A comparative pharmacokinetics study of Ashwagandha (Withania somnifera) Root Extract sustained-release capsules: an open-label, randomized, two treatment, two-sequence, two period, single-dose crossover clinical study

Background: In this open-label, randomized, balanced, two-treatment, two-sequence, two-period, crossover, single-dose oral comparative pharmacokinetics study, the pharmacokinetics, safety, and tolerability of test product ‘ashwagandha (Withania somnifera)’ root extract sustained release capsule 300 mg (Prolanza™), each containing 15 mg withanolides (administered dose: 2×15 mg) was compared with that of a reference product (organic KSM-66 ashwagandha extract [vegan] capsule, each containing 15 mg withanolides [administered dose: 2×15 mg]).Methods: Total 14 healthy men were randomized to receive either the test or the reference product as a single dose of 2 capsules in sequence, administered under fasting conditions. Plasma concentrations of total withanolides, withanolide A and 12-deoxywithastramonolide were measured using validated liquid chromatography–mass spectroscopy/mass spectroscopy.Results: The test product had higher relative absorption, better relative bioavailability, and longer elimination half-life indicating a sustained-release profile compared to reference. Specifically, the relative bioavailability of the test formulation was 12, 44, and 11 times higher for total withanolides, withanolide A and 12-deoxywithastramonolide, respectively. No adverse events were reported during the study.Conclusions: The sustained-release profile of the test product, compared to reference product, will provide more long-lasting therapeutic effects from a single daily dose (Retrospectively applied on Clinical Trials Registry - India [CTRI]. Application reference number: REF/2020/03/032408). The study reports the unique sustained release formulation of Withania somnifera (Ashwagandha) root extract. The pharmacokinetic study also reports for first time, the successful plasma estimation of withanolide A and 12-deoxywithastraamonolide, the major phytoactives of ashwagandha.

Tissue Culture Studies in Withania Somnifera (L.) Dunal

Withania somnifera is an important medicinal herb that has been widely used for the treatment of different clinical conditions. The overall medicinal properties of Withania somnifera make it a viable therapeutic agent for addressing anxiety, cancer, microbial infection, immunomodulation, and neurodegenerative disorders. Biochemical constituents of Withania somnifera like withanolide A, withanolide D, withaferin A and withaniamides play an important role in its pharmacological properties. Proteins like Withania somnifera glycoprotein and withania lectin like-protein possess potent therapeutic properties like antimicrobial, anti-snake venom poison and antimicrobial. In this review, we have tried to present different pharmacological properties associated with different extract preparations, phytochemical constituents and protein component of Withania somnifera. Future insights in this direction have also been highlighted.

Synthesis, In-Vitro and In-Silico Evaluation of Silver Nanoparticles with Root Extract of Withania somnifera for Antibacterial Activity via Binding of Penicillin-Binding Protein-4

Background: Metal Nanoparticles (NPs) have been widely used for various applications in biomedical sciences, including in drug delivery, and as therapeutic agents, but limited owing to their toxicity towards the healthy tissue. This warrants an alternative method, which can achieve the desired activity with much reduced or no toxicity. Being a biological product, Withania somnifera (W. somnifera) is environment friendly, besides being less toxic as compared to metal-based NPs. However, the exact mechanism of action of W. somnifera for its antibacterial activities has not been studied so far. Objective: To develop “silver nanoparticles with root extract of W. somnifera (AgNPs-REWS)” for antimicrobial and anticancer activities. Furthermore, the analysis of their mechanism of action will be studied. Methods: Using the in-silico approach, the molecular docking study was performed to evaluate the possible antibacterial mechanism of W. somnifera phytochemicals such as Anaferine, Somniferine, Stigmasterol, Withaferin A, Withanolide- A, G, M, and Withanone by the inhibition of Penicillin- Binding Protein 4 (PBP4). Next, we utilized a bottom-up approach for the green synthesis of AgNPs- REWS, performed an in-detail phytochemical analysis, confirmed the AgNPs-REWS by SEM, UVvisible spectroscopy, XRD, FT-IR, and HPLC. Eventually, we examined their antibacterial activity. Results: The result of molecular docking suggests that WS phytochemicals (Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone) possess the higher binding affinity toward the active site of PBP4 as compared to the Ampicillin (-6.39 kcal/mol) reference molecule. These phytochemicals predicted as potent inhibitors of PBP4. Next, as a proof-of-concept, AgNPs- REWS showed significant antibacterial effect as compared to crude, and control; against Xanthomonas and Ralstonia species. Conclusion: The in-silico and molecular docking analysis showed that active constituents of W. somnifera such as Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone possess inhibition potential for PBP4 and are responsible for the anti-bacterial property of W. somnifera extract. This study also establishes that AgNPs via the green synthesis with REWS showed enhanced antibacterial activity towards pathogenic bacteria.

Integrated System Pharmacology and In Silico Analysis Elucidating Neuropharmacological Actions of Withania somnifera in the Treatment of Alzheimer’s Disease

Background: Withania somnifera (WS), also referred to as Medhya Rasayana (nootropic or rejuvenating), has traditionally been prescribed for various neurological ailments, including dementia. Despite substantial evidence, pharmacological roles of WS, neither as nootropic nor as an antidementia agent, are well-understood at the cellular and molecular levels. Objectives: We aimed at elucidating the pharmacological action mechanisms of WS root constituents against Alzheimer’s Disease (AD) pathology. Methods: Various bioinformatics tools and resources, including DAVID, Cytoscape, NetworkAnalyst and KEGG pathway database were employed to analyze the interaction of WS root bioactive molecules with the protein targets of AD-associated cellular processes. We also used a molecular simulation approach to validate the interaction of compounds with selected protein targets. Results: Network analysis revealed that β-sitosterol, withaferin A, stigmasterol, withanolide A, and withanolide D are the major constituents of WS root that primarily target the cellular pathways such as PI3K/Akt signaling, neurotrophin signaling and toll-like receptor signaling and proteins such as Tropomyosin receptor Kinase B (TrkB), Glycogen Synthase Kinase-3β (GSK-3β), Toll-Like Receptor 2/4 (TLR2/4), and β-secretase (BACE-1). Also, the in silico analysis further validated the interaction patterns and binding affinity of the major WS compounds, particularly stigmasterol, withanolide A, withanolide D and β-sitosterol with TrkB, GSK-3β, TLR2/4, and BACE-1. Conclusion: The present findings demonstrate that stigmasterol, withanolide A, withanolide D and β-sitosterol are the major metabolites that are responsible for the neuropharmacological action of WS root against AD-associated pathobiology, and TrkB, GSK-3β, TLR2/4, and BACE-1 could be the potential druggable targets.

In Silico studies of Natural compounds that inhibit SARS-CoV-2 Nucleocapsid Nsp1/Nsp3 proteins mediated Viral Replication and Pathogenesis

Highly Transmissible and pathogenic coronavirus that emerged in late December of 2019 caused Severe acute respiratory syndrome (SARS-CoV-2), which challenged human health and public safety. Severity of the disease depends on the viral load and the type of mutation that occurred in the coronavirus. Nonstructural proteins like, Nsp1, Nsp3, Nsp12 and Nsp13 including other viral proteins plays important role during viral replication life cycle. Viral Replication initiated by hacking the host cellular mechanism either by synergy or by suppression using nucleocapsid proteins of the virus. Spike (S) protein of the SARS-CoV-2 uses angiotensin-converting enzyme II (ACE2) and TRMPSS as a cell entry. Once virus enters host cell, nucleocapsid proteins along with its genome is releases from endosomes into cytosol of the host cell. Ca2+/CaM (Calmodulin)/Calcineurin complex of the host cell plays important role during viral replication which is mediated by nucleocapsid proteins of the virus. Nsp1/Nsp3 nonstructural proteins triggers synergetic activity with CD147/CyPA/HSPG pathway and TRMP2/ADPr/Ca+2 mediated Ca2+/CaM (Calmodulin)/Calcineurin synthesis and free radicle generation in mitochondria leading to viral replication and severe chemokine activation pathways. Docking studies were carried out to inhibit Cyclophilin A and TRMP2 proteins as drug targets. Natural compounds like Withanolide A, Columbin, Cucurbitacin E, Boswellic acid along with Cyclosporines, Vitamin E and N-Acetyl cysteine (NAC) were selected as ligands to study docking studies. Withanolide A and Cyclosporines had shown good inhibition activity against Cyclophilin A, whereas Columbin, Boswellic acid, Cucurbitacin E, Vitamin E and N-Acetyl cysteine (NAC) had shown inhibitory activity against TRMP2. Thus, we suggest conducting further studies to conclude above pathways mechanism and inhibitory effect of natural compounds against the Nsp1/Nsp3 mediated pathways Invitro and In vivo.

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 (18.29 mg/g) and roots were observed highest WIA (19.63 mg/g) and WIB (1.36 mg/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.