Bioactive Withanolides Research Articles

An Integrated Strategy of UHPLC-ESI-MS/MS Combined with Bioactivity-Based Molecular Networking for Identification of Antitumoral Withanolides from Athenaea fasciculata (Vell.) I.M.C. Rodrigues & Stehmann.

Athenaea fasciculata, a Brazilian native species from the Solanaceae family, is recognized as a promising source of bioactive withanolides, particularly Aurelianolide A and B, which exhibit significant antitumoral activities. Despite its potential, research on the chemical constituents of this species remains limited. This study aimed to dereplicate extracts and partitions of A. fasciculata to streamline the discovery of bioactive withanolides. Using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), various extracts-including n-hexane, methanol, and ethanol-were analyzed, and their mass spectrometry data were processed through the GNPS platform for the generation of molecular networking. The results indicated that crude extracts displayed comparable cytotoxicity against Jurkat cells, by treatment at 150 µg/mL, while alcoholic extracts achieved approximately 80% inhibition of K562 cells and K562-Lucena 1 at the same concentration. Notably, the dichloromethane partition exhibited the highest cytotoxicity across leukemia cell lines, particularly against Jurkat cells (IC50 = 14.34 µg/mL). A total of 22 compounds were annotated by manual inspection and different libraries, with six of them demonstrating significant cytotoxic effects. This research underscores the therapeutic potential of A. fasciculata and highlights the effectiveness of integrating advanced analytical methods in drug discovery, paving the way for further exploration of its bioactive compounds.

Withanolides production by the endophytic fungus Penicillium oxalicum associated with Withania somnifera (L.) Dunal.

Withanolides are steroidal lactones with diverse bioactive potential and their production from plant sources varies with genotype, age, culture conditions, and geographical region. Endophytic fungi serve as an alternative source to produce withanolides, like their host plant, Withania somnifera (L.) Dunal. The present study aimed to isolate endophytic fungi capable of producing withanolides, characterization and investigation of biological activities of these molecules. The methanolic fungal crude extract of one of the fungal isolates WSE16 showed maximum withanolide production (219mg/L). The fungal isolate WSE16 was identified as Penicillium oxalicum based on its morphological and internal transcribed spacer (ITS) sequence analysis and submitted in NCBI (accession number OR888725). The methanolic crude extract of P. oxalicum was further purified by column chromatography, and collected fractions were assessed for the presence of withanolides. Fractions F3 and F4 showed a higher content of withanolides (51.8 and 59.1mg/L, respectively) than other fractions. Fractions F3 and F4 exhibited antibacterial activity against Staphylococcus aureus with an IC50 of 23.52 and 17.39µg/ml, respectively. These fractions also showed antioxidant activity (DPPH assay with IC50 of 39.42 and 38.71µg/ml, superoxide anion scavenging assay with IC50 of 41.10 and 38.84µg/ml, and reducing power assay with IC50 of 42.61 and 41.40µg/ml, respectively) and acetylcholinesterase inhibitory activity (IC50 of 30.34 and 22.05µg/ml, respectively). The withanolides present in fraction 3 and fraction 4 were identified as (20S, 22R)-1a-Acetoxy-27-hydroxywitha-5, 24-dienolide-3b-(O-b-D-glucopyranoside) and withanamide A, respectively, using UV, FTIR, HRMS, and NMR analysis. These results suggest that P. oxalicum, an endophytic fungus isolated from W. somnifera, is a potential source for producing bioactive withanolides.

UPLC-Q-Orbitrap-MS/MS-Guided Isolation of Bioactive Withanolides from the Fruits of Physalis angulata.

Physalis angulata Linn. is an exotic Amazonian fruit that is commonly recognized as wild tomato, winter cherry, and gooseberry. While its fruit is known to contain many nutrients, such as minerals, fibers, and vitamins, few papers have investigated withanolide derivatives from its fruits. UPLC-Q-Orbitrap-MS/MS, which produces fragmentation spectra, was applied for the first time to guide the isolation of bioactive withanolide derivatives from P. angulata fruits. As a result, twenty-six withanolide derivatives, including two novel 1,10-secowithanolides (1 and 2) and a new derivative (3), were obtained. Compounds 1 and 2 are rare rearranged 1,10-secowithanolides with a tetracyclic 7/6/6/5 ring system. All structures were assigned through various spectroscopic data and quantum chemical calculations. Nine withanolide derivatives exhibited significant inhibitory effects on three tumor cell lines with IC50 values of 0.51-13.79 μM. Moreover, three new compounds (1-3) exhibited potential nitric oxide inhibitory effects in lipopolysaccharide-stimulated RAW264.7 cells (IC50: 7.51-61.8 μM). This investigation indicated that fruits of P. angulata could be applied to treat and prevent cancer and inflammatory-related diseases due to their potent active withanolide derivatives.

Physalin A Induces Apoptosis and Autophagy in Hepatocellular Carcinoma via Inhibition of PI3K/Akt Signaling Pathway.

Physalin A (PA) is a bioactive withanolide with multiple pharmacological properties and has been indicated to be cytotoxic to hepatocellular carcinoma (HCC) cell line HepG2. This study aims to explore the mechanisms underlying PA antitumor activity in HCC. HepG2 cells were exposed to various concentrations of PA. Cell counting kit-8 assay and flow cytometry were implemented for evaluating cell viability and apoptosis, respectively. Immunofluorescence staining was utilized for detecting autophagic protein LC3. Western blotting was employed for measuring levels of autophagy-, apoptosis- and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling-related proteins. A xenograft mouse model was established to verify the antitumor activity of PA in vivo. PA impaired HepG2 cell viability, and triggered apoptosis as well as autophagy. Inhibiting autophagy augmented PA-evoked HepG2 cell apoptosis. PA repressed PI3K/Akt signaling in HCC cells and activating PI3K/Akt reversed PA-triggered apoptosis and autophagy. PA treatment inhibited tumor growth in tumor-bearing mice. PA triggers HCC cell apoptosis and autophagy by inactivating PI3K/Akt signaling.

In silico evaluation of pharmacokinetics properties of withanolides and simulation of their biological activities against Alzheimer’s disease

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

Physalin A alleviates intervertebral disc degeneration via anti-inflammatory and anti-fibrotic effects.

The incidence of intervertebral disc degeneration (IVDD) is a common degenerative disease with inflammation, decreased autophagy, and progression of fibrosis as its possible pathogenesis. Physalin A (PA) is a widely studied anti-inflammatory drug. However, its therapeutic effects on IVDD remain unexplored. Therefore, we aimed to explore the therapeutic potential of PA in IVDD progression. In vivo, we investigated PA bioactivity using a puncture-induced IVDD rat model. IVDD signals and height changes were detected using X-ray, micro-CT, and MRI, and structural and molecular lesions using histological staining and immunohistochemistry of intervertebral disc sections. In vivo, interleukin-1 beta (IL-1β) and TGF-β1 were employed to establish inflammation fibrotic nucleus pulposus (NP) cells. The PA effect duration, concentration, influence pathways, and pathological changes in IVDD treatment were elucidated using western blotting, real-time PCR, and immunofluorescence. PA exerted significant effects on IVDD remission due to anti-inflammation, fibrosis reduction, and autophagy enhancement. In vitro, PA improved inflammation by blocking the NF-κB and MAPK pathways, whereas it promoted autophagy via the PI3K/AKT/mTOR pathway and affected fibrotic progression by regulating the SMAD2/3 pathway. Moreover, PA improved the disc degeneration process in IVDD model. PA exhibited significant anti-inflammatory and anti-fibrotic effects and improved autophagy in vivo and in vitro IVDD models, thus effectively relieving IVDD progression, indicating it is a promising agent for IVDD treatment. This study successfully reveals that PA, a natural bioactive withanolide, effectively relieved IVDD progression via inflammation inhibition, fibrosis reduction, and autophagy enhancement, indicating it is a promising agent for IVDD treatment.

Mulberroside A alleviates osteoarthritis via restoring impaired autophagy and suppressing MAPK/NF-κB/PI3K-AKT-mTOR signaling pathways

Osteoarthritis (OA) is a trauma-/age-related degenerative disease characterized by chronic inflammation as one of its pathogenic mechanisms. Mulberroside A (MA), a natural bioactive withanolide, demonstrates anti-inflammatory properties in various diseases; however, little is known about the effect of MA on OA. We aim to examine the role of MA on OA and to identify the potential mechanisms through which it protects articular cartilage. Invitro, MA improved inflammatory response, anabolism, and catabolism in IL-1β-induced OA chondrocytes. The chondroprotective effects of MA were attributed to suppressing the MAPK, NF-κB, and PI3K-AKT-mTOR signaling pathways, as well as promoting the autophagy process. Invivo, intra-articular injection of MA reduced the cartilage destruction and reversed the change of anabolic and catabolic-related proteins in destabilized medial meniscus (DMM)-induced OA models. Thus, the study indicates that MA exhibits a chondroprotective effect and might be a promising agent for OA treatment.

Structural Characterization of Withanolide Glycosides from the Roots of Withania somnifera and Their Potential Biological Activities.

Withania somnifera (Solanaceae), commonly known as “ashwagandha”, is an ayurvedic medicinal plant that has been used for promoting good health and longevity. As part of our ongoing natural product research for the discovery of bioactive phytochemicals with novel structures, we conducted a phytochemical analysis of W. somnifera root, commonly used as an herbal medicine part. The phytochemical investigation aided by liquid chromatography-mass spectrometry (LC/MS)-based analysis led to the isolation of four withanolide glycosides (1–4), including one new compound, withanoside XII (1), from the methanol (MeOH) extract of W. somnifera root. The structure of the new compound was determined by nuclear magnetic resonance (NMR) spectroscopic data, high-resolution (HR) electrospray ionization (ESI) mass spectroscopy (MS), and electronic circular dichroism (ECD) data as well as enzymatic hydrolysis followed by LC/MS analysis. In addition, enzymatic hydrolysis of 1 afforded an aglycone (1a) of 1, which was identified as a new compound, withanoside XIIa (1a), by the interpretation of NMR spectroscopic data, HR-ESIMS, and ECD data. To the best of our knowledge, the structure of compound 2 (withagenin A diglucoside) was previously proposed by HRMS and MS/MS spectral data, without NMR experiment, and the physical and spectroscopic data of withagenin A diglucoside (2) are reported in this study for the first time. All the isolated compounds were evaluated for their anti-Helicobacter pylori, anti-oxidant, and anti-inflammatory activities. In the anti-Helicobacter pylori activity assay, compound 2 showed weak anti-H. pylori activity with 7.8% inhibition. All the isolated compounds showed significant ABTS radical scavenging activity. However, all isolates failed to show inhibitory activity against nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. This study demonstrated the experimental support that the W. somnifera root is rich in withanolides, and it can be a valuable natural resource for bioactive withanolides.

Phospholipid Based Colloidal Nanocarriers for Enhanced Solubility and Therapeutic Efficacy of Withanolides

The bioactive withanolides of Withania somnifera (WS) are reported to treat acute stress-induced anxiety and chronic stress-induced depression. However, its biopharmaceutical attributes such as poor water solubility, poor permeability, and lower bioavailability need to be tuned to improve the therapeutic efficacy. Hence, we hypothesized and designed the present study to enhance withanolides' solubility and therapeutic efficacy by fabricating their naturosomal delivery using phospholipid complexation (PLC). Moreover, investigation of the effect of PLC on solubility and colloidal stability of withanolides encapsulated in naturosomes (WNs) was another objective of the present investigation. Naturosomes were developed and optimized by employing Quality by Design, further characterized for demonstrating their physicochemical and functional properties. The solubility study of WNs exhibited a ∼14-fold and ∼1.3-fold increase in aqueous solubility than the pure WS and physical mixture (PM) with a phospholipid, respectively. The results of dynamic light scattering and zeta potential (−37.30 mV) of WNs has proven its colloidal stability with controlled size particles. These colloidal WNs were transformed in dry powder and retained their stability upon lyophilization. Likewise, the in-vitro dissolution studies exhibited >95% release of withanolides from WNs in comparison with WS (∼27%) or the PM (∼29%). The ex-vivo permeation studies demonstrated significant improvement in the permeation of WNs (>74%) in comparison to the WS (∼16%) or the PM (∼18%). Withanolides encapsulated naturosomes prepared by PLC could be an alternative strategy to enhance the solubility and improve bioavailability and efficacy.

Early selective strategies for higher yielding bio-economic Indian ginseng based on genotypic study through metabolic and molecular markers

Applying biotechnological tools to the selection of higher-yielding bioeconomic crops is a promising and remarkable means of reducing the burden on production on a global scale. In the present study, 25 germplasms of Indian ginseng (Withania somnifera (L.) Dunal) were examined for their genetic diversity by using morphological, biochemical, and molecular markers for twenty plant growth traits. The properties of plant growth differed significantly in the maximum genotypes of Indian ginseng, the markers of randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat (ISSR) showed considerable diversity between the genotypes. The combined unweighted pair group technique with arithmetic mean (UPGMA) dendrogram of morphological, biochemical, and molecular markers grouped all 25 genotypes into two main clusters at 0.61 coefficient value. In addition to this, secondary metabolite profiling by high-performance liquid chromatography (HPLC), there were high variations for withanolide B (WL-B), withanoside-V (WS-V), wedelolactone (WDL), withanoside-IV (WS-IV), and withaferin A (WF-A) content between different genotypes. For the total alkaloid and withanolide concentration in the roots and leaves, high heritability with an increased genetic gain was observed, indicating that selection based on these traits could be an effective method in breeding programs. Furthermore, the path coefficient analysis showed a direct positive impact of the total root fiber, WL-B (leaves), WF-A (leaves), WS-IV (roots), WDL (roots), and the total alkaloid content on the dry root yield. High content of WDL, a high-quality bioactive withanolide, was also described for the first time in the genotype UWS23. These properties can further be exploited to improve the dry root yield in W. somnifera genotypes. The outcomes of the present study also provide an essential foundation for the selection of high-yielding bioeconomic varieties that could be utilized to improve Ashwagandha breeding programs.

Physalin A Inhibits MAPK and NF-κB Signal Transduction Through Integrin αVβ3 and Exerts Chondroprotective Effect.

Osteoarthritis (OA) is a common articular ailment presented with cartilage loss and destruction that is common observed in the elderly population. Physalin A (PA), a natural bioactive withanolide, exerts anti-inflammatory residences in more than a few diseases; however, little is known about its efficacy for OA treatment. Here, we explored the therapeutic effects and potential mechanism of PA in mouse OA. After the in vitro administration of PA, the expression of inflammation indicators including inducible nitric oxide synthase and cyclooxygenase-2 was low, indicating that PA could alleviate the IL-1β-induced chondrocyte inflammation response. Moreover, PA reduced IL-1β-induced destruction of the extracellular matrix by upregulating the gene expression of anabolism factors, including collagen II, aggrecan, and sry-box transcription factor 9, and downregulating the gene expression of catabolic factors, including thrombospondin motif 5 and matrix metalloproteinases. In addition, the chondroprotective effect of PA was credited to the inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Furthermore, in vivo experiments showed that intra-articular injection of PA could alleviate cartilage destruction in a mouse OA model. However, the anti-inflammatory, anabolism enhancing, catabolism inhibiting, and MAPK and NF-κB signaling pathway inhibiting properties of PA on IL-1β-induced chondrocytes could be reversed when integrin αVβ3 is knocked down by siRNA. In conclusion, our work demonstrates that PA exhibits a chondroprotective effect that may be mediated by integrin αVβ3. Thus, PA or integrin αVβ3 might be a promising agent or molecular target for the treatment of OA.

Molecular modeling studies of the effects of withaferin A and its derivatives against oncoproteins associated with breast cancer stem cell activity

Cancer stem cells (CSCs) in breast cancers are considered to be a major cause of tumor recurrence and metastasis, which lead to chemotherapeutic failures and poor survival rates among breast cancer patients. Hence, this study attempted to evaluate the anti-tumor potential of a bioactive withanolide compound, withaferin A (WFA), against the molecular targets that maintain the stemness of breast cancer stem cells, using in silico molecular modeling approach. The crystal structures of potential breast cancer stem cell targets—HSP70, HSP90, NFκB, and BRCA1 were retrieved from PDB. The 2D structures of WFA, its analogs, along with doxorubicin and vinblastine were retrieved from the PubChem database. WFA and withaferin A diacetate exhibited strong receptor-ligand interactions for the target macromolecules. Their binding energy values ranged between –55.241 and –40.250 kcal/mol. Compared to doxorubicin, WFA markedly inhibited the growth and decreased the viability of MCF-7-derived breast cancer stem cells, with an IC50 value of 1.476 μM for 48 h. Therefore, our results indicate that WFA and its derivatives could be promising anti-tumor agents against breast cancer stem cells. Further in vitro studies are warranted in order to gain a greater understanding of the action of this compound in a physiological setting.

Withanolides from Withania somnifera Ameliorate Neutrophil Infiltration in Endotoxin-Induced Peritonitis by Regulating Oxidative Stress and Inflammatory Cytokines.

Identification of novel anti-inflammatory strategies are needed to avoid the side effects associated with the currently available therapies. Use of anti-inflammatory herbal remedies is gaining attention. The purpose of the present investigation was to evaluate the pharmacological potential of the withanolide-rich root extracts of the medical plant Withania somnifera (L.) Dunal using in vivo and in vitro models of endotoxin-induced inflammation and oxidative stress. The pharmacological effects of W. somnifera root extracts were evaluated using a mouse model of endotoxin (lipopolysaccharide)-induced peritonitis and various relevant human cell lines. HPLC analysis of the W. somnifera root extracts identified the presence of various bioactive withanolides. In vivo challenge of mice with endotoxin resulted in the infiltration of various leukocytes, specifically neutrophils, along with monocytes and lymphocytes into the peritoneal cavity. Importantly, prophylactic treatment with W. somnifera inhibited the migration of neutrophils, lymphocytes, and monocytes and decreased the release of interleukin-1β, TNF-α, and interleukin-6 cytokines into the peritoneal cavity as identified by ELISA. Liver (glutathione peroxidase, glutathione, glutathione disulfide, superoxide dismutase, malondialdehyde, myeloperoxidase) and peritoneal fluid (nitrite) biochemical analysis revealed the antioxidant profile of W. somnifera. Similarly, in human HepG2 cells, W. somnifera significantly modulated the antioxidant levels. In THP-1 cells, W. somnifera decreased the secretion of interleukin-6 and TNF-α. In HEK-Blue reporter cells, W. somnifera inhibited TNF-α-induced nuclear factor-κB/activator protein 1 transcriptional activity. Our findings suggest the pharmacological effects of root extracts of W. somnifera rich in withanolides inhibit neutrophil infiltration, oxidative hepatic damage, and cytokine secretion via modulating the nuclear factor-κB/activator protein 1 pathway.

Closed polybag foliar methyl-jasmonate treatment: New technology for rapid enhancement of bioactive withanolide biosynthesis in field-grown plants of Withania somnifera

Withania somnifera (L.) Dunal, is an important medicinal plant with tremendous use in the herbal drug industry. Withanolides are the major bioactive metabolites present in the leaves of W. somnifera. However, due to the low abundance of withanolides, it is difficult to fulfil the market demand. The rationale of this study was to develop a simple technology for enhanced withanolide biosynthesis in field-grown plants of W. somnifera. Exogenous elicitor-treatment to the field-grown plants is an effective strategy to increase the production of many plant secondary metabolites. In this study, a high-performance liquid chromatography method was developed for the rapid separation of withanolides from the leaves along with phenolic acid and flavonoids. Field-grown plants of W. somnifera were directly used for methyl jasmonate elicitor treatment using a novel closed polybag approach. In this approach, branches were covered with a polybag and then MeJa was injected using a specially designed vial. Methyl-jasmonate (20μM) treatment for 4 h resulted in 2.2-fold, 1.9-fold, 2.0-fold, and 2.2-fold increase in withaferin A, withanolide A, withanolide D, and withanone production, respectively, over the control treatment. Methyl-jasmonate -treatment increased the transcript levels of four key withanolide biosynthetic genes, namely 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, and cycloartenol synthase. Methyl-jasmonate -treated leaf extracts and isolated pure withanolides also showed a significant cytotoxic effect against selected human breast cancer cell line. These results suggest that MeJa-treatment using a closed polybag system is an effective and easy-to-perform elicitation strategy for the enhanced production of bioactive withanolides using field-grown plants of W. somnifera. Farmers and industry can easily adapt this technology to meet the growing demand for bioactive withanolides.

UPLC-MS/MS Identification and Quantification of Withanolides from Six Parts of the Medicinal Plant Datura Metel L.

Withanolides from six parts (flower, leaf, stem, root, seed, and peel) of Datura metel L. (D metel L.) obtained from ten production areas in China were identified and quantified by UPLC-MS/MS. A total of 85 withanolides were characterized for the first time using the UPLC-Q-TOF-MS/MS system. Additionally, a simultaneous, rapid and accurate measurement method was developed for the determination of 22 bioactive withanolides from ten production areas with the UPLC-Q-TRAP-MS/MS system. The results show the total withanolide content is highest in the leaves (155640.0 ng/g) and lowest in the roots (14839.8 ng/g). Compared with other production areas, the total content of plants from Dujiangyan was the highest at 82013.9 ng/g (value range of ten areas: 82013.9–42278.5 ng/g). The results also show significant differences in the distribution of withanolides in the different plant parts, as well as across different production areas. This is a breakthrough report providing a simultaneous qualitative and quantitative analysis of 22 withanolides in D. metel L. It could be the basis for the more rational use of various parts of D. metel L., and the expansion of medicinal resources. This work also lays a solid foundation for research on the quality control of D. metel L.

Potential of Organic Nutrient Sources for Improving Yield and Bioactive Principle of Ashwagandha (Withania Somnifera) through Enhanced Soil Fertility and Biological Functions

ABSTRACTAshwagandha (Withania somnifera L. Dunal) is an industrially important medicinal herb and root and is a rich source of bioactive withanolides. To investigate the impacts of different organic nutrient management practices on ashwagandha yield and quality, a field experiment was conducted for 2 consecutive years. The experiment was carried out in randomized bock designed (RBD) with treatments comprised of different organic manures (farmyard manure, vermicompost, and castor cake) and microbial consortium along with recommended doses of fertilizer and the control. The root yield parameters (root girth, length, and fresh and dry root weight) of ashwagandha were influenced significantly by the treatments containing organic manures and microbial consortium alone or in combination. The highest fresh (1505 kg ha−1) and dry (767 kg ha−1) root yields as well as total withanolide contents (0.947 mg g−1) were recorded under the treatment receiving castor cake + microbial consortium (T7) followed by application of vermicompost + microbial consortium (T8). Significant improvements in soil fertility status (mineral N, available P and S) and soil biochemical parameters (microbial biomass and enzymes) were observed under the treatments containing castor cake and vermicompost. A positive correlation coefficient (< 0.01) was found between dry root yield, withanolide content and soil properties (r = 0.34–0.64). Thus, the results indicate that castor cake and vermicompost could be promising nutrient source for production of quality medicinal herb by improving soil properties.

Drier Climatic Conditions Increase Withanolide Content of Withania coagulans Enhancing Its Inhibitory Potential Against Human Prostate Cancer Cells.

Prostate cancer is one of the major causes of cancer-related deaths in men and there is a growing interest in identifying natural compounds for its management. We analyzed bioactive withanolides in Withania coagulans from 11 different sites in Pakistan and evaluated the antiprostate cancer activities of leaf extracts from two sites with the greatest amounts. Total withanolide concentration differed by ~ 17-fold between sites, ranging from 1.01 ± 0.01mg/g dry weight (mean ± SE) at Jand to 16.83 ± 0.02mg/g at Mohmand Agency. Different tissues varied in their total withanolide content with roots having the least (0.42 ± 0.07mg/g dry weight) and leaves the most (2.45 ± 0.45mg/g). We found strong inverse correlations between site annual precipitation versus withanolide amounts in fruits (r = - 0.84, P = 0.001), leaves (r = - 0.88, P < 0.001), roots (r = - 0.91, P < 0.001), and total (r = - 0.89, P < 0.001), but not stems (r = - 0.20, P = 0.556). Extracts made from Mianwali and Mohmand Agency leaves possessed high anticancer activity in terms of increased induction of apoptosis and decreased cell viability, cell proliferation, invasion, and migration of different prostate cancer cell lines. These results are useful for the selection of withanolide-rich germplasm with potent anticancer properties.

Two sulfonate metabolites of physalin A in rats

1. Physalin A is a bioactive withanolide isolated from the natural plant Physalis alkekengi var. franchetii (Solanaceae), a common traditional Chinese herbal medicine. This study aims to investigate the metabolites of physalin A in vivo.2. Two metabolites (M1 and M2) were characterized as sulfonate metabolites in the feces obtained from rats treated with physalin A orally at a dose of 15 mg/kg/day for 3 days, by application of a UPLC-Q/TOF-MS method. Furthermore, formation of the two sulfonate metabolites was verified by chemical synthesis and NMR, including 1H NMR, 13C NMR and two-dimensional NMR. The structures of M1 and M2 were identified to be 3α-sulfo-2,25β,27-trihydrophysalin A and 3α,27-disulfo-2,25α-dihydrophysalin A, respectively.3. In summary, this study indicated that physalin A could be biotransformed to sulfonate metabolites with strong polarity, which contributed to the elimination of physalin A. A rare metabolic pathway has been revealed in this study.

Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling.

The signal transducers and activators of transcription 3 (STAT3) signaling pathway plays critical roles in the pathogenesis and progression of various human cancers, including non-small cell lung cancer (NSCLC). In this study, we aimed to evaluate the therapeutic potential of physalin A, a bioactive withanolide derived from Physalis alkekengi var. francheti used in traditional Chinese medicine, was evaluated in human NSCLC cells. Its and determined whether it effect oninhibited both constitutive and induced STAT3 activity, through repressing the phosphorylation levels of JAK2 and JAK3, resulting in anti-proliferation and pro-apoptotic effects on NSCLC cells was also determined, and. theThe antitumor effects of physalin A were also validated usingin an in vivo mouse xenograft models of NSCLC cells. Physalin A had anti-proliferative and pro-apoptotic effects in NSCLC cells with constitutively activated STAT3; it also suppressed both constitutive and induced STAT3 activity by modulating the phosphorylation of JAK2 and JAK3. Furthermore, physalin A abrogated the nuclear translocation and transcriptional activity of STAT3, thereby decreasing the expression levels of STAT3, its target genes, such as Bcl-2 and XIAP. Knockdown of STAT3 expression by small interfering RNA (siRNA) significantly enhanced the pro-apoptotic effects of physalin A in NSCLC cells. Moreover, physalin A significantly suppressed tumor xenograft growth. Thus, as an inhibitor of JAK2/3-STAT3 signaling, physalin A, has potent anti-tumor activities, which may facilitate the development of a therapeutic strategy for treating NSCLC.

Cold stress affects antioxidative response and accumulation of medicinally important withanolides in Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal (Indian ginseng) is a high value medicinal plant. It synthesizes a large array of biologically active withanolides. In this study, two month old seedlings of AGB002 (wild genotype) and AGB025 (cultivated genotype) of W. somnifera were subjected to cold stress (4°C) under controlled environment. Plants were analyzed for three medicinally important secondary metabolites (withanolide A, withanone and withaferin A), lipid peroxidation (MDA), cell injury, superoxide radical (O2−) accumulation and anti-oxidative enzymes activities such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). Increases in the titers of superoxide anion and MDA were observed from day 1 to day 7 in both genotypes, although the increase on the first day of exposure was significantly higher. Enzymatic activities of SOD, CAT, APX and GR also showed an increasing trend in both genotypes and reached a maximum on day 7 of the cold temperature exposure; however, this increase was higher in AGB002 than AGB025. Withanolide A (WS-1) in the roots of both genotypes significantly decreased on the first day of cold exposure and then showed a recovery until day 7. WS-1 was not detected in the leaves of either genotype. Withanone (WS-2) content in the leaves also decreased towards the end of the cold period in both genotypes. Cold stress also elicited the accumulation of WS-2 in AGB025, but was not detectable in control seedlings. At maturity, WS-2 was also detected in control plants. Furthermore, a significant increase in the leaf withaferin A (WS-3) content was recorded from day 1 to day 7 of the cold exposure in both the genotypes, suggesting the possible involvement of withanolides in cold-protection. AGB002 showed comparatively higher accumulation of antioxidant enzymes and selected marker withanolides than AGB025, indicating that AGB002 is better adapted to cold than AGB025. It could be inferred from these observations that cold stress induces bioactive withanolide accumulation in W. somnifera as a mechanism for scavenging reactive oxygen species (ROS). These studies also provide an impetus for enhancing the withanolide accumulation in W. somnifera using controlled environment technology.