Withanolide A Research Articles

Nano-cubosomes of the phyto-active principle in Withania somnifera: LC-MS-NMR, anti-microbial, and insights of the anti-neuropathic and anti-inflammatory mechanism

Withania somnifera (W. somnifera) has a long history of safety in the amelioration of neuro-active ailments. The current study aims to explore Withania somnifera phyto-active principle anti-microbial, ant-neuropathic, and anti-inflammatory activities, and to modify these activities utilizing nano-cubosomes exploiting their mechanisms of action. Bio-guided fractionation technique was utilized, to identify the most phyto-active compound, using LC-MS-NMR online technique and biological models of diabetes, neuropathy, and inflammation. In-vitro antibacterial activity was also monitored. The HbA1c, in-vivo antioxidant (serum-catalase, TBARS, and GSH), serum insulin, and pro-inflammatory serum cytokines (TNF alpha, IL-six, and IL-ten) levels have been assessed to establish the anti-neuropathic and anti-inflammatory mechanisms. The nano-cubosomal formulations (CUB 1–3) were utilized to improve the W. somnifera most active compound efficacy. W. somnifera has shown ten major peaks; coagulin Q (10.2 %), dihydrowithanolide A (2.4 %), dihydrowithaferin D (1.8 %), physagulin D (7.6 %), withanoside V (2.3 %), withanolide A (WDA, 10.3 %), withafrin A (4.9 %), withaferin D (7.7 %), withanone 9 (9.9 %), withanolide D (4.8 %). The bio-guided fractionation technique utilizing LC-MS-NMR technique has proved that withanolide A (WDA) is the most phyto-active compound in W. somnifera. The latter has shown better results than WDA, which might be due to other effective compounds in Ws. However, CUB 3 (WDA nano-cubosomes dispersion) has shown more prominent anti-diabetic, anti-neuropathic, anti-inflammatory, and anti-bacterial potentials than Ws and WDA. Thus, CUB 3 modified WDA activity, and improved its efficacy. The normalization of HbA1c levels, increased insulin secretagogue potential, and the amelioration of the oxidative-stress may be the underlying Ws, WDA, and CUB 3 antidiabetic neuropathy mechanism. Moreover, the Ws, WDA, and CUB 1–3 anti-inflammatory mechanism might be due to the amelioration of the pro-inflammatory serum cytokines (decreasing TNF alpha and IL-six levels and increasing IL-ten). Thus, CUB 3 might be a powerful tool in augmenting Withania somnifera activity as an oral drug-delivery system and improving its efficacy against neuropathy and inflammation.

Advances in Sardinian Withania somnifera (L.) Dunal crops through phytochemical and biological approaches

Withania somnifera (L.) Dunal is widely used in the Indian traditional system of medicine to promote general health, wellness, and longevity. Its pharmacological properties are attributed to a group of molecules called withanolides, among which Withaferin A holds great interest for its anti-carcinogenic action. For this reason, numerous studies in recent years have focused on different metabolic or genetic engineering solutions to increase its yield. Here, we present the Sardinian chemotype of Withania somnifera as a potential crop for the extraction of Withaferin A. W. somnifera was cultivated from Sardinian wild germplasm collected in the northeast of the island. After 18 months, the leaves and the roots were collected and their methanolic extract was analyzed by HPLC. 0.3 mg/g DW of Withanolide A (WA), 1.0 mg/g DW of Withanolide B (WB) and 17.7 mg/g DW of Withaferin A (WF) were detected in the leaf sample, while lower values were detected in the roots (0.1 mg/g WF, 0.3 WA mg/g, 0.1 mg/g WB, 0.2 mg/g WO). This research not only confirms the high Withaferin A content found in the wild population leaves, but shows how they are reproducible in cultivated specimens, highlighting Sardinian W. somnifera leaves as a potential source of high-content Withaferin A products. Finally, we focused on the leaves extract by characterizing the phenolic and flavonoid content, as well as the in-vitro antioxidant capacity by DPPH and ABTS assays, revealing a significant amount of phenolic compounds and a related free radical scavenging activity. The leaves extract was further characterized for its anti-aging properties for potential cosmetic application, by the inhibition of tyrosinase, elastase, and collagenase enzymes.

Physiological and biochemical traits positively modulate tissue-specific withanolides and untargeted metabolites in Withania somnifera (L.) dunal under salinity stress

Withania somnifera (L.) Dunal (Ashwagandha) has been used in herbal medicines worldwide and in the Indian traditional medicinal system for 3000 years. It is a member of the Solanaceae family distributed across Asia, Africa, Australia, and Europe. Its bioactive secondary metabolite (withanolide) biosynthesis is sensitive to salinity stress, though the mechanism remains unexplored. Therefore, we investigated the effect of Sodium chloride (NaCl) on growth, photosynthesis, biochemical traits, tissue-specific withanolide, and untargeted metabolites in W. somnifera. Ashwagandha plants were raised in pots containing soil mixture and treated with different NaCl concentrations (0 as control, 10, 30, and 50 mM) for one month inside the greenhouse. NaCl stress significantly enhanced withaferin A (WFA) (3.79 mg/g), withanolide A (WA) (0.51 mg/g), and withanone (WN) (0.022 mg/g) at 50 mM NaCl groups in the shoot. Similarly, in the root, a significant increase in WFA (0.19 mg/g) and WN (0.0016 mg/g) were observed at 10 mM, WA (0.059 mg/g) at 30 mM, and withanolide B (WB) (0.013 mg/g) at 50 mM NaCl groups compared to control. LC-MS-based untargeted metabolite profiling revealed 37 differentially accumulated metabolites in all groups. Maximum abundance of glycyl-hydroxyproline (8X) followed by tyrosyl-valine (2X) and 3-hydroxy-beta-ionone (2X) were recorded at 50 mM NaCl groups compared to the control. This study showed for the first time that low NaCl stress enhances the biosynthesis of tissue-specific withanolides through physio-biochemical and metabolites adjustment. Overall, we demonstrated a multifaceted approach for cultivating medicinal crops in salt-affected areas with enhanced bioactive metabolites for healthcare and pharmaceutical industries.

Effect of Withanolide-A on Mice Subjected to Simulated Chronic Jetlag/Shift Work

Objective: Circadian rhythm is maintained by central and peripheral clocks that regulate an organism’s physiological functions. Frequent trans-meridian travel, an irregular sleep-wake cycle, light exposure during the rest period, rotating shift work, and other factors cause desynchronization of central and peripheral clocks, impairing social and occupational behavior. Withanolide-A (WA) is used for its powerful nootropic and neuroprotective properties.Methods: Effects of 13 days consecutive pre-dosing of WA (10 μmol/kg/day) on circadian rhythm disruption caused due to 9 h advance-delay chronic jetlag light-dark shift was studied on re-entrainment rate analyzed through locomotor activity recording of mice using the ClockLab setup, learning and memory studied through Y-maze and Barnes maze tests. In the hippocampus, gene expression of <i>PER1</i> and <i>BDNF</i> was assessed using reverse transcription-quantitative polymerase chain reaction. The effect of WA treatment was also studied on superoxide dismutase, catalase, reactive oxygen species (ROS), and lipid peroxidation in hippocampal tissue.Results: The results showed that WA treatment leads to faster re-entrainment and better performance during Y-maze and Barnes maze tests. <i>PER1</i> and <i>BDNF</i> genes expression was upregulated in the treated group compared to chronic jetlag exposure groups in the hippocampus and suprachiasmatic nuclei. Moreover, the WA-treated group produced fewer ROS and superoxide radicals, and there was also less lipid peroxidation in the treated group.Conclusion: These results suggest that the WA may act as a chronobiotic and neuroprotective agent as treated group mice took fewer transient cycles to re-entrain after chronic light-dark shift. Neuroprotective property is supported by learning-memory behavioral tests, gene studies and biochemical assays.

Withanolide-A Accelerates Re-entrainment of Circadian Locomotor Activity Rhythm and Preserves Spatial Memory in Mice Following Acute Phase Inversion of Light-Dark Cycles

Objective: Circadian rhythms are present almost in all living organisms and synchronized with the zeitgebers (time givers), especially lightdark (LD) cycles. The jetlag and rotating shift work schedules lead to desynchronization of the circadian clock with zeitgebers which may cause various health problems. Studies have shown that withanolide-A (WA) is a potent nootropic and neuroprotective agent found in Withania somnifera. Thus, the objective was to study the effect of WA on the circadian locomotor activity rhythm following the phase inversion of 12:12 h LD cycle in mice and its impact on the spatial memory in mice.Methods: Adult male mice were housed in the chronocubicle and recorded circadian locomotor activity rhythm by using ClockLab setup. The experimental mice were administered with WA (10 μmol/kg/day) for 13 consecutive days at about ZT15 and then evoked an acute phase inversion of 12:12 h light-dark cycle. Further, the Barnes maze test was performed to check the effect of WA on spatial memory following the phase inversion.Results: The results showed that WA-treated group took significantly fewer transient cycles (4.66+0.66) for re-entrainment than the control (9.5+0.43) and vehicle-treated (8.33+1.28) mice following the phase inversion. Similarly, WA-treated mice traveled less distance to reach the target zone in the Barnes maze compared to vehicle and control groups.Conclusion: These results suggest that the WA may act as chronobiotic and neuroprotective agent since it decreased the transient cycles for re-entrainment and retaining spatial memory following the phase inversion.

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 relevanceWithania 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 studyEndothelial 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 methodsTransverse 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. ResultsFingerprinting 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). ConclusionAltogether 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.

Red laser-mediated alterations in seed germination, growth, pigments and withanolide content of Ashwagandha [Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal, generally well-known as Ashwagandha, is part of Indian traditional medicinal systems like Ayurveda, Siddha, and Unani for over 3000 years for treating an array of disorders. The chief bioactive component of this plant is the withanolides, a group of C28-steroidal lactone triterpenoids. These compounds are present in very low concentrations and hence cell culture methods have been used to enhance their production. Low-level laser irradiation has been reported to have elicited the seed germination, agronomical characters, biosynthesis of bioactive compounds in some plants. Therefore, the objective of the study was to investigate the effect of red (He-Ne) laser irradiation on seed germination, growth characters, pigment contents and withanolide content in W. somnifera. The seeds were inoculated onto two different combinations of Murashige and Skoog (MS) media and incubated for germination. The highest germination percentage was observed in ½ MS with pH 6.5 and GA3 presoaking followed by ½ MS with different pH. Four different doses of Helium-Neon (He-Ne) laser (10, 15, 20 and 25 J/cm2) were used to irradiate the seeds at 632.8 nm and germinated in vitro on ½ MS with pH 6.5. The maximum germination percentage, 63.88% was noted from seeds irradiated with 25 J/cm2 (P = 0.04). The highest total length of 13.33 cm was observed in the seedlings irradiated with 25 J/cm2 groups (P = 0.008). The highest total chlorophyll content of 329.5 μg/g fresh weight (FW) was observed for seedlings irradiated with 15 J/cm2 (P = 0.02) and the highest carotenoid content of 49.6 μg/g FW was observed for 25 J/cm2 treated seedlings. Further, primary root length was measured and found to be highest (11.14 cm) in seedlings irradiated with 10 J/cm2 and the highest number of lateral roots were observed for 15 and 25 J/cm2 groups. The significant amount of Withanolide A (WA) 0.52 μg/g dry weight (DW) and 0.60 μg/g DW was noted in 15 (P = 0.01) and 20 J/cm2 (P = 0.002) groups, respectively than control. The present investigation thus reveals the positive impact of red laser on the germination of seeds, growth characters and withanolide contents under in vitro environment.

Induction of stress resistance and extension of lifespan in Chaenorhabditis elegans serotonin-receptor knockout strains by withanolide A

IntroductionApproximately 300 million people worldwide suffer from depression. The COVID-19 crisis may dramatically increase these numbers. Severe side effects and resistance development limit the use of standard antidepressants. The steroidal lactone withanolide A (WA) from Withania somnifera may be a promising alternative. Caenorhabditis elegans was used as model to explore WA's anti-depressive and anti-stress potential. MethodsC. elegans wildtype (N2) and deficient strains (AQ866, DA1814, DA2100, DA2109 and MT9772) were used to assess oxidative, osmotic or heat stress as measured by generation of reactive oxygen species (ROS), determination of lifespan, and mRNA expression of serotonin receptor (ser-1, ser-4, ser-7) and serotonin transporter genes (mod-5). The protective effect of WA was compared to fluoxetine as clinically established antidepressant. Additionally, WA's effect on lifespan was determined. Furthermore, the binding affinities and pKi values of WA, fluoxetine and serotonin as natural ligand to Ser-1, Ser-4, Ser-7, Mod-5 and their human orthologues proteins were calculated by molecular docking. ResultsBaseline oxidative stress was higher in deficient than wildtype worms. WA and fluoxetine reduced ROS levels in all strains except MT9772. WA and fluoxetine prolonged survival times in wildtype and mutants under osmotic stress. WA but not fluoxetine increased lifespan of all heat-stressed C. elegans strains except DA2100. Furthermore, WA but not fluoxetine extended lifespan in all non-stressed C. elegans strains. WA also induced mRNA expression of serotonin receptors and transporters in wildtype and mutants. WA bound with higher affinity and lower pKi values to all C. elegans and human serotonin receptors and transporters than serotonin, indicating that WA may competitively displaced serotonin from the binding pockets of these proteins. ConclusionWA reduced stress and increased lifespan by ROS scavenging and interference with the serotonin system. Hence, WA may serve as promising candidate to treat depression.

Traditional Horticulture Practices Increase the Production of Selected Withanolides in Withania Somnifera (L.) Dunal-A RP-UFLC Analysis.

The study evaluates the effect of two traditional horticulture treatments mentioned in Vrikshayurveda, a text from ancient India on the science of plant life, namely Kunapa jala (KJ) and Pancha gavya (PG) on the production of Withaferin A (WFA), withanolide A (WIA) and Withanolide B (WIB) in Withania somnifera (L) Dunal. Leaves and roots of W. somnifera were collected from different treated groups viz. control, KJ, PG, farmyard manure (FYM) and inorganic fertilizer (NPK). Reverse phase ultra-flow liquid chromatography (RP-UFLC) method was developed, validated for simultaneous detection of WFA, WIA and WIB. Statistical analysis of data was performed by ANOVA and tested for significance by the Dunnett multiple comparison test and data were expressed as mean±standard deviation (SD). Results revealed, leaves possessed highest WFA content and roots possessed highest content of WIA and WIB. PG treated leaves were observed highest WFA (18.29mg/g) and roots were observed highest WIA (19.63mg/g) and WIB (1.36mg/g). Conclusively, RP-UFLC method for simultaneous detection of withanolides has been developed and validated to evaluate the effect of traditional horticulture treatments. It is concluded that the enhanced production of withanolides can be achieved by the application of PG when compared to NPK application.

Withanolide-A treatment exerts a neuroprotective effect via inhibiting neuroinflammation in the hippocampus after pilocarpine-induced status epilepticus

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

Withanolide a penetrates brain via intra-nasal administration and exerts neuroprotection in cerebral ischemia reperfusion injury in mice

1. Withanolide A (WA), a major constituent phytochemical of the Ayurvedic herb Withania somnifera reportedly combats neurodegeneration in Alzheimer’s disease and Parkinson’s disease. But no study has yet reported the ability of WA in crossing the blood–brain barrier (BBB). The present study analyses the brain penetration ability of WA after intra-nasal administration and assesses its neuroprotective ability in cerebral ischemia-reperfusion injury in adult mice model.2. Brain penetration of WA after intranasal administration in cortex and cerebellum was assessed using HPLC-UV. Three different doses (1 mg/kg, 5 mg/kg and 10 mg/kg) of the phytochemical were used to study the neuroprotective ability of WA by evaluating the brain damage, changes in cerebral neurotransmitter levels and brain tissue morphology.3. Intranasal administration of the phytochemical facilitates its penetration in the cortex and cerebellum. Post-treatment with WA significantly reduced cerebral infarction, restored BBB disruption and cerebral oedema. The WA post-treatment also lowered the ischemia-induced elevated neurotransmitter and biochemical levels in brain compartments. The highest dose (10 mg/kg) of WA also markedly reduced the morphological damages, apoptotic and necrotic cell death in brain tissue occurring due to cerebral ischemia pathophysiology.4. Intra-nasal administration enables brain penetration of WA and allows the phytochemical to exert neuroprotective ability in the global cerebral ischemia model.

Extraction Optimization for Phenolic- and Withanolide-Rich Fractions from Withania somnifera Roots: Identification and Quantification of Withaferin A, 12-Deoxywithastromonolide, and Withanolide A in Plant Materials and Marketed Formulations Using a Reversed-Phase HPLC-Photodiode Array Detection Method.

Background: Both the roots and leaves of Withania somnifera are products of commerce. They contain active compounds of therapeutic value and mostly different withanolides. Several pharmacological activities of W. somnifera have links to one or more withanolides. The presence of phenolic compounds in extracts could play a vital role in the reduction of blood glucose levels in diabetic subjects. Objective: The present study was carried out for the selection of a solvent to prepare extracts rich in phenolics, withaferin A (WA), 12-deoxywithastromonolide (12WD), and withanolide A (WDA). A simple, rapid HPLC method was also developed for the identification and quantification of WA, 12WD, and WDA. Methods: The extraction efficiency of aqueous alcoholic solvents including hexane, chloroform, ethyl acetate, and methanol were compared for three selected withanolides and total phenolic content. The contents of WA, 12WD, and WDA and total phenolics were determined in the extracts. The quality of nine formulations containing W. sominfera were also compared in terms of the content of WA, 12WD, and WDA and total phenolics. Results: The maximum extract yield and the total withanolide and phenolic content were obtained from aqueous alcoholic compositions at 50:50 (v/v), 70:30 (v/v), and 100:0 (v/v), respectively. In the case of organic solvents, chloroform and ethyl acetate yielded the highest concentrations of phenolics and three withanolides, respectively. The total phenolic content in formulations was in the range of 1.84-3.13%, and total withanolide content showed wide variability. Conclusions: The outcome of the present investigation could be utilized for the selection of extraction solvents to prepare W. somnifera-enriched extracts and their quality monitoring by using the developed and validated HPLC-Photodiode array detection method. Highlights: A process for preparation of phenolics and withanolides (withaferin A, 12-deoxywithastramonolide and withanolide A) enriched extracts of Withania somnifera. Simple and rapid HPLC method was also developed and validated as per the ICH guidelines for identification and quantification of three major withanolides. The developed HPLC method was applied to analyze the quality of extracts and marketed herbal products (mono, as well as poly constituents). Optimized extraction process could be utilized for upscaling process development in preparation of enriched extracts from Withania somnifera, crop improvement, bio-prospection studies and quality control.

Determination of permeability, plasma protein binding, blood partitioning, pharmacokinetics and tissue distribution of Withanolide A in rats: A neuroprotective steroidal lactone.

Preclinical Research & Development Withanolide A (WA), a steroidal lactone is a major bioactive constituent of Withania somnifera (L.) with remarkable neuropharmacological activity. In this study, we investigated the permeability, plasma protein binding (PPB), blood partitioning, intravenous (i.v.), and oral pharmacokinetics as well as i.v. tissue distribution (TD) of pure WA in a rat model. The PPB, RBCs partitioning, and permeability of WA were determined by Ultra Performance Liquid Chromatography (UPLC) method. However, the pharmacokinetics and TD of WA were evaluated by validated and sensitive liquid chromatography coupled mass spectrometry (LC-ESI-MS/MS) method. The PPB and permeability of WA were determined by equilibrium dialysis and parallel artificial membrane permeability assay method, respectively. The results demonstrated that WA has high PPB and passive permeability. Furthermore, WA was found to have fast equilibration between RBCs and plasma. Following i.v. (2 mg/kg) and per-oral (25 mg/kg) administration of WA, the max concentration (Cmax ) in plasma was found as 85.53 ± 6.54 and 48.04 ±5.78 ng/mL, respectively. The TD study results indicated that WA has a rapid and wide TD. The maximum concentration in various tissues was found in following order: Clung > Cliver > Ckidney ≈ Cspleen > Cheart > Cbrain . The preclinical in vitro, as well as pharmacokinetics and TD results, are anticipated to support the future preclinical and clinical application of WA.

Altered muscarinic receptor expression in the cerebral cortex of epileptic rats: restorative role of Withania somnifera.

Temporal lobe epilepsy involves a sequence of events that can lead to neurotransmitter signalling alterations. There are many herbal extracts considered to be alternative therapeutic methods to manage epilepsy. In this study, we investigated the effect of Withania somnifera (WS) root extract and withanolide A (WA) in the management of temporal lobe epilepsy. Confocal imaging of TOPRO-3-stained cortical sections showed severe damage in the epileptic brain. We also observed a reduced antioxidant potential and increased peroxide levels in the epileptic test group of rats. Oxidative stress resulted in the down-regulation of CREB, NF-κB, and TNF-α, and with up-regulation of the apoptotic factors caspases 8 and 3 and Bax in the epileptic group. Epileptic condition also resulted in increased muscarinic receptor binding and mRNA expression in the cerebral cortex. Withania somnifera and withanolide A significantly reversed the altered muscarinic receptor expression and reversed the oxidative stress and resultant derailment in cell signalling. Thus our studies suggest that Withania somnifera and withanolide A play important roles in central muscarinic receptor functional balance and activation of the antioxidant system in the cerebral cortex in temporal lobe epilepsy. These findings can be of immense therapeutic significance for managing epilepsy.

Accumulation of three important bioactive compounds in different plant parts of Withania somnifera and its determination by the LC-ESI-MS-MS (MRM) method.

A comprehensive experiment was conducted to study the accumulation pattern and determination of three important bioactive compounds namely withaferin-A (WA), 12-deoxywithastramonolide (WO) and withanolide-A (WD) and its determination by the liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) method in root, stem, fruits and leaves of Withania somnifera. A rapid and sensitive LC-ESI-MS-MS method was developed and validated for the determination of these three important bioactive compounds, having same molecular weight. The multiple reaction monitoring method was established by two transitions for each analyte and intense transition used for quantification. Separation of the three analytes was achieved within a run time of 5 min on an RP-18 column using a mobile phase consisting of acetonitrile and 0.1% acetic acid in water in an isocratic condition. The developed method was validated as per the ICH guidelines. The developed method was found to be suitable for identification and quantification of WA, WO and WD in different plant parts such as roots, stems, fruits and leaves of W. somnifera. The accumulation of WA was highest in leaves samples (8.84 ± 0.37 mg/g) and it was 2.23, 5.85 and 27.26 times higher than its concentration in fruits, stems and roots, respectively. WO and WD contents were highest (0.44 ± 0.016 and 0.72 ± 0.016 mg/g, respectively) in root.

Withania somnifera: a pre-clinical study on neuroregenerative therapy for stroke.

Stroke management exerts insurmountable societal and economic burden on the patient as well as their caregivers. In the year 2010 alone, the direct and indirect costs of stroke care amounted to 36.5 billion dollars (Go et al., 2014). Despite concentrated efforts to develop a safe, effective drug for stroke, we have not discovered one since the introduction of recombinant tissue plasminogen activator (rtPA)—the standalone FDA-approved therapy for stroke. While rtPA is highly effective, it needs to be given within 3–4.5 hours of the onset of stroke symptoms (Zivin, 2009). This is often complicated by the delay in the commencement of treatment due to preliminary inclusion parameters that are required to be ascertained before rtPA administration.

β-Amyloid1-42, HIV-1Ba-L (clade B) infection and drugs of abuse induced degeneration in human neuronal cells and protective effects of ashwagandha (Withania somnifera) and its constituent Withanolide A.

Alzheimer's disease (AD) is characterized by progressive dysfunction of memory and higher cognitive functions with abnormal accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles throughout cortical and limbic brain regions. Withania somnifera (WS) also known as ‘ashwagandha’ (ASH) is used widely in Ayurvedic medicine as a nerve tonic and memory enhancer. However, there is paucity of data on potential neuroprotective effects of ASH against β-Amyloid (1–42) (Aβ) induced neuropathogenesis. In the present study, we have tested the neuroprotective effects of Methanol: Chloroform (3:1) extract of ASH and its constituent Withanolide A (WA) against Aβ induced toxicity, HIV-1Ba-L (clade B) infection and the effects of drugs of abuse using a human neuronal SK-N-MC cell line. Aβ when tested individually, induced cytotoxic effects in SK-N-MC cells as shown by increased trypan blue stained cells. However, when ASH was added to Aβ treated cells the toxic effects were neutralized. This observation was supported by cellular localization of Aβ, MTT formazan exocytosis, and the levels of acetylcholinesterase activity, confirming the chemopreventive or protective effects of ASH against Aβ induced toxicity. Further, the levels of MAP2 were significantly increased in cells infected with HIV-1Ba-L (clade B) as well as in cells treated with Cocaine (COC) and Methamphetamine (METH) compared with control cells. In ASH treated cells the MAP2 levels were significantly less compared to controls. Similar results were observed in combination experiments. Also, WA, a purified constituent of ASH, showed same pattern using MTT assay as a parameter. These results suggests that neuroprotective properties of ASH observed in the present study may provide some explanation for the ethnopharmacological uses of ASH in traditional medicine for cognitive and other HIV associated neurodegenerative disorders and further ASH could be a potential novel drug to reduce the brain amyloid burden and/or improve the HIV-1 associated neurocognitive impairments

Impaired motor learning attributed to altered AMPA receptor function in the cerebellum of rats with temporal lobe epilepsy: Ameliorating effects of Withania somnifera and withanolide A

The aim of this study was to investigate the effect of Withania somnifera (WS) extract, withanolide A (WA), and carbamazepine (CBZ) on cerebellar AMPA receptor function in pilocarpine-induced temporal lobe epilepsy (TLE). In the present study, motor learning deficit was studied by rotarod test, grid walk test, and narrow beam test. Motor learning was significantly impaired in rats with epilepsy. The treatment with WS and WA significantly reversed the motor learning deficit in rats with epilepsy when compared with control rats. There was an increase in glutamate content and IP3 content observed in rats with epilepsy which was reversed in WS- and WA-treated rats with epilepsy. alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor dysfunction was analyzed using radiolabeled AMPA receptor binding assay, AMPA receptor mRNA expression, and immunohistochemistry using anti-AMPA receptor antibody. Our results suggest that there was a decrease in Bmax, mRNA expression, and AMPA receptor expression indicating AMPA receptor dysfunction, which is suggested to have contributed to the motor learning deficit observed in rats with epilepsy. Moreover, treatment with WS and WA resulted in physiological expression of AMPA receptors. There was also alteration in GAD and GLAST expression which supplemented the increase in extracellular glutamate. The treatment with WS and WA reversed the GAD and GLAST expression. These findings suggest that WS and WA regulate AMPA receptor function in the cerebellum of rats with TLE, which has therapeutic application in epilepsy.

Lack of the cytochrome P450 3A interaction of methanolic extract of Withania somnifera, Withaferin A, Withanolide A and Withanoside IV

Aims: Withania somnifera is widely employed as a rejuvenator and expected to promote physical health and increase longevity. The aim of the present research work was to evaluate Cytochrome P450 3A (CYP3A) interaction of Withania somnifera . Materials and Methods: In vitro CYP3A interaction of methanolic extract of Withania somnifera (WS) and its principal phytoconstituents: Withaferin-A (WA), Withanolide-A (WL-A) and Withanoside-IV (WS-IV) were investigated in rat and human liver microsomes. In vivo CYP3A interaction potential was investigated by administering methanolic extract of WS orally at a dose of 500 mg/kg in female Wistar rats. Sildenafil citrate was used to index the activity of CYP3A. Results: IC 50 values of methanolic extract of Withania somnifera, WA, WL-A, WS-IV were found to be 200 μg/ml, >20 μM, >64 μM and >64 μM for CYP3A both in rats and humans, respectively. When sildenafil citrate was orally co-administered with methanolic extract of WS and compared with orally administered sildenafil citrate alone, the area under plasma concentration time (AUC) curve and C max did not significantly differ as compared to the group which received rifampicin orally (positive control). Conclusions: Results suggested that methanolic extract of WS, WA, WL-A, WS-IV showed no in vitro CYP3A inhibition in rats and humans. Methanolic extract of WS did not significantly alter the pharmacokinetics of sildenafil citrate in rats; indicating its safety when co-administered with other drugs that are substrates of CYP3A. Thus the results indicate the lesser likelihood of drug herb interactions when concomitantly administered with CYP3A substrates.

Oxidative Stress Induced NMDA Receptor Alteration Leads to Spatial Memory Deficits in Temporal Lobe Epilepsy: Ameliorative Effects of Withania somnifera and Withanolide A

In the present study we investigate the effect of Withania somnifera (WS) root extract and Withanolide A (WA) in restoring spatial memory deficit by inhibiting oxidative stress induced alteration in glutamergic neurotransmission. We demonstrate significant cellular loss in hippocampus of epileptic rats, visualized through decreased TOPRO stained neurons. Impaired spatial memory was observed in epileptic rats after Radial arm maze test. Treatment with WS and WA has resulted in increased number of TOPRO stained neurons. Enhanced performance of epileptic rats treated with WS and WA was observed in Radial arm maze test. The antioxidant activity of WS and WA was studied using superoxide dismutase (SOD) and Catalase (CAT) assays in the hippocampus of experimental rats. The SOD activity and CAT activity decreased significantly in epileptic group, treatment with WS and WA significantly reversed the enzymatic activities to near control. Real time gene expression studies of SOD and GPx showed significant up-regulation in epileptic group compared to control. Treatment with WS and WA showed significant reversal to near control. Lipid peroxidation quantified using TBARS assay, significantly increased in epileptic rats. Treatment with WS and WA showed significant reversal to near control. NMDA receptor expression decreased in epileptic rats. The treatment with WS and WA resulted in physiological expression of NMDA receptors. This data suggests that oxidative stress effects membrane constitution resulting in decreased NMDA receptor density leading to impaired spatial memory. Treatment with WS and WA has ameliorated spatial memory deficits by enhancing antioxidant system and restoring altered NMDA receptor density.