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.

Physalin H ameliorates LPS-induced acute lung injury via KEAP1/NRF2 axis.

Physalin H (PH), a withanolide isolated from Physalisangulata 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-1ß, 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.

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.

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.

Pharmacokinetics and bioequivalence of Withania somnifera (Ashwagandha) extracts - A double blind, crossover study in healthy adults.

Introduction: Withania somnifera (WS) or ashwagandha is an adaptogenic plant used extensively in traditional medicines and as a food supplement. Despite a long history of use and numerous clinical trials, the human pharmacokinetics of withanolides, the active phytochemicals in WS extracts, have not been fully evaluated. This study evaluated the oral pharmacokinetics and bioequivalence of active withanolides in human plasma after administration of a single dose of two commercial ashwagandha extracts containing equal amounts of total withanolides. Methods: This randomized, double-blind, single-dose crossover study of 16 healthy human volunteers evaluated the acute oral bioavailability of withanolides and the bioequivalence of two WS extracts, WS-35 and WS-2.5. WS-35 was standardized to total withanolides not less than 40% comprising not less than 35% withanolide glycosides and WS-2.5 was standardized to 2.5% withanolides. The clinical dosages were normalized to 185 mg of total withanolide in each extract at the bioequivalent dosages. The pharmacokinetic parameters of withanolide A, withanoside IV, withaferin A, and total withanolides were quantified in the blood plasma using a validated LC-MS/MS method. Results: The half-life, C-max, and mean residence time of the total withanolides were 5.18, 5.62 and 4.13 times significantly higher and had lower systemic clearance with WS-35 than with WS-2.5 extract. Considering the plasma AUC 0-inf of total withanolides per mg of each WS extract administered orally, WS-35 was 280.74 times more bioavailable than WS-2.5. Conclusion: The results of this study highlight the importance of withanolide glycosides in improving the pharmacokinetics of WS extracts. Owing to its superior pharmacokinetic profile, WS-35, with 35% withanolide glycosides, is a promising candidate for further studies on Withania somnifera. Clinical trial registration: CTRI/2020/10/028397 [registered on:13/10/2020] (Trial prospectively registered) http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=42149&EncHid=&userName=CTRI/2020/10/028397.

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.

Estimating the production of withaferin A and withanolide A in Withania somnifera (L.) dunal using aquaponics for sustainable development in hill agriculture.

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.

Antibacterial activity of withanolides and their structure-activity relationship.

Two new withanolides, (17R,20S,22R)-4ß-acetoxy-5ß,6ß-epoxy-19,27-dihydroxy-1-oxo-witha-2,24-dienolide (withalongolide A 4-acetate (5) and (17R,20S,22R)-5ß,6ß-epoxy-27-hydroxy-1,4-dioxo-witha-24-enolide (9), and seven known withanolides with normal structure (1-4, 6-8) were isolated from aerial parts of Cuatresia colombiana. Several semisynthetic derivatives were prepared from the natural metabolites withaferin A and jaborosalactone 38. The compounds were fully characterized by a combination of spectroscopic methods (1D and 2D NMR and MS). The compounds isolated from C. colombiana, sixteen withanolides previously isolated from different Solanaceae species with different skeletons and semisynthetic derivatives were evaluated for their antibacterial activity against a selected panel of Gram-positive and Gram-negative bacteria. According to the bioactivity against S. aureus and E. faecalis, the compounds evaluated were divided into three groups: compounds with high activity (MIC 0.063 mM), compounds with moderate activity (0.5 mM > MIC > 0.125 mM) and non-active compounds (MIC ≥1 mM); in addition, some structure-activity relationship keys could be inferred.

Withania somnifera extracts induced attenuation of HIV-1: a mechanistic approach to restrict viral infection.

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.

Golden berry leaf extract containing withanolides suppresses TNF-α and IL-17 induced IL-6 expression in HeLa Cells.

Inflammation, characterized by the overexpression of IL-6 in various tissues, has been reported as a symptom of coronavirus disease 2019. In this study, we established an experimental system for overexpression of IL-6 in HeLa cells stimulated by TNF-α and IL-17, along with identification of anti-inflammatory materials and components from local agricultural, forestry, and fishery resources. We constructed a library of extracts from natural sources, of which 111 samples were evaluated for their anti-inflammatory activities. The MeOH extract of Golden Berry (Physalis peruviana L) leaf was found to exhibit strong anti-inflammatory properties (IC50 = 4.97 µg/mL). Preparative chromatography identified two active constituents, 4ß-hydroxywithanolide E (4ß-HWE) (IC50 = 183 nM) and withanolide E (WE) (IC50 = 65.1 nM). Withanolides are known anti-inflammatory ingredients of Withania somnifera, an Ayurvedic herbal medicine. P. peruviana leaves containing 4ß-HWE and WE should be considered as useful natural resources for anti-inflammatory products.

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 promising chemotherapeutic candidates from plants against human adult T-cell leukemia/lymphoma (VIII): six new withanolides from Physalis philadelphica.

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-4ß, 16ß, 20R-trihydroxy-1-oxowitha-2-en-5ß, 6 ß -epoxy-22,26-olide (1), 4ß, 7ß,20R-trihydroxy-1-oxowitha-2-en-5ß, 6ß -epoxy-22,26-olide (2), 17ß,20 S-dihydroxywithanone (3), 2,3-dihydro-3ß-methoxy-23ß-hydroxywithaphysacarpin (4), 3-O-(4-rhamnosyl)glucosyl-physalolactone B (5), and 17R, 20R, 22S, 23S, 24R, 25R-4ß, 5α, 6ß, 20ß, 22α -tetrahydroxy-16ß, 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.

Physapruin A Exerts Endoplasmic Reticulum Stress to Trigger Breast Cancer Cell Apoptosis via Oxidative Stress.

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.

Talasteroid, a new withanolide from the marine-derived fungus Talaromyces stollii.

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.

Molecular dynamics simulations reveal the inhibitory mechanism of Withanolide A against α-glucosidase and α-amylase.

Diabetes mellitus (DM) is a global chronic disease characterized by hyperglycemia and insulin resistance. The unsavory severe gastrointestinal side-effects of synthetic drugs to regulate hyperglycemia have warranted the search for alternative treatments to inhibit the carbohydrate digestive enzymes (e.g. α-amylase and α-glucosidase). Certain phytochemicals recently captured the scientific community's attention as carbohydrate digestive enzyme inhibitors due to their low toxicity and high efficacy, specifically the Withanolides-loaded extract of Withania somnifera. That said, the present study evaluated in silico the efficacy of Withanolide A in targeting both α-amylase and α-glucosidase in comparison to the synthetic drug Acarbose. Protein-ligand interactions, binding affinity, and stability were characterized using pharmacological profiling, high-end molecular docking, and molecular-dynamic simulation. Withanolide A inhibited the activity of α-glucosidase and α-amylase better, exhibiting good pharmacokinetic properties, absorption, and metabolism. Also, Withanolide A was minimally toxic, with higher bioavailability. Interestingly, Withanolide A bonded well to the active site of α-amylase and α-glucosidase, yielding the lowest binding free energy of -82.144 ± 10.671 kcal/mol and -102.1043 ± 11.231 kcal/mol compared to the Acarbose-enzyme complexes (-63.220 ± 13.283 kcal/mol and -82.148 ± 10.671 kcal/mol). Hence, the findings supported the therapeutic potential of Withanolide A as α-amylase and α-glucosidase inhibitor for DM treatment.Communicated by Ramaswamy H. Sarma.

Antitumor Potential of Withanolide Glycosides from Ashwagandha (Withania somnifera) on Apoptosis of Human Hepatocellular Carcinoma Cells and Tube Formation in Human Umbilical Vein Endothelial Cells.

Hepatocellular carcinoma (HCC) is the fastest-growing tumor capable of spreading to other organs via blood vessels formed by endothelial cells. Apoptosis and angiogenesis-targeting therapies are attractive for cancer treatment. In this study, we aimed to study the in vitro cytotoxicity of Withania somnifera against human HCC (HepG2) cells, identify potential antitumoral withanolide glycosides from the active fraction, and elucidate cytotoxic molecular mechanisms of identified bioactive compounds. W. somnifera (Solanaceae), well-known as 'ashwagandha', is an Ayurvedic medicinal plant used to promote health and longevity, and the MeOH extract of W. somnifera root exhibited cytotoxicity against HepG2 cells during initial screening. Bioactivity-guided fractionation of the MeOH extract and subsequent phytochemical investigation of the active n-BuOH-soluble fraction resulted in the isolation of five withanolide glycosides (1-5), including one new metabolite, withanoside XIII (1), aided by liquid chromatography-mass spectrometry-based analysis. The new compound structure was determined by 1D and 2D nuclear magnetic resonance spectroscopy, high-resolution electrospray ionization mass spectroscopy, electronic circular dichroism, and enzymatic hydrolysis. In addition, withanoside XIIIa (1a) was identified as the new aglycone (1a) of 1. Isolated withanolide glycosides 1-5 and 1a were cytotoxic toward HepG2 cells; withagenin A diglucoside (WAD) (3) exhibited the most potent cytotoxicity against HepG2 cells, with cell viability less than 50% at 100 µM. WAD cytotoxicity was mediated by both extrinsic and intrinsic apoptosis pathways. Treatment with WAD increased protein expression levels of cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, Bcl-2-associated X protein (Bax), and cleaved poly(ADP-ribose) polymerase (cleaved PARP) but decreased expression levels of B-cell lymphoma 2 (Bcl-2). Moreover, WAD inhibited tubular structure formation in human umbilical vein endothelial cells (HUVECs) by inhibiting the protein expression of vascular endothelial growth factor receptor 2 and its downstream pathways, including extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). These effects were also enhanced by co-treatment with ERK and PI3K inhibitors. Overall, these results indicate that WAD (3) induced HepG2 apoptosis and inhibited HUVEC tube formation, suggesting its potential application in treating liver cancers.

Differential Gene Expression and Withanolides Biosynthesis During in vitro and ex vitro Growth of Withania somnifera (L.) Dunal.

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.

High performance thin layer chromatography based chemo profiling of Ashvagandharishta and its antidepressant activity.

Withania somnifera (L.) has long been used as a traditional rasayana herb against a variety of human ailments. This research presents a high performance thin layer chromatography based chemo profiling of Ashvagandharishta and its antidepressant activity. The in-house formulation was made using a fermentation process according to the Indian Pharmacopoeia. Physiochemical standardization of the formulation was performed using different quality control parameters such as total ash, acid insoluble ash, alcohol soluble extract value and water soluble extract value. A column chromatography and high performance thin layer chromatography method was used to isolate and estimation of withanolide-A, withaferin-A & ß-sitosterol from the root of W. somnifera. In addition. The antidepressant effect of different formulations were carried out by force swimming test in albino mice. The thiobarbituric acid reactive substances (TBARS) and Glutathione (GSH) assay was used to find out the oxidative stress. W. somnifera root has been standardized macroscopically, microscopically, physico-chemically according to the guidelines of the Ayurvedic Pharmacopoeia. The qualitative and quantitative analysis was performed using high performance thin layer chromatography and it was performed on each formulation and found the content of withanolide-A and -sitosterol in the in-house formulation is higher while withaferin-A is rather contained in the decoction. The antidepressant effect showed that the immobility time was lowest in the case of the standard formulation followed by in house formulation, while the increase in glutathione and the reduction in thiobarbituric acid reactive substances levels revealed the antioxidant nature of the formulation. In conclusion, based on the above results, we can conclude that Ashvagandharishta could be a breakthrough for the treatment of depression in the future.

Cloning and homologous characterization of geranylgeranyl pyrophosphate synthase (GGPPS) from Withania somnifera revealed alterations in metabolic flux towards gibberellic acid biosynthesis.

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.