-protective potential of a vesicular system of a standardized extract of a new chemotype of Withania somnifera Dunal (NMITLI118RT+) against cerebral stroke in rats.

Withania somnifera Dunal is an Indian medicinal plant with significant pharmacological properties, such as adaptogenic, anti-inflammatory, anti-oxidant, anti-platelet, anti-hypertensive, hypoglycemic and hypolipidemic effects. Several chemotypes of W. somnifera include NMITLI-101, NMITLI-118 and NMITLI-128. The present work elaborates the optimization and development of a liposomal delivery system for efficient delivery of NMITLI118RT+ [a standardized ethanolic extract of a new chemotype of W. somnifera Dunal (NMITLI-118) roots] against cerebral stroke in rats. Liposomal systems were prepared using thin-film hydration method and characterized on the basis of size, zeta potential, physical stability, FT-IR, DSC-TGA analysis and surface morphological studies by TEM. NMITLI118RT+ and its formulations (NMITLI118RT+LF) were evaluated for biological activity utilizing middle cerebral artery occlusion model in rats. The Z average of the developed liposomal formulation was about 142.6 ± 0.09 nm with a zeta potential of -31.20 ± 1.0 mV. Results of TEM revealed spherical particles in the range of 200 nm. The entrapment efficiency was found to be 94.603 ± 2%. The formulation was found to be physically stable over a 3-week period. Results were suggestive of the fact that both NMITLI118RT+ and its delivery system possess significant neuroprotective activity in cerebral ischemia. The liposomal system largely exhibits better performance over NMITLI118RT+ precisely in the post-treatment group. The present studies could elucidate the successful development of a delivery system for NMITLI118RT+ and demonstrate their beneficial neuro-protective potential in overcoming and reversing the consequences of I/R injury following stroke.

Stability indicating studies on NMITLI 118RT+ (standardized extract of withania somnifera dunal).

BACKGROUND: Withania somnifera Dunal (Ashwagandha) is an Indian medicinal plant of great medicinal value; used in many clinically proven conditions. NMITLI-118RT+ is a candidate drug under a Council of Scientific and Industrial Research (CSIR) networking project. It is a chemotype of W. somnifera's root extract, which has been used for the present study. OBJECTIVES: The present investigation aims to develop and validate a simple isocratic reverse phase-high performance liquid chromatography (RP-HPLC) system for the detection and estimation of Withanolide A (marker compound) and its analytical application for stability indicating studies on NMITLI-118RT+. MATERIAL AND METHODS: A validated RP-HPLC method for Withanolide A was established on a Waters HPLC system and the same was used on NMITLI-118RT+ for quantification and fingerprinting purposes, and for establishing forced degradation, isothermal stress tests, and drug-excipient testing protocols as per International Conference on Harmonization (ICH) guidelines. RESULTS: A validated method was established, which could detect the marker at a retention time of around 6.3 minutes, with a linearity range of 2-100 µg/mL, by varying the amounts of the said marker, which were estimated in four different batches of NMITLI-118RT+. Photostability as per ICH guidelines suggested a slight loss of the active constituent and maximum degradation was afforded with alkali followed by acid, and then peroxide, in the forced degradation studies. In the drug-excipient studies, the maximum amount of active constituent could be detected in the samples with ethyl cellulose and the least with hydroxy propyl cellulose. CONCLUSION: The method developed here was simple and rapid. The various stability indicating studies carried out in the present investigation would be useful for formulation development and were suggestive of deciding the recommended storage conditions for NMITLI-118RT+.

Influence of plant growth regulators on callus mediated regeneration and secondary metabolites synthesis in Withania somnifera (L.) Dunal.

Withania somnifera (L.) Dunal, is an important medicinal plant being the source of extremely important compounds like withanolides and withaferin. Influence of different plant growth regulators (PGRs) were evaluated for induction of callus, callus mediated regeneration and production of secondary metabolites in them. Explants for callusing were collected from plants grown in vitro and maximum callusing (98 %) was obtained on MS medium supplemented with a combination of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5 mg l(-1)) and kinetin (KN) (0.2 mg l(-1)). Among different types of calli, best shoot regeneration was observed on green, compact calli produced on MS medium with a combination of 6-benzylamino purine (BAP) and indole butyric acid (IBA). MS medium supplemented with BAP (2 mg l(-1)) showed highest frequency (98 %) of shoot bud regeneration. The micro-shoots were efficiently rooted on MS media supplemented with 0.5 mg l(-1) IBA. Rooted plants were transferred to soil-vermi-compost (1:3; w/w) medium in greenhouse for acclimatization. Presence of withanolide A and withaferin A in calli was validated through high performance thin layer chromatography (HPTLC). It was interesting to observe that the PGRs showed significant influence on the secondary metabolites production in callus and 2,4-D having the least effect. Histological studies revealed the origin of shoot tip in the callus during regeneration.

Comparative antioxidant potential of Withania somnifera based herbal formulation prepared by traditional and non-traditional fermentation processes.

BACKGROUND: Ashwagandharishtha is a liquid polyherbal formulation traditionally prepared by fermentation process using the flowers of Woodfordia fruticosa. It contains roots of Withania somnifera as a major crude drug. Alcohol generated during the fermentation causes the extraction of water insoluble phytoconstituents. Yeasts present on the flowers are responsible for this fermentation. METHODS: Total nine formulations of ashwagandharishtha were prepared by fermentation process using traditional Woodfordia fruticosa flowers (ASG-WFS) and using yeasts isolated from the same flowers. During fermentation, kinetic of alcohol generation, sugar consumption, changes in pH and withanolides extraction were studied. All the formulations were tested for in vitro antioxidant potential by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, hydrogen peroxide scavenging and total reducing power assay. The results were compared with standard ascorbic acid. RESULTS: Traditional formulation (ASG-WFS) showed the highest activity (p < 0.001) relative to other formulations and standard ascorbic acid. ASG-WFS showed significant (DPPH) free radical scavenging (78.75%) and hydrogen peroxide scavenging (69.62%) at the concentration of 1000 µg/mL and 100 µg/mL, respectively. CONCLUSION: Traditional process is the best process for preparing ashwagandharishtha to obtain significant antioxidant activity.