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 (219 mg/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 59.1 mg/L, respectively) than other fractions. Fractions F3 and F4 exhibited antibacterial activity against Staphylococcus aureus with an IC50 of 23.52 and 17.39 µg/ml, respectively. These fractions also showed antioxidant activity (DPPH assay with IC50 of 39.42 and 38.71 µg/ml, superoxide anion scavenging assay with IC50 of 41.10 and 38.84 µg/ml, and reducing power assay with IC50 of 42.61 and 41.40 µg/ml, respectively) and acetylcholinesterase inhibitory activity (IC50 of 30.34 and 22.05 µg/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.

Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia.

In our previous studies, we showed that the generation of ovarian tumors in NSG mice (immune-compromised) resulted in the induction of muscle and cardiac cachexia, and treatment with withaferin A (WFA; a steroidal lactone) attenuated both muscle and cardiac cachexia. However, our studies could not address if these restorations by WFA were mediated by its anti-tumorigenic properties that might, in turn, reduce the tumor burden or WFA's direct, inherent anti-cachectic properties. To address this important issue, in our present study, we used a cachectic model induced by the continuous infusion of Ang II by implanting osmotic pumps in immunocompetent C57BL/6 mice. The continuous infusion of Ang II resulted in the loss of the normal functions of the left ventricle (LV) (both systolic and diastolic), including a significant reduction in fractional shortening, an increase in heart weight and LV wall thickness, and the development of cardiac hypertrophy. The infusion of Ang II also resulted in the development of cardiac fibrosis, and significant increases in the expression levels of genes (ANP, BNP, and MHCß) associated with cardiac hypertrophy and the chemical staining of the collagen abundance as an indication of fibrosis. In addition, Ang II caused a significant increase in expression levels of inflammatory cytokines (IL-6, IL-17, MIP-2, and IFNγ), NLRP3 inflammasomes, AT1 receptor, and a decrease in AT2 receptor. Treatment with WFA rescued the LV functions and heart hypertrophy and fibrosis. Our results demonstrated, for the first time, that, while WFA has anti-tumorigenic properties, it also ameliorates the cardiac dysfunction induced by Ang II, suggesting that it could be an anticachectic agent that induces direct effects on cardiac muscles.

Withaferin A Inhibits Liver Cancer Tumorigenesis by Suppressing Aerobic Glycolysis through the p53/IDH1/HIF-1α Signaling Axis.

BACKGROUND: The energy supply of certain cancer cells depends on aerobic glycolysis rather than oxidative phosphorylation. Our previous studies have shown that withaferin A (WA), a lactone compound derived from Withania somnifera, suppresses skin carcinogenesis at least partially by stabilizing IDH1 and promoting oxidative phosphorylation. Here, we have extended our studies to evaluate the anti-tumor effect of WA in liver cancer. METHODS: Differential expression of glycolysis-related genes between liver cancer tissues and normal tissues and prognosis were verified using an online database. Glycolysis-related protein expression was detected using western blot after overexpression and knockdown of IDH1 and mitochondrial membrane potential assay based on JC-1, and mitochondrial complex I activity was also detected. The inhibitory effect of WA on the biological functions of HepG2 cells was detected along with cell viability using MTT assay, scratch assay, clone formation assay, glucose consumption and lactate production assay. Western blot and qRT-PCR were used to detect the expression of proteins and genes related to IDH1, p53 and HIF1α signaling pathways. RESULTS: We first identified that IDH1 expression was downregulated in human liver cancer cells compared to normal liver cells. Next, we found that treatment of HepG2 cells with WA resulted in significantly increased protein levels of IDH1, accompanied by decreased levels of several glycolytic enzymes. Furthermore, we found that WA stabilized IDH1 proteins by inhibiting the degradation by the proteasome. The tumor suppressor p53 was also upregulated by WA treatment, which played a critical role in the upregulation of IDH1 and downregulation of the glycolysis-related genes. Under hypoxic conditions, glycolysis-related genes were induced, which was suppressed by WA treatment, and IDH1 expression was still maintained at higher levels under hypoxia. CONCLUSION: Taken together, our results indicated that WA suppresses liver cancer tumorigenesis by p53-mediated IDH1 upregulation, which promotes mitochondrial respiration, thereby inhibiting the HIF-1α pathway and blocking aerobic glycolysis.

A novel combinatorial approach using sulforaphane- and withaferin A-rich extracts for prevention of estrogen receptor-negative breast cancer through epigenetic and gut microbial mechanisms.

Estrogen receptor-negative [ER(-)] mammary cancer is the most aggressive type of breast cancer (BC) with higher rate of metastasis and recurrence. In recent years, dietary prevention of BC with epigenetically active phytochemicals has received increased attention due to its feasibility, effectiveness, and ease of implementation. In this regard, combinatorial phytochemical intervention enables more efficacious BC inhibition by simultaneously targeting multiple tumorigenic pathways. We, therefore, focused on investigation of the effect of sulforaphane (SFN)-rich broccoli sprouts (BSp) and withaferin A (WA)-rich Ashwagandha (Ash) combination on BC prevention in estrogen receptor-negative [ER(-)] mammary cancer using transgenic mice. Our results indicated that combinatorial BSp + Ash treatment significantly reduced tumor incidence and tumor growth (~ 75%) as well as delayed (~ 21%) tumor latency when compared to the control treatment and combinatorial BSp + Ash treatment was statistically more effective in suppressing BC compared to single BSp or Ash intervention. At the molecular level, the BSp and Ash combination upregulated tumor suppressors (p53, p57) along with apoptosis associated proteins (BAX, PUMA) and BAX:BCL-2 ratio. Furthermore, our result indicated an expressional decline of epigenetic machinery HDAC1 and DNMT3A in mammary tumor tissue because of combinatorial treatment. Interestingly, we have reported multiple synergistic interactions between BSp and Ash that have impacted both tumor phenotype and molecular expression due to combinatorial BSp and Ash treatment. Our RNA-seq analysis results also demonstrated a transcriptome-wide expressional reshuffling of genes associated with multiple cell-signaling pathways, transcription factor activity and epigenetic regulations due to combined BSp and Ash administration. In addition, we discovered an alteration of gut microbial composition change because of combinatorial treatment. Overall, combinatorial BSp and Ash supplementation can prevent ER(-) BC through enhanced tumor suppression, apoptosis induction and transcriptome-wide reshuffling of gene expression possibly influencing multiple cell signaling pathways, epigenetic regulation and reshaping gut microbiota.

Leptin combined with withaferin A boost posthemorrhagic neurogenesis via activation of STAT3/SOCS3 pathway.

Neurogenesis as a potential strategy to improve the consequences of intracerebral hemorrhage (ICH). The current study investigates the effects of withaferin A (WFA) in combination with leptin (LEP) on ICH and neurogenesis mechanisms. LEP levels were dramatically reduced on days 7 and 14 following ICH insults in mice, but continuous WFA therapy significantly improved the potency of intrinsic LEP on day 14 after ICH. Furthermore, WFA combined with LEP enhances intrinsic neurogenesis and lessen motor deficits and long-term cognitive outcomes after ICH. In parallel, leptin deficiency in ob/ob mice limits enhancement of neurogenesis following ICH in response to WFA combined with LEP treatment. Importantly, the functional recovery conferred by WFA combined with LEP after ICH was inhibited by neurogenesis suppression. Mechanistically, this study unveiled that the signal transducer and activator of transcription-3 (STAT3) / suppressor of cytokine signaling-3 (SOCS3) pathway is a critical signaling pathway through which WFA combined with LEP treatment promotes intrinsic neurogenesis after ICH. Collectively, the results of this study elucidate the neuroprotective effects of WFA and LEP in ICH, and highlight a potential approach for ICH cell therapy.

Novel Withanolides from Tubocapsicum anomalum Suppress Triple-Negative Breast Cancer by Triggering Apoptosis and p53-ASCT2-SLC7A11-Mediated Ferroptosis.

Triple-negative breast cancer (TNBC) is a malignant breast cancer. There is an urgent need for effective drugs to be developed for TNBC. Tubocapsicum anomalum (T. anomalum) has been reported to have an anti-tumor effect, and six novel withanolides were isolated from it and designated as TAMEWs. However, its anti-TNBC effect is still unknown. The results of an MTT assay indicated a higher sensitivity of TNBC cells to TAMEWs compared to other cells. TAMEWs induced apoptosis via mitochondrial dysfunction. They caused increased levels of lipid ROS and Fe2+, with downregulation of GSH and cystine uptake, and it has been confirmed that TAMEWs induced ferroptosis. Additionally, the results of Western blotting indicate that TAMEWs significantly decrease the expressions of ferroptosis-related proteins. Through further investigation, it was found that the knockdown of the p53 gene resulted in a significant reversal of ferroptosis and the expressions of its associated proteins SLC7A11, ASCT2, and GPX4. In vivo, TAMEWs suppressed TNBC growth with no obvious damage. The IHC results also showed that TAMEWs induced apoptosis and ferroptosis in vivo. Our findings provide the first evidence that TAMEWs suppress TNBC growth through apoptosis and ferroptosis.

The Potential Pharmacological Effects of Natural Product Withaferin A in Cancer: Opportunities and Challenges for Clinical Translation.

Cancer is one of the biggest health concerns with a complex pathophysiology. Currently, available chemotherapeutic drugs are showing deleterious side effects, and tumors often show resistance to treatment. Hence, extensive research is required to develop new treatment strategies to fight against cancer. Natural resources from plants are at the forefront of hunting novel drugs to treat various types of cancers. Withaferin A (WA) is a naturally occurring withanolide, a biologically active component obtained from the plant Ashwagandha. Various in vitro and in vivo oncological studies have reported that Withaferin A (WA) has shown protection from cancer. WA shows its activity by inhibiting the growth and proliferation of malignant cells, apoptosis, and inhibiting angiogenesis, metastasis, and cancer stem cells (CSCs). In addition, WA also showed chemo- and radio-sensitizing properties. Besides the beneficiary pharmacological activities of WA, a few aspects like pharmacokinetic properties, safety, and toxicity studies are still lacking, hindering this potent natural product from entering clinical development. In this review, we have summarized the various pharmacological mechanisms shown by WA in in vitro and in vivo cancer studies and the challenges that must be overcome for this potential natural product's clinical translation to be effective.

Combined untargeted metabolomics and network pharmacology approaches to reveal the therapeutic role of withanolide B in psoriasis.

Psoriasis is a refractory inflammatory skin disorder in which keratinocyte hyperproliferation is a crucial pathogenic factor. Up to now, it is commonly acknowledged that psoriasis has a tight connection with metabolic disorders. Withanolides from Datura metel L. (DML) have been proved to possess anti-inflammatory and anti-proliferative properties in multiple diseases including psoriasis. Withanolide B (WB) is one of the abundant molecular components in DML. However, existing experimental studies regarding the potential effects and mechanisms of WB on psoriasis still remain lacking. Present study aimed to integrate network pharmacology and untargeted metabolomics strategies to investigate the therapeutic effects and mechanisms of WB on metabolic disorders in psoriasis. In our study, we observed that WB might effectively improve the symptoms of psoriasis and alleviate the epidermal hyperplasia in imiquimod (IMQ)-induced psoriasis-like mice. Both network pharmacology and untargeted metabolomics results suggested that arachidonic acid metabolism and arginine and proline metabolism pathways were linked to the treatment of psoriasis with WB. Meanwhile, we also found that WB may affect the expression of regulated enzymes 5-lipoxygenase (5-LOX), 12-LOX, ornithine decarboxylase 1 (ODC1) and arginase 1 (ARG1) in the arachidonic acid metabolism and arginine and proline metabolism pathways. In summary, this paper showed the potential metabolic mechanisms of WB against psoriasis and suggested that WB would have greater potential in psoriasis treatment.

Withaferin A, a natural thioredoxin reductase 1 (TrxR1) inhibitor, synergistically enhances the antitumor efficacy of sorafenib through ROS-mediated ER stress and DNA damage in hepatocellular carcinoma cells.

BACKGROUND: Sorafenib (Sora), a multi-target tyrosine kinase inhibitor, is widely recognized as a standard chemotherapy treatment for advanced hepatocellular carcinoma (HCC). However, drug resistance mechanisms hinder its anticancer efficacy. Derived from Withania somnifera, Withaferin A (WA) exhibits remarkable anti-tumor properties as a natural bioactive compound. This study aimed to examine the mechanisms that underlie the impacts of Sora and WA co-treatment on HCC. METHODS: Cell proliferation was evaluated through colony formation and MTT assays. Flow cytometry was employed to determine cellular apoptosis and reactive oxygen species (ROS) levels. The evaluation of apoptosis-related protein levels, DNA damage, and endoplasmic reticulum stress was conducte utilizing IHC staining and western blotting. Moreover, the caspase inhibitor Z-VAD-FMK, ATF4 siRNA, ROS scavenger N-acetyl cysteine (NAC), and TrxR1 shRNA were used to elucidate the underlying signaling pathways. To validate the antitumor effects of Sora/WA co-treatment, in vivo experiments were ultimately executed using Huh7 xenografts. RESULTS: Sora/WA co-treatment demonstrated significant synergistic antitumor impacts both in vivo and in vitro. Mechanistically, the enhanced antitumor impact of Sora by WA was achieved through the inhibition of TrxR1 activity, resulting in ROS accumulation. Moreover, ROS generation induced the activation of DNA damage and endoplasmic reticulum (ER) stress pathways, eventually triggering cellular apoptosis. Pre-treatment with the antioxidant NAC significantly inhibited ROS generation, ER stress, DNA damage, and apoptosis induced by Sora/WA co-treatment. Additionally, the inhibition of ATF4 by small interfering RNA (siRNA) attenuated Sora/WA co-treatment-induced apoptosis. In vivo, Sora/WA co-treatment significantly suppressed tumor growth in HCC xenograft models and decreased TrxR1 activity in tumor tissues. CONCLUSION: Our study suggests that WA synergistically enhances the antitumor effect of Sora, offering promising implications for evolving treatment approaches for HCC.

Phyto-therapeutic potential of Withania somnifera: Molecular mechanism and health implications.

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.

A Homodimer of Withaferin A Formed by Base-Promoted Elimination of Acetic Acid from 27-O-Acetylwithaferin A Followed by a Diels-Alder Reaction.

Treatment of 27-O-acetylwithaferin A (2) with the non-nucleophilic base, 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), afforded 5ß,6ß-epoxy-4ß-hydroxy-1-oxo-witha-2(3),23(24),25(27)-trienolide (3) and 4, a homodimer of withaferin A resulting from a Diels-Alder [4 + 2] type cycloaddition of the intermediate α,ß-dimethylene-δ-lactone (9). Structures of 3 and 4 were elucidated using HRMS and 1D and 2D NMR spectroscopic data. The structure of 4 was also confirmed by single crystal X-ray crystallographic analysis of its bis-4-O-p-nitrobenzoate (8). Formation of withaferin A homodimer (4) as the major product suggests regio- and stereoselectivity of the Diels-Alder [4 + 2] cycloaddition reaction of 9. Acetylation of 2-4 afforded their acetyl derivatives 5-7, respectively. Compounds 2-4 and 6-8 were evaluated for their cytotoxic activities against four prostate cancer (PC) cell lines (LNCaP, 22Rv1, DU-145, and PC-3) and normal human foreskin fibroblast (HFF) cells. Significantly, 4 exhibited improved activity compared to the other compounds for most of the tested cell lines.

Enhanced production of withaferin A from the hairy root culture of Withania somnifera via synergistic effect of Methyl jasmonate and ß-cyclodextrin.

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of ß-cyclodextrin (ß-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (µ = 0.036 day-1) and growth index (GI = 9.18), and the shortest doubling time (Td = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM ß-CD + 100 µM MeJA (9.57 mg/g DW) and 5.0 mM ß-CD + 100 µM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined ß-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and ß-CD on withaferin A production.

Withanolides from the active extract of Physalis angulate and their anti-hepatic fibrosis effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalis angulata L., a traditional Chinese medicine called "Kuzhi" in China, was used traditionally to treat liver diseases (eg. icterus, hepatitis) as well as malaria, asthma, and rheumatism. AIM OF THE STUDY: Our study aimed to investigate the withanolides with anti-hepatic fibrosis effect from P. angulate. MATERIALS AND METHODS: Withanolides were obtained from the EtOH extract of P. angulate by bioassay-molecular networking analysis-guided isolation using column chromatography and normal/reversed-phase semipreparative HPLC. The structures of new withanolides were elucidated by combinations of spectroscopic techniques with NMR and ECD calculations. MTT cell viability assay, AO/EB staining method, cell wound healing assay, ELISA and Western blot experiments were employed to evaluate the anti-hepatic fibrosis activity and to uncover related mechanism. Molecular docking analysis and cellular thermal shift assay were used to evaluate and verify the interaction between the active withanolides and their potential targets. RESULTS: Eight unreported withanolides, withagulides A-H (1-8), along with twenty-eight known ones were obtained from P. angulate. Withanolides 6, 9, 10, 24, 27, and 29-32 showed marked anti-hepatic fibrosis effect with COL1A1 expression inhibition above 50 %. Physalin F (9), the main component in the active fraction, significantly decreased the TGF ß1-stimulated expressions of collagen I and α-SMA in LX-2 cells. Mechanism study revealed that physalin F exerted its anti-hepatic fibrosis effect via the PI3K/AKT/mTOR signaling pathway. CONCLUSION: This study suggested that withanolides were an important class of natural products with marked anti-hepatic fibrosis effect. The main withanolide physalin F might be a promising candidate for hepatic fibrosis treatment. The work provided experimental foundation for the use of P. angulate to treat hepatic fibrosis.

Withanolides: Promising candidates for cancer therapy.

Natural products have played a significant role throughout history in the prevention and treatment of numerous diseases, particularly cancers. As a natural product primarily derived from various medicinal plants in the Withania genus, withanolides have been shown in several studies to exhibit potential activities in cancer treatment. Consequently, understanding the molecular mechanism of withanolides could herald the discovery of new anticancer agents. Withanolides have been studied widely, especially in the last 20 years, and attracted the attention of numerous researchers. Currently, over 1200 withanolides have been classified, with approximately a quarter of them having been reported in the literature to be able to modulate the survival and death of cancer cells through multiple avenues. To what extent, though, has the anticancer effects of these compounds been studied? How far are they from being developed into clinical drugs? What are their potential, characteristic features, and challenges? In this review, we elaborate on the current knowledge of natural compounds belonging to this class and provide an overview of their natural sources, anticancer activity, mechanism of action, molecular targets, and implications for anticancer drug research. In addition, direct targets and clinical research to guide the design and implementation of future preclinical and clinical studies to accelerate the application of withanolides have been highlighted.

Withania somnifera root extract inhibits MGO-induced skin fibroblast cells dysfunction via ECM-integrin interaction.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal, known as Ashwagandha, has long been used in traditional medicine in Ayurveda, India, a representative adaptogen. The main active constituents of W. somnifera are withanolides, and the root is often used as a medicine with a wide range of pharmacological activities, which can be used to treat insomnia, neurasthenia, diabetes mellitus and skin cancer. AIM OF THE STUDY: Whole-component qualitative and quantitative analyses were performed on W. somnifera. We explored the ameliorative effect of the adaptogen representative plant W. somnifera on the senescence events of MGO-injured fibroblasts and its action mechanism and verified the hypotheses that WS can inhibit the accumulation of AGEs and regulate the dynamic balance among the components of the ECM by modulating the expression of integrin ß1 receptor; as a result, WS maintains cellular behavioural and biological functions in a normal range and retards the aging of skin from the cellular level. MATERIALS AND METHODS: In this study, the components of WS were first qualitatively and quantitatively analysed by HPLC fingerprinting and LC-MS detection. Second, a model of MGO-induced injury of CML-overexpressing fibroblasts was established. ELISA was used to detect CML expression and the synthesis of key extracellular matrix ECM protein components COL1, FN1, LM5 and TNC synthesis; CCK-8 was used to detect cell viability; EDU was used to detect cell proliferation capacity; fluorescence was used to detect cell adhesion capacity; and migration assay were used to detect cell migration capacity; qRT-PCR was used to detect the regulatory pathway TGF-ß1 and MMP-2, MMP-9 in ECMs; immunofluorescence was used to detect the expression of ITGB1; and WB was used to detect the expression of COL1, FN1, LM5, Tnc, TGF-ß1, MMP-2, MMP-9 and ITGB1. RESULTS: In total, 27 active ingredients were analysed from WS, which mainly consisted of withanolide components, such as withaferin A and withanolide A. Based on the model of MGO-induced fibroblast senescence injury, WS significantly inhibited CML synthesis. By up-regulating the expression of integrin ß1, it upregulated the expression of the TGF-ß1 gene, which is closely related to the generation of ECMs, downregulated the expression of the MMP-2 and MMP-9 genes, which are closely related to the degradation of ECMs, maintained the dynamic balance of the four types of ECMs, and improved cell viability as well as proliferation, migration and adhesion abilities. CONCLUSIONS: WS can prevent cellular behavioural dysfunction and delay skin ageing by reducing the accumulation of CML, upregulating the expression of the ITGB1 receptor, maintaining the normal function of ECM-integrin receptor interaction and preventing an imbalance between the production and degradation of protein components of ECMs. The findings reported in this study suggest that WS as a CML inhibitor can modulate ECM-integrin homeostasis and has great potential in the field of aging retardation.

Study of Drug Targets Associated With Oncogenesis and Cancer Cell Survival and the Therapeutic Activity of Engineered Ashwagandha Extract Having Differential Withanolide Constitutions.

Ashwagandha (Withania somnifera) has gained worldwide popularity for a multitude of health benefits inclusive of cancer-preventive and curative effects. Despite numerous research data supporting the benefits of this wonder herb, the actual use of ashwagandha for cancer treatment in clinics is limited. The primary reason for this is the inconsistent therapeutic outcome due to highly variable composition and constitution of active ingredients in the plant extract impacting ashwagandha's pharmacology. We investigate here an engineered yield: an ashwagandha extract (Oncowithanib) that has a unique and fixed portion of active ingredients to achieve consistent and effective therapeutic activity. Using the MCF7 cell line, Oncowithanib was studied for its anti-neoplastic efficacy and drug targets associated with cell cycle regulation, translation machinery, and cell survival and apoptosis. Results demonstrate a dose-dependent decline in Oncowithanib-treated MCF7 cell viability and reduced colony-forming ability. Treated cells showed increased cell death as evidenced by enhancement of Caspase 3 enzyme activity and decreased expressions of cell proliferation markers such as Ki67 and Aurora Kinase A. Oncowithanib treatment was also found to be associated with expressional suppression of key cellular kinases such as RSK1, Akt1, and mTOR in MCF7 cells. Our findings indicate that Oncowithanib decreases MCF7 cell survival and propagation, and sheds light on common drug targets that might be good candidates for the development of cancer therapeutics. Further in-depth investigations are required to fully explore the potency and pharmacology of this novel extract. This study also highlights the importance of the standardization of herbal extracts to get consistent therapeutic activity for the disease indication.

Withanolides from Physalis angulata var. villosa and the Relative Configurational Revision of Some Known Analogs.

Physalis angulata var. villosa is a plant possessing abundant withanolides, but in-depth research is lacking. In our ongoing study of P. angulata var. villosa, 15 previously undescribed withanolides (1-15), along with 21 known analogs (16-36), were isolated from the whole plant. The structures of the withanolides (1-15) were elucidated based on analysis of their 1D and 2D NMR, HRESIMS, and ECD data. Additionally, the application of γ-gauche effects with the help of ROESY correlations led to the formulation of empirical rules for withanolides with 14-OH/15-OAc to rapidly determine the 14-OH orientations, making it possible to propose configurational revisions of 19 previously reported analogs (1'-19'). Withanolides 1, 4-6, and 10 showed potent cytotoxic activities against three human cancer cell lines (HCT-116, MDA-MB-231, and A549).

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.

Screening of alkaloids and withanolides isolated from Solanaceae plants for antifungal properties against non-wild type Sporothrix brasiliensis.

Antifungal resistance has often been found in animal sporotrichosis in Southern Brazil. The biological potential of compounds from plants of the Solanaceae family against infectious diseases is known, however, it is still unknown against Sporothrix brasiliensis. This study evaluated the anti-Sporothrix brasiliensis activity, synergism, cytotoxicity, and action mechanism of steroidal lactones (withanolides) and alkaloids isolated from these plants. Pure compounds of withanolide D (WNOD), physalin F (PHYF), withanicandin (WNIC), nicandin B (NICB), solasonine (SSON), and solamargine (SMAR) were tested against 12 Sporothrix brasiliensis isolated from cats (n = 11) and dogs (n = 2) through M38-A2 CLSI. For the compounds with the best activity, a checkerboard assay for synergism, sorbitol protection, and ergosterol effect for action mechanism; and MTT test for cytotoxicity were performed. The withanolides WNOD, PHYF, WNIC, and NICB were not antifungal, but SSON (MIC 0.125-1 mg/mL) and SMAR (MIC 0.5-1 mg/mL) were both fungistatic and fungicidal (MFC 0.5-1 mg/mL for both) against wild-type (WT) and non-WT isolates. The activity of SSON and SMAR was indifferent when combined with itraconazole. In the mechanism of action, cell wall and plasma membrane by complexation with ergosterol seemed to be two target structures of SSON and SMAR. SSON was selected for cytotoxicity, whose cell viability in MDBK cells ranged from 28.85 % to 101.75 %, and was higher than 87.49 % at concentrations ≤0.0015 mg/ml. Only the steroidal alkaloids SSON and SMAR were active against non-WT isolates, being promising antifungal candidates for the treatment of feline and canine sporotrichosis with low susceptibility to itraconazole.

Cytotoxic withanolides from the stems and leaves of Physalis ixocarpa.

The stems and leaves of the tomatillo (Physalis ixocarpa or Physalis philadelphica) were considered agricultural waste during the processing of tomatillo fruits. However, their potential value for utilization has not yet been explored. The investigation resulted in the isolation of a total of 29 withanolides, out of which 15 never reported. These newly discovered withanolides were then tested for their cytotoxicity against eight different human tumor cell lines. Compounds 2-3, 6-7, 17, 19, and 25-27 displayed encouraging cytotoxic effects. Given the potent inhibitory activity of physagulin C (25) on the proliferation of HepG2 cells in vitro, further investigation was conducted to determine its molecular mechanism. Physagulin C inhibited epithelial-mesenchymal transition (EMT) process through the down-regulation of the JAK2/STAT3 and PI3K/AKT/mTOR pathways. Withanolides presenting in the stems and leaves of tomatillo make the plant possess potential commercial importance. Therefore, tomatillos could be commercialized worldwide in the food and pharmaceutical industries.