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Withanolides are naturally occurring steroidal lactones found in certain species of the Withania genus, especially Withania somnifera (commonly known as Ashwagandha). These compounds have gained considerable attention due to their wide range of therapeutic properties and potential applications in modern medicine. To meet the rapidly growing demand for withanolides, innovative approaches such as in vitro culture techniques and synthetic biology offer promising solutions. In recent years, synthetic biology has enabled the production of engineered withanolides using heterologous systems, such as yeast and bacteria. Additionally, in vitro methods like cell suspension culture and hairy root culture have been employed to enhance withanolide production. Nevertheless, one of the primary obstacles to increasing the production of withanolides using these techniques has been the intricacy of the biosynthetic pathways for withanolides. The present article examines new developments in withanolide production through in vitro culture. A comprehensive summary of viable traditional methods for producing withanolide is also provided. The development of withanolide production in heterologous systems is examined and emphasized. The use of machine learning as a potent tool to model and improve the bioprocesses involved in the generation of withanolide is then discussed. In addition, the control and modification of the withanolide biosynthesis pathway by metabolic engineering mediated by CRISPR are discussed. [ABSTRACT FROM AUTHOR]

Withania somnifera, the plant named Indian ginseng, Ashwagandha, or winter cherry, has been used since ancient times to cure various health ailments. Withania somnifera is rich in constituents belonging to chemical classes like alkaloids, saponins, flavonoids, phenolic acids, and withanolides. Several chemotypes were identified based on their phytochemical composition and credited for their multiple bioactivities. Besides, exhibiting neuroprotective, immunomodulatory, adaptogenic, anti‐stress, bone health, plant has shown promising anti‐cancer properties. Several withanolides have been reported to play a crucial role in cancer; they target cancer cells by different mechanisms such as modulating the expression of tumor suppressor genes, apoptosis, telomerase expression, and regulating cell signaling pathway. Though, many treatments are available for cancer; however, to date, no assured reliable cure for cancer is made available. Additionally, synthetic drugs may lead to development of resistance in time; therefore, focus on new and natural drugs for cancer therapeutics may prove a longtime effective alternative. This current report is a comprehensive combined analysis upto 2023 with articles focused on bio‐activities of plant Withania somnifera from various sources, including national and international government sources. This review focuses on understanding of various mechanisms and pathways to inhibit uncontrolled cell growth by W. somnifera bioactives, as reported in literature. This review provides a recent updated status of the W. somnifera on pharmacological properties in general and anti‐cancer in particular and may provide a guiding resource for researchers associated with natural product‐based cancer research and healthcare management. [ABSTRACT FROM AUTHOR]

Withania somnifera, a valuable medicinal plant, synthesizes low molecular weight secondary metabolites known as withanolides with beneficial activities towards several diseases. The study of gene expression profiles and the development of EST datasets have developed an understanding of biosynthetic pathways for withanolides to some extent. However, molecular mechanisms regulating this biosynthetic pathway have yet to be studied in detail. In the present study, two transcription factors, WsMYBL1 and WsMYBL2, with contrasting differences in expression in root and leaf tissues, have been used to demonstrate their role in the biosynthesis of withanolides. Using the Virus-Induced Gene Silencing Approach (VIGS), the silencing of both transcription factors modulated plant phenotype and the expression of genes involved in the terpenoid pathway. This led to changes in total and specific withanolide content in the leaf and root tissues of silenced plants compared to the control. In conclusion, our study suggests the role of WsMYBL1 and WsMYBL2 as negative regulators of the withanolides biosynthesis pathway in Withania somnifera. [ABSTRACT FROM AUTHOR]

Abstract Background Several anti-retroviral drugs are available against Human immunodeficiency virus type-1, but have multiple adverse side effects. Hence, there is an incessant compulsion for effectual anti-retroviral agents with minimal or no intricacy. Traditionally, natural products have been the most successful source for the development of new medications. Withania somnifera, also known as Ashwagandha, is the utmost treasured medicinal plant used in Ayurveda, which holds the potential to give adaptogenic, immunomodulatory, and antiviral effects. However, its effect on HIV-1 replication at the cellular level has never been explored. Herein, we focused on the anti-HIV-1 activity and the probable mechanism of action of hydroalcoholic and aqueous extracts of Withania somnifera roots and its phytomolecules. Methods The cytotoxicity of the extracts was determined through MTT assay, while the in vitro anti-HIV-1 activity was assessed in TZM-bl cells against the HIV-1 strains of X4 and R5 subtypes. Results were confirmed in peripheral blood mononuclear cells, using the HIV-1 p24 antigen assay. Additionally, the mechanism of action was determined through the Time of Addition assay, which was further validated through the series of enzymatic assays, i.e. HIV-1 Integrase, Reverse transcriptase, and Protease assays. To explore the role of the identified active metabolites of Withania somnifera in antiretroviral activity, molecular docking analyses were performed against these key HIV-1 replication enzymes. Results The hydroalcoholic and aqueous extracts of Withania somnifera roots were found to be safer at the sub-cytotoxic concentrations and exhibited their ability to inhibit replication of two primary isolates of HIV-1 through cell-associated and cell-free assays, in dose-dependent kinetics. Several active phytomolecules found in Withania somnifera successfully established hydrogens bonds in the active binding pocket site residues responsible for the catalytic activity of HIV replication and therefore, signifying their role in the attenuation of HIV-1 infection as implied through the in silico molecular docking studies. Conclusions Our research identified both the hydroalcoholic and aqueous extracts of Withania somnifera roots as potent inhibitors of HIV-1 infection. The in silico analyses also indicated the key components of Withania somnifera with the highest binding affinity against the HIV-1 Integrase by 12-Deoxywithastramonolide and 27-Hydroxywithanone, HIV-1 Protease by Ashwagandhanolide and Withacoagin, and HIV-1 Reverse transcriptase by Ashwagandhanolide and Withanolide B, thereby showing possible mechanisms of HIV-1 extenuation. Overall, this study classified the role of Withania somnifera extracts and their active compounds as potential agents against HIV-1 infection.

Withanolides are naturally occurring steroidal lactones found in certain species of the Withania genus, especially Withania somnifera (commonly known as Ashwagandha). These compounds have gained considerable attention due to their wide range of therapeutic properties and potential applications in modern medicine. To meet the rapidly growing demand for withanolides, innovative approaches such as in vitro culture techniques and synthetic biology offer promising solutions. In recent years, synthetic biology has enabled the production of engineered withanolides using heterologous systems, such as yeast and bacteria. Additionally, in vitro methods like cell suspension culture and hairy root culture have been employed to enhance withanolide production. Nevertheless, one of the primary obstacles to increasing the production of withanolides using these techniques has been the intricacy of the biosynthetic pathways for withanolides. The present article examines new developments in withanolide production through in vitro culture. A comprehensive summary of viable traditional methods for producing withanolide is also provided. The development of withanolide production in heterologous systems is examined and emphasized. The use of machine learning as a potent tool to model and improve the bioprocesses involved in the generation of withanolide is then discussed. In addition, the control and modification of the withanolide biosynthesis pathway by metabolic engineering mediated by CRISPR are discussed.

A new withanolide, talasteroid (1), and a known steroid (2), along with eight meroterpenoids (3–10), were obtained from the rice culture of the marine-derived fungus Talaromyces stollii HBU-115. The structure of 1 including its absolute configuration was determined by extensive 1 D and 2 D NMR spectroscopy, and single-crystal X-ray diffraction analysis. Compound 1 represents the first withanolide featuring a 4-substituted 2,3-dimethyl-2-butenolide ring in the side chain. The isolated compounds were evaluated for their antimicrobial and antioxidant activities. [ABSTRACT FROM AUTHOR]

Introduction: Humanity is suffering from huge and severe difficulties, including changes in climate, soil degradation, scarcity of water and the security of food and medicines, among others. The aquaponics system acts as a closed loop consisting of aquaculture elements and hydroponics, which may contribute to addressing these problems. The aquaponics method is quickly expanding as the requirement to increase the production of sustainable herbal products, including medicinal compounds and foods, in freshwater systems and replenish phosphorous reserves shrinks. Methods: The current work is designed to increase the production of the antioxidants withaferin A and withanolide A in two varieties (Jawahar-20 and Poshita) of W. somnifera using the aquaponics technique. Total 100 seedlings (one month old) grown in soil initially were taken to be grown in aquaponics for a time period of 6 months. And 100 seedlings were placed in pots containing soil as control for study after six months. Results: It was observed that the higher content of withaferin A was analyzed in the root and stem samples of Jawahar-20 and Poshita from the six-month-old plant of W. somnifera. The maximum content of withanolide A was examined in the root samples of the six month-old plants of Poshita (1.879 mg/g) and Jawahar-20 (1.221 mg/g). While the 6 month old Poshita seedling grown in soil recorded less withaferin A (0.115 ± 0.009b) and withanolide A (0.138 ± 0.008d). Discussion: It is concluded that Poshita was found to be more promising for the enhanced production of withaferin A and withanolide A in the aquaponics system. Moreover, the root was observed as the best source for the production of withaferin A and withanolide A and the best age of the plant is 2 years for the production compounds in medicinal plants with futuristic perspective to hill agriculture integrated farming. compounds. Thus aquaponics can be an effective approach with enhanced yield of bioactive compounds in medicinal plants with futuristic perspective to hill agriculture and integrated farming. [ABSTRACT FROM AUTHOR]

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature peripheral T-lymphocytes caused by human T-cell leukemia virus type I (HTLV-I). There are an estimated 5–20 million HTLV-1-infected individuals worldwide. Conventional chemotherapeutic regimens used against other malignant lymphomas have been administered to patients with ATL, but the therapeutic outcomes of acute and lymphoma-type ATL remain extremely poor. In the course of our screening program for novel chemotherapeutic candidate compounds from plants against two human T-cell leukemia virus I-infected T-cell lines (MT-1 and MT-2), we screened 16 extracts obtained from different parts of 7 Solanaceae plants. We identified that the extracts of Physalis pruinosa and P. philadelphica showed potent anti-proliferative activity in MT-1 and MT-2 cells. In our previous study, we have isolated withanolides from extract of aerial parts of P. pruinosa and examined their structure–activity relationships. In addition, we are also investigating further structure–activity relationships about other withanolides from Solanaceae plants (Withania somnifera, Withania coagulans, Physalis angulate, Nicandra physalodes, Petunia hybrida, and Solanum cilistum). In this study, we attempted to isolate their active compounds against MT-1 and MT-2 from extracts of P. philadelphica. We identified 13 withanolides, including six newly isolated compounds [24R, 25S-4β, 16β, 20R-trihydroxy-1-oxowitha-2-en-5β, 6 β -epoxy-22,26-olide (1), 4β, 7β,20R-trihydroxy-1-oxowitha-2-en-5β, 6β -epoxy-22,26-olide (2), 17β,20 S-dihydroxywithanone (3), 2,3-dihydro-3β-methoxy-23β-hydroxywithaphysacarpin (4), 3-O-(4-rhamnosyl)glucosyl-physalolactone B (5), and 17R, 20R, 22S, 23S, 24R, 25R-4β, 5α, 6β, 20β, 22α -tetrahydroxy-16β, 23-diepoxy-1-oxowitha-2-en-26, 23-olide (6)], from the extract and examined the structure–activity relationships. The 50% effective concentration of withaphysacarpin (compound 7) [MT-1: 0.10 µM and MT-2: 0.04 µM] was comparable to that of etoposide [MT-1: 0.08 µM and MT-2: 0.07 µM]. Therefore, withanolides might be promising candidates for the treatment of ATL. [ABSTRACT FROM AUTHOR]

Background: Several anti-retroviral drugs are available against Human immunodeficiency virus type-1, but have multiple adverse side effects. Hence, there is an incessant compulsion for effectual anti-retroviral agents with minimal or no intricacy. Traditionally, natural products have been the most successful source for the development of new medications. Withania somnifera, also known as Ashwagandha, is the utmost treasured medicinal plant used in Ayurveda, which holds the potential to give adaptogenic, immunomodulatory, and antiviral effects. However, its effect on HIV-1 replication at the cellular level has never been explored. Herein, we focused on the anti-HIV-1 activity and the probable mechanism of action of hydroalcoholic and aqueous extracts of Withania somnifera roots and its phytomolecules. Methods: The cytotoxicity of the extracts was determined through MTT assay, while the in vitro anti-HIV-1 activity was assessed in TZM-bl cells against the HIV-1 strains of X4 and R5 subtypes. Results were confirmed in peripheral blood mononuclear cells, using the HIV-1 p24 antigen assay. Additionally, the mechanism of action was determined through the Time of Addition assay, which was further validated through the series of enzymatic assays, i.e. HIV-1 Integrase, Reverse transcriptase, and Protease assays. To explore the role of the identified active metabolites of Withania somnifera in antiretroviral activity, molecular docking analyses were performed against these key HIV-1 replication enzymes. Results: The hydroalcoholic and aqueous extracts of Withania somnifera roots were found to be safer at the sub-cytotoxic concentrations and exhibited their ability to inhibit replication of two primary isolates of HIV-1 through cell-associated and cell-free assays, in dose-dependent kinetics. Several active phytomolecules found in Withania somnifera successfully established hydrogens bonds in the active binding pocket site residues responsible for the catalytic activity of HIV replication and therefore, signifying their role in the attenuation of HIV-1 infection as implied through the in silico molecular docking studies. Conclusions: Our research identified both the hydroalcoholic and aqueous extracts of Withania somnifera roots as potent inhibitors of HIV-1 infection. The in silico analyses also indicated the key components of Withania somnifera with the highest binding affinity against the HIV-1 Integrase by 12-Deoxywithastramonolide and 27-Hydroxywithanone, HIV-1 Protease by Ashwagandhanolide and Withacoagin, and HIV-1 Reverse transcriptase by Ashwagandhanolide and Withanolide B, thereby showing possible mechanisms of HIV-1 extenuation. Overall, this study classified the role of Withania somnifera extracts and their active compounds as potential agents against HIV-1 infection. [ABSTRACT FROM AUTHOR]

Due to the increasing dependency of the global population on herbal-based medicines, enhanced production of secondary metabolites has become utmost necessary. Several scientists have revealed the role of NaCl as an abiotic elicitor for in-planta enhancement of phytochemical compounds. Thus, we have micropropagated Withania somnifera from callus tissues in varying concentrations of BAP hormone and found that MS media supplemented with 2 mg L−1 is best suited for maximum yield. Further, the microshoots were treated with varying concentrations (50–250 mM) of NaCl to assess morphological, physico-biochemical, and molecular characteristics. Higher concentrations of NaCl significantly hampered the morphological and photosynthetic attributes of microshoots. A significant enhancement in the proline, phenol, and sugar contents was recorded and the highest accumulation was observed by 139.32%, 53.2%, and 134.94% respectively with 250 mM treated shoots. Free radical scavenging activity and malondialdehydelevel were also up-regulated by 1.44 and 1.75-fold respectively. Similarly, anti-oxidants enzymatic activities were increased linearly. Contrary to this, the highest production of flavonoid (49%) and withaferin-A (97.86%) was limited only upto 150 mM NaCl. Higher accumulation of withanolide was further attested by the Real-time PCR analysis of related genes which revealed their maximum expression by 150 mM salt treatment. Conversely, the expression of photosynthetic-related genes significantly declined after increasing doses of NaCl. These outcomes depicted that a moderate dose of NaCl can act as an elicitor for enhanced secondary metabolites of W. somnifera without compromising its growth significantly. Key Message: Micropropagated shoots of Withania somnifera in 2 mg L−1 BAP, responded differentially in different NaCl concentrations with maximum secondary metabolism (withaferin A) at 150 mM NaCl. [ABSTRACT FROM AUTHOR]

IntroductionHumanity is suffering from huge and severe difficulties, including changes in climate, soil degradation, scarcity of water and the security of food and medicines, among others. The aquaponics system acts as a closed loop consisting of aquaculture elements and hydroponics, which may contribute to addressing these problems. The aquaponics method is quickly expanding as the requirement to increase the production of sustainable herbal products, including medicinal compounds and foods, in freshwater systems and replenish phosphorous reserves shrinks.MethodsThe current work is designed to increase the production of the antioxidants withaferin A and withanolide A in two varieties (Jawahar-20 and Poshita) of W. somnifera using the aquaponics technique. Total 100 seedlings (one month old) grown in soil initially were taken to be grown in aquaponics for a time period of 6 months.And 100 seedlings were placed in pots containing soil as control for study after six months.ResultsIt was observed that the higher content of withaferin A was analyzed in the root and stem samples of Jawahar-20 and Poshita from the six-month-old plant of W. somnifera. The maximum content of withanolide A was examined in the root samples of the six month-old plants of Poshita (1.879 mg/g) and Jawahar-20 (1.221 mg/g). While the 6 month old Poshita seedling grown in soil recorded less withaferin A (0.115 ± 0.009b) and withanolide A (0.138 ± 0.008d).DiscussionIt is concluded that Poshita was found to be more promising for the enhanced production of withaferin A and withanolide A in the aquaponics system. Moreover, the root was observed as the best source for the production of withaferin A and withanolide A and the best age of the plant is 2 years for the production compounds in medicinal plants with futuristic perspective to hill agriculture integrated farming. compounds. Thus aquaponics can be an effective approach with enhanced yield of bioactive compounds in medicinal plants with futuristic perspective to hill agriculture and integrated farming.

Physalis plants are commonly used traditional medicinal herbs, and most of their extracts containing withanolides show anticancer effects. Physapruin A (PHA), a withanolide isolated from P. peruviana, shows antiproliferative effects on breast cancer cells involving oxidative stress, apoptosis, and autophagy. However, the other oxidative stress-associated response, such as endoplasmic reticulum (ER) stress, and its participation in regulating apoptosis in PHA-treated breast cancer cells remain unclear. This study aims to explore the function of oxidative stress and ER stress in modulating the proliferation and apoptosis of breast cancer cells treated with PHA. PHA induced a more significant ER expansion and aggresome formation of breast cancer cells (MCF7 and MDA-MB-231). The mRNA and protein levels of ER stress-responsive genes (IRE1α and BIP) were upregulated by PHA in breast cancer cells. The co-treatment of PHA with the ER stress-inducer (thapsigargin, TG), i.e., TG/PHA, demonstrated synergistic antiproliferation, reactive oxygen species generation, subG1 accumulation, and apoptosis (annexin V and caspases 3/8 activation) as examined by ATP assay, flow cytometry, and western blotting. These ER stress responses, their associated antiproliferation, and apoptosis changes were partly alleviated by the N-acetylcysteine, an oxidative stress inhibitor. Taken together, PHA exhibits ER stress-inducing function to promote antiproliferation and apoptosis of breast cancer cells involving oxidative stress. [ABSTRACT FROM AUTHOR]

Withania somnifera belongs to the family of Solanaceae. It is widely used by the locals, especially in India, as a medicinal plant. W. somnifera is rich in alkaloid and steroidal lactone that account for various pharmacological activities. The present study aimed to review all the evidence on the ethnobotanical perspective of W. somnifera in some countries. This review also analyses the bioactive compounds that account for the pharmacological activities. The online medical literature databases such as ScienceDirect, PubMed, and Google Scholar were used to search articles up to March 2022. W. somnifera is widely used in Asian and African countries like India and South Africa, Pakistan, Egypt, Jordan, and Lesotho. In India, W. somnifera is prepared by boiling and crushing the leaves and roots to make a tonic poultice, juice, and paste to treat bacterial infections and bruises. Numerous in vivo and in vitro studies have demonstrated that W. somnifera exerts pharmacological effects such as anti-Parkinson’s, anti-Alzheimer’s, cardioprotective, antidiabetic, antiarthritic, and antibacterial properties. Withaferin A and withanolide are the major bioactive compounds contributing to the pharmacological effects. W. somnifera is a valuable plant that has been used in traditional medicine systems for a long time and is supported by its wide range of pharmacological activities. The extensive medicinal uses of W. somnifera are a sign of its great potential.

Introduction and purpose: Ashwagandha (fam. Solanaceae) also known as Withania somnifera, is an herb commonly used in traditional Ayurvedic medicine. It is sometimes called Indian ginseng or winter cherry and it is used for its extensive health benefits for millennia as a Rasayana. Ashwagadha supplementation is playing an increasing role not only as an adaptogen, but is beneficial in the context of health in individuals struggling with obesity. This is a growing field of research and the aim of this brief review is to provide an update on the positive effects of Ashwagandha supplementation on the health of those dealing with obesity. Description of the state of knowledge: Ashwagandha has a wide range of positive effects, most common are sedative, anti-stress, immunomodulatory and anti-cancer effects. The literature also draws attention to other results such as binding insulin sensitivity, increasing endurance, effects on adipogenesis and lipogenesis. Ashwagandha lowers blood cortisol levels, inhibits fat accumulation and the expression of genes involved into excessive fat tissue accumulation. Summary: Regular intake of Ashwagandha is associated with health benefits in people struggling with obesity. As an adaptogen with adipogenic potential, Ashwagandha has great potential to participate in future adjunctive obesity therapy and the prevention of other similar and obesity-related diseases, but further research is needed in this area.

A new compound, 5β,6β-epoxy-20α,24β,25α-trihydroxy-1,3-dioxowitha-2-enolide ( 1 ), and two known compounds, gracillin ( 2 ) and 5α-hydroxylaxogenin ( 3 ), have been isolated from the aerial part of Saponaria officinalis . High-resolution mass spectrometry and 1D and 2D NMR spectroscopy analysis data were used to establish the structure of the new compound. The antimicrobial activity of the new compound against some opportunistic strains of microorganisms and its cytotoxicity against two cancer cell lines and two healthy cell lines in vitro were studied. 5β,6β-Epoxy-20α,24β,25α-trihydroxy-1,3-dioxowitha-2-enolide was found to be most active against three bacterial strains: Staphylococcus aureus , Bacillus subtilis , and Pseudomonas aeruginosa , with inhibition zone diameters of 21.04 ± 0.10 mm, 22.08 ± 0.12 mm, and 18.12 ± 0.13 mm, respectively. Compound 1 also showed significant cytotoxicity against HEp-2 and HeLa cell lines (IC 50 = 23.2 μM and 54.9 μM, respectively).

Biosynthetic gene clusters (BGCs) are sets of often heterologous genes that are genetically and functionally linked. Among eukaryotes, BGCs are most common in plants and fungi and ensure the coexpression of the different enzymes coordinating the biosynthesis of specialized metabolites. Here, we report the identification of a withanolide BGC in Physalis grisea (ground-cherry), a member of the nightshade family ( Solanaceae ). A combination of transcriptomic, epigenomic, and metabolic analyses revealed that, following a duplication event, this BGC evolved two tissue-specifically expressed subclusters, containing several pairs of paralogs that contribute to related but distinct biochemical processes; this subfunctionalization is tightly associated with epigenetic features and the local chromatin environment. The two subclusters appear strictly isolated from each other at the structural chromatin level, each forming a highly self-interacting chromatin domain with tissue-dependent levels of condensation. This correlates with gene expression in either above- or below-ground tissue, thus spatially separating the production of different withanolide compounds. By comparative phylogenomics, we show that the withanolide BGC most likely evolved before the diversification of the Solanaceae family and underwent lineage-specific diversifications and losses. The tissue-specific subfunctionalization is common to species of the Physalideae tribe but distinct from other, independent duplication events outside of this clade. In sum, our study reports on an instance of an epigenetically modulated subfunctionalization within a BGC and sheds light on the biosynthesis of withanolides, a highly diverse group of steroidal triterpenoids important in plant defense and amenable to pharmaceutical applications due to their anti-inflammatory, antibiotic, and anticancer properties., Competing Interests: Competing interests statement:The authors declare no competing interest.

Withania somnifera (L.) Dunal (WS), also known as Ashwagandha, is an Indian herb classified as Rasayana in Ayurveda owing to its rejuvenating and health-promoting activities. A sensitive and robust high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of withanoside IV (WS4), withanoside V (WS5), withaferin A (WFA), and kaempferol-based glucoside (KRG) in the roots and aerial parts of WS. The present rapid HPTLC method reports the separation and simultaneous quantification of three diverse classes of WS as withanolide (WFA), withanosides (WS4 and WS5) with an analytical marker flavonoid glycoside (KRG) for effective detection, of the aerial parts separating them from roots. In normal-phase TLC plates, these markers showed good resolution in ethyl acetate‒chloroform‒methanol‒water (8:3:4.4:1.8, V/V) solvent system. Densitometric analysis was performed for KRG (at 254 nm), WFA, WS4, and WS5 (at 540 nm after derivatization) with characterization by high-performance thin-layer chromatography‒tandem mass spectrometry (HPTLC‒MS/MS). The method was found linear (R2 > 0.99) for KRG and WFA (200‒1000 ng/band) and WS4‒WS5 (400‒2000 ng/band) with excellent recoveries (80‒100%) for all compounds. In this HPTLC based assessment, three compounds of WS could be rapidly detected for quality control distinguishing from other Withania and plant species reported as adulterants. Additionally, this method separated pigment-based compounds at 366 nm in chemical fingerprinting in WS aerial parts samples. Therefore, the presence of KRG as an analytical marker with these pigment zones can identify aerial parts, leaves, stems, fruits with calyx, and fruits in the HPTLC analysis, separating them from root samples. Hence, this HPLTC method was found robust and economical for the estimation and surety of the quality of WS samples in quantitative and qualitative analysis. [ABSTRACT FROM AUTHOR]

Background: The present study was designed with the aim of minimizing the incorporation of the drugs in a polyherbal antidiabetic formulation so the load of the drugs to the patient can be avoided and also helps in standardization. Materials and Methods: Molecular docking-based screening of potential herbal leads of M. charantia, (Drug A), G. sylvestre, (Drug B) and W. somnifera, (Drug C) were performed with intent to prioritize the ratio of herbal extracts. Lyophilised hydro-alcohol (50%) extracts of above three drugs were combined in different ratios as HA: Hydroalcoholic extracts combination A, HB: Hydroalcoholic extracts combination B and HC: Hydroalcoholic extracts combination C. The study of 21 days, carried out on alloxan-induced diabetic Wistar rats to compare its anti-diabetic effect with the marketed herbal formulation (MHF). The biochemical parameters studied were serum glucose level, lipid profile and liver glycogen content along with the body weight measurement. Results: The proposed Hydroalcoholic extracts combination B (HB, 1000 mg/kg per oral) possess antihyperglycemic effect with improvement in the abnormal lipid profile as observed in the diabetic experimental animals. Moreover, the formulation also reported check on the loss of body weight when compared with untreated diabetic rats. The observations were found to be at par with standard drug, metformin (500 mg/kg). Conclusion: Our findings suggest the proposed formulation have antihyperglycemic and anti-hyperlipidimic potentials. Furthermore, its effect on chronic diabetes and its complications is needed to be explored. Based on the docking results it has been clearly indicated that the antidiabetic effect of the used herbal extract is because of the presence of Withanolide-A and Charantin. [ABSTRACT FROM AUTHOR]

Main conclusion: Overexpression of a novel geranylgeranyl pyrophosphate synthase gene (WsGGPPS) in planta resulted in increased levels of gibberellic acid and decrease in withanolide content. Withania somnifera (L.) Dunal, the herb from family Solanaceae is one of the most treasured medicinal plant used in traditional medicinal systems owing to its unique stockpile of pharmaceutically active secondary metabolites. Phytochemical and pharmacological studies in this plant were well established, but the genes affecting the regulation of biosynthesis of major metabolites were not well elucidated. In this study cloning and functional characterization of a key enzyme in terpenoid biosynthetic pathway viz. geranylgeranyl pyrophosphate synthase (EC 2.5.1.29) gene from Withania somnifera was performed. The full length WsGGPPS gene contained 1,104 base pairs that encode a polypeptide of 365 amino acids. The quantitative expression analysis suggested that WsGGPPS transcripts were expressed maximally in flower tissues followed by berry tissues. The expression levels of WsGGPPS were found to be regulated by methyl jasmonate (MeJA) and salicylic acid (SA). Amino acid sequence alignment and phylogenetic studies suggested that WsGGPPS had close similarities with GGPPS of Solanum tuberosum and Solanum pennellii. The structural analysis provided basic information about three dimensional features and physicochemical parameters of WsGGPPS protein. Overexpression of WsGGPPS in planta for its functional characterization suggested that the WsGGPPS was involved in gibberellic acid biosynthesis. [ABSTRACT FROM AUTHOR]

Ashwagandha (Withania somnifera L. Dunal) is a medicinally important plant with withanolides as its major bioactive compounds, abundant in the roots and leaves. We examined the influence of plant growth regulators (PGRs) on direct organogenesis, adventitious root development, withanolide biosynthetic pathway gene expression, withanolide contents, and metabolites during vegetative and reproductive growth phases under in vitro and ex vitro conditions. The highest shooting responses were observed with 6-benzylaminopurine (BAP) (2.0 mg L–1) + Kinetin (KIN) (1.5 mg L–1) supplementation. Furthermore, BAP (2.0 mg L–1) + KIN (1.5 mg L–1) + gibberellic acid (GA3) (0.5 mg L–1) exhibited better elongation responses with in vitro flowering. Half-strength MS medium with indole-3-butyric acid (IBA) (1.5 mg L–1) exhibited the highest rooting responses and IBA (1.0 mg L–1) with highest fruits, and overall biomass. Higher contents of withaferin A (WFA) [∼8.2 mg g–1 dry weight (DW)] were detected in the reproductive phase, whereas substantially lower WFA contents (∼1.10 mg g–1 DW) were detected in the vegetative phase. Cycloartenol synthase (CAS) (P = 0.0025), sterol methyltransferase (SMT) (P = 0.0059), and 1-deoxy-D-xylulose-5-phosphate reductase (DXR) (P = 0.0375) genes resulted in a significant fold change in expression during the reproductive phase. The liquid chromatography-mass spectrometry (LC-MS) analysis revealed metabolites that were common (177) and distinct in reproductive (218) and vegetative (167) phases. Adventitious roots cultured using varying concentrations of indole-3-acetic acid (IAA) (0.5 mg L–1) + IBA (1.0 mg L–1) + GA3 (0.2 mg L–1) exhibited the highest biomass, and IAA (0.5 mg L–1) + IBA (1.0 mg L–1) exhibited the highest withanolides content. Overall, our findings demonstrate the peculiarity of withanolide biosynthesis during distinct growth phases, which is relevant for the large-scale production of withanolides. [ABSTRACT FROM AUTHOR]

Lysine specific demethylase 1 (LSD1), which is overexpressed in several human cancers and acts as a demethylase of histone 3, lysine 4 and lysine 9, has become an attractive therapeutic target for cancer therapy. Based on our previous systematic studies, withanolides are important secondary metabolites mostly from the Solanaceae family of plants, which are crucial agents for cancer treatment. Here, withanolides were characterized as LSD1 inhibitors, especially withaferin A, with an IC 50 value of 3.04 μM. In vitro bioactivity assays and virtual molecular docking indicated that withaferin A could inhibit MDA-MB-231 cell migration by inhibiting intracellular LSD1 activity. These findings provide a new withanolide-based natural molecular skeleton for LSD1 inhibitors with potential antitumor activity. [Display omitted] • Lysine specific demethylase 1 (LSD1) is a target for cancer therapy. • Natural withanolides are first characterized as LSD1 inhibitors. • Withaferin A can inhibit LSD1 activity with IC 50 value of 3.04 μM. • Withaferin A also can inactive LSD1 activity in MDA-MB-231 cells. • Withaferin A may inhibit breast cancer cell proliferation and migration. [ABSTRACT FROM AUTHOR]

The transcription factor MYB is expressed predominantly in hematopoietic progenitor cells, where it plays an essential role in the development of most lineages of the hematopoietic system. In the myeloid lineage, MYB is known to cooperate with members of the CCAAT box/enhancer binding protein (C/EBP) family of transcription factors. MYB and C/EBPs interact with the co-activator p300 or its paralog CREB-binding protein (CBP), to form a transcriptional module involved in myeloid-specific gene expression. Recent work has demonstrated that MYB is involved in the development of human leukemia, especially in acute T-cell leukemia (T-ALL) and acute myeloid leukemia (AML). Chemical entities that inhibit the transcriptional activity of the MYB-C/EBPβ-p300 transcription module may therefore be of use as potential anti-tumour drugs. In searching for small molecule inhibitors, studies from our group over the last 10 years have identified natural products belonging to different structural classes, including various sesquiterpene lactones, a steroid lactone, quinone methide triterpenes and naphthoquinones that interfere with the activity of this transcriptional module in different ways. This review gives a comprehensive overview on the various classes of inhibitors and the inhibitory mechanisms by which they affect the MYB-C/EBPβ-p300 transcriptional module as a potential anti-tumor target. We also focus on the current knowledge on structure-activity relationships underlying these biological effects and on the potential of these compounds for further development. [ABSTRACT FROM AUTHOR]

Nephrotic syndrome, a multifaceted medical condition characterized by significant proteinuria, has recently prompted a reorientation of research efforts toward B-cell-mediated mechanisms. This shift underscores the pivotal role played by B-cells in its pathogenesis. The aim of this study was to explore potential therapeutic pathways, with specific attention given to compounds found in Physalis angulata , including withanolides, such as physalins, which constitute one of the five distinct withanolide subgroups identified in Physalis angulata . Furthermore, the study assessed the monoclonal antibody belimumab, designed to target B-cell activating factor (BAFF) and its associated receptors (TACI, BCMA, and BAFF-R). Various research techniques were employed, encompassing data mining, bioactivity analysis, Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiling, molecular modeling, and docking studies. Withanolide was demonstrated as a potential inhibitor for the protein BAFF, showing a binding energy of -7.1 kcal/mol. Physalin F emerged as the leading candidate inhibitor for the protein TACI, with a binding energy of -8.3 kcal/mol. Similarly, withanolide was identified as the top inhibitor candidate for the protein BCMA, exhibiting a binding energy of -7.0 kcal/mol. The most favorable interaction with BAFF-R was physalin F, which displayed a binding energy of -8.0 kcal/mol. Moreover, molecular dynamic simulation suggested that physalin F was able to maintain protein stability, hence being a good inhibitor candidate for BAFF-R and TACI proteins. The results of this investigation demonstrated substantial promise, indicating that these withanolides and withaphysalin A compounds derived from Physalis angulata offer alternative avenues for B-cell targeting. Consequently, this study presents opportunities for pioneering treatments in the management of nephrotic syndrome., Competing Interests: All the authors declare that there are no conflicts of interest., (© 2024 The Author(s).)

[Display omitted] • 75 withanolides and structures were identified in literature and databases. • QSAR models predicted physicochemical, pharmacokinetic and toxicity properties. • In silico prediction correlated well with published data. Ashwagandha is a medicinal plant used in traditional Asian medicines as an adaptogen to promote both physical and mental health. Withanolides are the major bioactive phytochemicals in Ashwagandha; they are a group of naturally occurring C 28 -steroidal lactones with an ergostane-based skeleton. Despite the broad use of Ashwagandha, data on the pharmacokinetic and toxicological properties of withanolides remain scarce. Here, 75 withanolides in Ashwagandha were identified in journal publications, databases, and monographs. In silico quantitative structure-activity relationship (QSAR) models were used to evaluate the physicochemical and pharmacokinetic properties as well as the acute toxicity of withanolides. Withanolides had high molecular weight and pKa, low aqueous solubility, and high lipophilicity. QSAR models also predicted high effective human jejunal permeability and plasma protein binding, tissue partitioning, extensive metabolism, hepatic uptake, and renal excretion for certain withanolides. In addition, two thirds of the withanolides evaluated had predicted median lethal dose (LD 50) under 100 mg/kg after oral administration in rats. These in silico results could be used to guide further testing and confirmed by in vitro and in vivo studies. [ABSTRACT FROM AUTHOR]

Withania coagulans (also known as Indian ginseng) is a medicinal plant traditionally used for purposes such as purification of blood, eradication of muscle wasting, prevention of failure to thrive in children, management of insomnia and exhaustion, and treatment of liver failure and diabetes mellitus. Despite increasing interest in these applications, a holistic overview of its medicinal value is lacking, and the underlying pharmacological basis remains to be examined. This study dissected extensive phytochemical and pharmacological information collected from international databases as well as local literature. Keywords related to medicinal uses, chemical compositions, and biological activities of W. coagulans were used for the bibliography search. Data were collected primarily from the last decade and used to build a systematic framework of its therapeutic potentials. To highlight the therapeutic significance of W. coagulans , an evidence-based framework of biological activities is built to delineate its pharmacological properties, which are cross-checked against its medicinal uses and phytochemical constituents. In particular, antioxidant, anti-inflammatory, immunomodulatory, antimicrobial, anticancer, antidiabetic, antihyperlipidemic, neuroprotective, cardioprotective, nephroprotective, hepatoprotective, reproductive system regulatory, and anthelmintic activities are outlined with emphasis on their therapeutic relevance to the plant. Several bioactive compounds, including withanolides (steroidal lactones), are identified as likely contributors to these activities, thereby supporting its medicinal value. The bioactivity framework built for W. coagulans , along with the critical interpretations of its phytochemical and pharmacological information in this study, provides a solid basis for its medicinal applications and offers insight into next phase of experimental and therapeutic discoveries. [Display omitted] • Withania coagulans has been traditionally used for a wide range of health benefits. • Alcoholic extracts of Withania fruits have shown various pharmacological activities. • Information from both international and local sources are dissected and integrated. • A holistic overview of pharmacological data is provided for critical interpretation. • A bioactivity framework is built to highlight ethnomedicinal and therapeutic value. [ABSTRACT FROM AUTHOR]

[Display omitted] Withanolides represent an important category of natural products with a steroidal lactone core. Many of them contain an α,β-unsaturated carbonyl moiety with a high reactivity toward sulfhydryl groups, including protein cysteine thiols. Different withanolides endowed with marked antitumor and anti-inflammatory have been shown to form stable covalent complexes with exposed cysteines present in the active site of oncogenic kinases (BTK, IKKβ, Zap70), metabolism enzymes (Prdx-1/6, Pin1, PHGDH), transcription factors (Nrf2, NFκB, C/EBPβ) and other structural and signaling molecules (GFAP, β-tubulin, p97, Hsp90, vimentin, Mpro, IPO5, NEMO, ...). The present review analyzed the covalent complexes formed through Michael addition alkylation reactions between six major withanolides (withaferin A, physalin A, withangulatin A, 4β-hydroxywithanolide E, withanone and tubocapsanolide A) and key cysteine residues of about 20 proteins and the resulting biological effects. The covalent conjugation of the α,β-unsaturated carbonyl system of withanolides with reactive protein thiols can occur with a large set of soluble and membrane proteins. It points to a general mechanism, well described with the leading natural product withaferin A, but likely valid for most withanolides harboring a reactive (electrophilic) enone moiety susceptible to react covalently with cysteinyl residues of proteins. The multiplicity of reactive proteins should be taken into account when studying the mechanism of action of new withanolides. Proteomic and network analyses shall be implemented to capture and compare the cysteine covalent-binding map for the major withanolides, so as to identify the protein targets at the origin of their activity and/or unwanted effects. Screening of the cysteinome will help understanding the mechanism of action and designing cysteine-reactive electrophilic drug candidates. [ABSTRACT FROM AUTHOR]

• PCF-EtOH ameliorates pulmonary fibrosis by inhibiting inflammation and ECM deposition. • PCF-EtOH inhibits the Wnt/β-catenin signaling pathway. • Withanolides and flavonoids are the predominant active constituents. • Physapubescin, a withanolide in PCF, shows potent effect against pulmonary fibrosis Pulmonary fibrosis is a chronic and advancing interstitial lung disease, and there is an urgent need for novel agents for its therapy. Physalis Calyx seu Fructus (PCF) has been utilized in traditional Chinese medicine to treat respiratory disorders with a long history, however, the therapeutic effect and mechanism of PCF against pulmonary fibrosis are still unclear. To assess therapeutic efficacy and underlying mechanism of 75 % ethanol extract of PCF (PCF-EtOH) against pulmonary fibrosis, as well as to discover active constituents in PCF. A bleomycin-stimulated mice model was established to assess potential therapy of PCF-EtOH against pulmonary fibrosis in vivo. A lipopolysaccharide-induced inflammatory model in RAW 264.7 cells and a transforming growth factor β1-induced fibrosis model in MRC-5 cells were established to assess potential therapy and mechanisms of purified constituents in PCF-EtOH. UPLC-MS/MS analysis was adopted to ascertain the constituents of PCF-EtOH. Network pharmacology was employed to forecast targets of PCF against pulmonary fibrosis. PCF-EtOH ameliorated bleomycin-induced pulmonary fibrosis through repressing inflammatory response and extracellular matrix deposition. Meanwhile, PCF-EtOH inhibited Wnt/β-catenin pathway through decreasing β-catenin nuclear accumulation and promoting phosphorylation. Furthermore, withanolides and flavonoids were presumed to be main active compounds of PCF against pulmonary fibrosis based on the network pharmacology. Importantly, we found an extensive presence of withanolides in PCF-EtOH. Physapubescin, a typical withanolide in PCF-EtOH, inhibited the inflammatory response, extracellular matrix deposition, and Wnt/β-catenin pathway. Notably, physapubescin demonstrated a more potent antifibrotic effect than pirfenidone, a clinically approved antifibrotic drug, in the tested model. Withanolides and flavonoids are responsible for the inhibitory effect of PCF-EtOH against pulmonary fibrosis. Withanolides may represent a class of promising therapeutic agents against pulmonary fibrosis, and an in-depth exploration is warranted to validate this proposition. [Display omitted] [ABSTRACT FROM AUTHOR]

Covid-19 is a disease caused by virus named SARC CoV type II of Corona virus family and is announced as Pandemic disease by WHO in the month of March 2020. Virus is spreading at exponential rate and causing more than 1.3 lack death globally today. Covid-19 has been announced as one of the biggest threat to the mankind in last 100 years. Antiviral drugs and the vaccines are under clinical phases, to combat this outbreak, it is essential to maintained good health condition with boosted immune activity. Ashwagandha has been a medicinal plant under Ayurvedic studies for various therapeutic uses since ancient times of India. It has been regarded as the most important plant as every part of the plant can serve as a remedial/curative alternative. Despite the versatility of the plant, scanty research has been done to explore its potential in creating varied healthcare and value-added allied products used as immune system booster in situation similar to Covid 19 outbreak. There is a huge lacuna in literature dedicated to documenting the prospective applications of Ashwagandha healthcare products. This work is an attempt to compile current literature with an eye on the possibility of creating healthcare and value addition products based on this versatile medicinal plant. The paper provides a historical perspective on the evolution of and investigations carried out to explore the applications of Ashwagandha as a medicinal plant. It the wake of a sudden interest in herbal plants, this work will act as a primer for the herbal-based solutions in the areas of cancer treatment, neural disorders, immune boosters especial for Covid 19 cause of viral outbreak, stress-relief, and rejuvenation. It also finds applications as cardio-respiratory endurance enhancer, anti-ageing, antioxidant, hypoglycemic, hypocholesterolemic and as an effective adaptogen. To reach out to a wider audience the paper concludes with open research issues, challenges in using Ashwagandha as a medicinal plant in the current scenario. [ABSTRACT FROM AUTHOR]

A phytochemical investigation of Physalis minima led to the isolation of six withanolides, including withanolide E (1), withaperuvin C (2), 4β-hydroxywithanolide E (3), 28-hydroxywithaperuvin C (4), physaperuvin G (5), and 4-deoxywithaperuvin (6). Their chemical structures were elucidated by 1 D-NMR and 2 D-NMR data, as well as comparison with the data reported in the literature. All isolated compounds were evaluated for their cytotoxic activity against HepG2, SK-LU-1, and MCF7 cancer cell lines. As the obtained results, compounds 1 and 3 displayed the strongest cytotoxicity against HepG2, SK-LU-1, and MCF7 cell lines with IC50 value ranging from 0.051 ± 0.004 to 0.86 ± 0.09 μg/mL. [ABSTRACT FROM AUTHOR]

In China, the fruits of Physalis alkekengi L. var. franchetii, which are conventionally utilized as edible berry, have attracted wide attention due to its significant biological activities. In the present study, phytochemical studies on the fruits of Physalis plants afforded six compounds, including two new withanolides (1–2) and four known agnologues (3–6). The inhibitory effects of these compounds on the formation of nitric oxide (NO) stimulated by lipopolysaccharide (LPS) in RAW264.7 macrophages were evaluated. Physapubescin M (1), with IC50 value of 1.58 μM, was selected for further study. The protein expression of COX‐2 and iNOS, and LPS‐induced production of cytokines (IL‐6, IL‐1β and TNF‐α) were reduced by physapubescin M (1) in a dose‐dependent way. In addition, transcriptomic analyses were conducted to profile gene expression alterations in LPS‐induced RAW264.7 cells upon treatment of physapubescin M (1) and the potential antiinflammatory mechanism of withnolides was mentioned. These results provide broad view to the underlying antiinflammatory mechanism of withnolides, and give a theoretical basis for the utilization of the fruits of P. alkekengi L. var. franchetii. [ABSTRACT FROM AUTHOR]

Obesity is a risk factor for many diseases, including type 2 diabetes and cardiovascular disease, and is related to the rising morbidity and mortality. Discovery of agents targeting adipogenesis, especially from natural sources, is important for the treatment of obesity. Here, we aimed to identify anti-adipogenic substances in methanol extracts of Physalis peruviana and to investigate their effect, along with underlying mechanisms. Activity-guided fractionation of the extract revealed 4β-hydroxywithanolide E (HWE) and withanolide E (WE) as the adipogenesis inhibitors. Both compounds suppressed mRNA expression of central adipogenic transcription factors, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer-binding protein α in the early stage of adipocyte differentiation. The inhibitory action of these two withanolides on adipogenesis was largely limited to this stage. The proliferation of preadipocytes was markedly suppressed by treatment with HWE and WE for 24 and 48 h in the differentiation medium, and cell-cycle arrest in the G0/G1 phase was observed. Therefore, our results suggested that withanolides from P. peruviana to be novel anti-adipogenic compounds that modulate mitotic clonal expansion. [ABSTRACT FROM AUTHOR]

• Physalin H treatment suppresses LPS-induced inflammation and oxidative stress. • Physalin H blocks the interaction between NRF2 and KEAP1 to inhibit the ubiquitination of NRF2. • Physalin H-mediated NRF2 nuclear translocation and activation plays a protective role in acute lung injury. Physalin H (PH), a withanolide isolated from Physalis angulata L. has been reported to have anti-inflammatory effect. However, its impact on acute lung injury (ALI) remains unexplored. In this study, we observed that PH significantly alleviated inflammation in LPS-stimulated macrophages by suppressing the release of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and down-regulating the expression of the inflammation-related genes. RNA sequencing analysis revealed a significant up-regulation of the NRF2 pathway by PH. Further investigation elucidated that PH attenuated the ubiquitination of NRF2 by impeding the interaction between NRF2 and KEAP1, thereby facilitating NRF2 nuclear translocation and up-regulating the expression of target genes. Consequently, it regulated redox system and exerted anti-inflammatory effect. Consistently, PH also significantly alleviated pathological damage and inflammation in LPS-induced ALI mice model, which could be reversed by administration of an NRF2 inhibitor. Collectively, these results suggest that PH ameliorates ALI by activating the KEAP1/NRF2 pathway. These findings provide a foundation for further development of pH as a new anti-inflammatory agent for ALI therapy. [ABSTRACT FROM AUTHOR]

Withania somnifera (Linn.) Dunal is a prominent Indian medicinal plant owing to its immense therapeutic potential. It has extensive genetic diversity, resulting in the availability of chemotypes and variations in phenotype. Around fifteen cultivars have been developed and released by various research institutes to meet the higher demand worldwide. However, the released varieties have not been characterized genetically. In this study, an attempt has been made to explore genetic diversity in cultivars and wild laces of W. somnifera using RAPD and ISSR, as they are the most popular techniques for genetic diversity assessment among species, populations, and varieties. The objective of the current study is to unveil the genetic diversity in 39 accessions of W. somnifera cultivars, consisting of seven commercial cultivars and three wild varieties, through Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) primers. The RAPD analysis generated a total of 141 bands, out of which 137 (96.90%) bands were polymorphic with an average band size of 15.66 ± 4.24, while ISSR primers yielded a total of 143 bands, out of which 132 were polymorphic (91.75%) with an average band size of 15.88 ± 4.22. For RAPD primers, the polymorphic information content (PIC) spans from 0.442 (OPA-02) to 0.860 (OPA-09), whereas with ISSR primers, the PIC ranges from 0.438 (UBC-880) to 0.898 (SOP-04). High PIC values were observed, indicating the informativeness of the markers. Analysis of molecular variance (AMOVA) revealed the highest percent variation of 72.43% with RAPD primers and of 65.60132% with ISSR primers among populations within groups, while among groups, the lowest percent variation of 4.35% (RAPD primers) and 10.26% (ISSR primers) was observed. The genetic distance among the 10 populations of W. somnifera ranged from 0.0375 to 0.6845 with RAPD markers and with ISSR markers ranged from 0.0698 to 0.6122, indicating that the differences among the 10 populations were considerably greater. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis with RAPD and ISSR primers divided the 39 accessions into two major clusters consisting of all the populations of seven commercially released varieties and three wild varieties, respectively. Among all the commercially released varieties, WSJ2 formed a separate subcluster with both RAPD and ISSR primers. The results of this study concluded that both RAPD-PCR and ISSR-PCR molecular marker techniques are appropriate for studying genetic diversity among and within W. somnifera varieties, comprising commercially developed cultivars and wild varieties. WSJ2 produced a discrete subcluster using both RAPD and ISSR primers, making it the most diversified variety, which can be investigated further phytochemically and pharmacologically to determine the most elite variety of W. somnifera. As a result of its adaptogenic characteristics and current study, ashwagandha has excellent prospects in traditional medicine, stress management, and potential medicinal uses. [Display omitted] • Ashwagandha is well-known medicinal herb due to vast therapeutic potential and genetic diversity. • Molecular marker are appropriate for genetic variability in Aswagandha. • Studies would help in identification of genes associated with secondary metabolite production. • WSJ2 formed a separate subcluster with both primers making it the most diverse variety. [ABSTRACT FROM AUTHOR]

Withania somnifera (Solanaceae), well-known as ‘Indian ginseng’ or ‘Ashwagandha’, is a medicinal plant that is used in Ayurvedic practice to promote good health and longevity. As part of an ongoing investigation for bioactive natural products with novel structures, we performed a phytochemical examination of the roots of W. somnifera employed with liquid chromatography–mass spectrometry (LC/MS)-based analysis. The chemical analysis of the methanol extract of W. somnifera roots using repeated column chromatography and high-performance liquid chromatography under the guidance of an LC/MS-based analysis resulted in a new withanolide, withasomniferol D (1). The structure of the newly isolated compound was elucidated by spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high-resolution (HR) electrospray ionization (ESI) mass spectroscopy, and its absolute configuration was established by electronic circular dichroism (ECD) calculations. The anti-adipogenic activities of withasomniferol D (1) were evaluated using 3T3-L1 preadipocytes with Oil Red O staining and quantitative real-time polymerase chain reaction (qPCR). We found that withasomniferol D (1) inhibited adipogenesis and suppressed the enlargement of lipid droplets compared to the control. Additionally, the mRNA expression levels of adipocyte markers Fabp4 and Adipsin decreased noticeably following treatment with 25 μM of withasomniferol D (1). Taken together, these findings provide experimental evidence that withasomniferol D (1), isolated from W. somnifera, exhibits anti-adipogenic activity, supporting the potential application of this compound in the treatment of obesity and related metabolic diseases.

Two new withanolides named physaminilides L ( 1 ) and M ( 2 ), together with four known ones ( 3-6 ) were isolated from the Physalis minima L. The structures were established by analysis of the HR ESIMS, IR and NMR spectroscopic data. The absolute configurations were determined through NOESY and ECD spectra. For compounds 1-5 assayed at 20  µ M and compound 6 at 10  µ M, inhibition rates of hepatic fibrosis were 22.19%, 15.29%, 37.07%, 9.27%, 12.45%, and 37.03%, respectively.

Breast cancer expresses clinically heterogeneous characteristics and requires multipurpose drug development for curing the different tumor subtypes. Many withanolides have been isolated from Physalis species showing anticancer effects, but the anticancer function of physapruin A (PHA) has rarely been investigated. In this study, the anticancer properties of PHA in breast cancer cells were examined by concentration and time-course experiments. In terms of cellular ATP content, PHA inhibited the proliferation of three kinds of breast cancer cells: MCF7 (estrogen receptor (ER)+, progesterone receptor (PR)+/−, human epidermal growth factor receptor 2 (HER2)−), SKBR3 (ER−/PR−/HER2+), and MDA-MB-231 (triple-negative). Moreover, PHA induced G2/M arrest in MCF7 and MDA-MB-231 cells. In terms of flow cytometry, PHA induced the generation of reactive oxygen species (ROS), the generation of mitochondrial superoxide, mitochondrial membrane potential depletion, and γH2AX-detected DNA damage in breast cancer MCF7 and MDA-MB-231 cells, which were suppressed by the ROS inhibitor N-acetylcysteine (NAC). In terms of flow cytometry and Western blotting, PHA induced apoptotic expression (annexin V, and intrinsic and extrinsic apoptotic signaling), which was suppressed by NAC and an apoptosis inhibitor (Z-VAD-FMK), in breast cancer cells. Therefore, PHA is a potential anti-breast-cancer natural product that modulates the oxidative-stress response, cell-cycle disturbance, apoptosis, and γH2AX-detected DNA damage.

Withaferin A (WFA), a Withania somnifera-derived triterpenoid, is an anticancer natural product. The anticancer effect of nonionizing radiation such as ultraviolet-C (UVC) as well as the combined treatment of UVC and WFA is rarely investigated. Low dose UVC and/or WFA treatments (12 J/m2 and/or 1 μM) were chosen to evaluate antioral cancer cell line effects by examining cytotoxicity, cell cycle disruption, apoptosis induction, and DNA damage. For two cancer cell lines (Ca9-22 and HSC-3), single treatment (UVC or WFA) showed about 80% viability, while a combined treatment of UVC/WFA showed about 40% viability. In contrast, there was noncytotoxicity to normal oral cell lines (HGF-1). Compared to single treatment and control, low dose UVC/WFA shows high inductions of apoptosis in terms of flow cytometric detections for subG1, annexin V, pancaspase changes as well as Western blotting for detecting cleaved poly (ADP-ribose) polymerase (c-PARP) and caspase 3 (c-Cas 3) and luciferase assay for detecting Cas 3/7 activity. Low dose UVC/WFA also showed high inductions of oxidative stress and DNA damage in terms of flow cytometric detections of reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX) generation, and membrane potential (MitoMP) destruction, γH2AX and 8-oxo-2’deoxyguanosine (8-oxodG) types of DNA damages. For comparison, low dose UVC/WFA show rare inductions of annexin V, Cas 3/7 activity, ROS, MitoSOX, and MitoMP changes to normal oral HGF-1 cells. Therefore, low dose UVC/WFA provides a novel selectively killing mechanism to oral cancer cells, suggesting that WFA is a UVC sensitizer to inhibit the proliferation of oral cancer cells.

Some withanolides, particularly the family of steroidal lactones, show anticancer effects, but this is rarely reported for withanolide C (WHC)—especially anti-breast cancer effects. The subject of this study is to evaluate the ability of WHC to regulate the proliferation of breast cancer cells, using both time and concentration in treatment with WHC. In terms of ATP depletion, WHC induced more antiproliferation to three breast cancer cell lines, SKBR3, MCF7, and MDA-MB-231, than to normal breast M10 cell lines. SKBR3 and MCF7 cells showing higher sensitivity to WHC were used to explore the antiproliferation mechanism. Flow cytometric apoptosis analyses showed that subG1 phase and annexin V population were increased in breast cancer cells after WHC treatment. Western blotting showed that cleaved forms of the apoptotic proteins poly (ADP-ribose) polymerase (c-PARP) and cleaved caspase 3 (c-Cas 3) were increased in breast cancer cells. Flow cytometric oxidative stress analyses showed that WHC triggered reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX) production as well as glutathione depletion. In contrast, normal breast M10 cells showed lower levels of ROS and annexin V expression than breast cancer cells. Flow cytometric DNA damage analyses showed that WHC triggered γH2AX and 8-oxo-2′-deoxyguanosine (8-oxodG) expression in breast cancer cells. Moreover, N-acetylcysteine (NAC) pretreatment reverted oxidative stress-mediated ATP depletion, apoptosis, and DNA damage. Therefore, WHC kills breast cancer cells depending on oxidative stress-associated mechanisms.

The withanolides are naturally occurring steroidal lactones found mainly in plants of the Solanaceae family. The subtribe Withaninae includes species like Withania sominifera , which are a source of many bioactive withanolides. In this work, we selected and evaluate the ADMET-related properties of 91 withanolides found in species of the subtribe Withaninae computationally, to predict the relationship between their structures and their pharmacokinetic profiles. We also evaluated the interaction of these withanolides with known targets of Alzheimer's disease (AD) through molecular docking and molecular dynamics. Withanolides presented favorable pharmacokinetic properties, like high gastrointestinal absorption, lipophilicity (logP ≤ 5), good distribution and excretion parameters, and a favorable toxicity profile. The specie Withania aristata stood out as an interesting source of the promising withanolides classified as 5-ene with 16-ene or 17-ene. These withanolides presented a favourable pharmacokinetic profile and were also highlighted as the best candidates for inhibition of AD-related targets. Our results also suggest that withanolides are likely to act as cholinesterase inhibitors by interacting with the catalytic pocket in an energy favorable and stable way.Communicated by Ramaswamy H. Sarma.