Tubocapsicum anomalum, a Chinese medicinal plant rich in anti-tumor withanolides, requires efficient extraction methods. In this paper, an HPLC method was first established for the detection of withanolides, and gradient elution was carried out using a methanol-water solvent system. It was found that the content of withanolides was the highest in the leaves of T. anomalum, followed by the stems and fruits, and almost none in the roots. During the actual picking process, the quantity of leaves collected was relatively small, while the number of stems was the highest. Therefore, the Box-Behnken response surface method was used to optimize the ultrasonic-assisted extraction process of withanolides from the stems of T. anomalum. The optimal extraction conditions were determined as follows: the liquid-solid ratio was 20:1, the extraction solvent was 70 % ethanol, the ultrasonic power was 250 W, the ultrasonic time was 40 min, and the ultrasonic temperature was 50 °C. Under these conditions, the average yields of tubocapsenolide A (Te-A) and tubocapsanolide A (Ta-A) can reach 2.87 ± 0.12 mg/g and 1.18 ± 0.05 mg/g, respectively. We further compared extraction rates of two withanolides from different parts of T. anomalum using ultrasonic and traditional extraction methods. Ultrasonic extraction significantly increased rates, with the highest yields from leaves, followed by stems and fruits. The results show that ultrasonic optimization can improve extraction rate, reduce time, lower costs, enhance quality, and increase yield. Therefore, the optimized ultrasonic-assisted extraction process was adopted to extract the aerial parts of T. anomalum and separate the components. After optimization, the extract underwent several chromatographic separations to isolate eight previously undescribed withanolides (1-8) and two artificial withanolides (9-10), in addition to fifteen known compounds (11-25). Their structures were established through extensive spectroscopic data analysis. The compounds were evaluated for their antiproliferative effects against multiple cancer cell lines, including human hepatocellular carcinoma cells (HepG2, Hep3B, and MHCC97-H), human lung cancer cells (A549), human fibro-sarcoma cancer cells (HT1080), human chronic myeloid leukemia cells (K562), and human breast cancer cells (MDA-MB-231 and MCF7). Compounds 1-3, 5, 7, 11, 13, 15-16, and 22 displayed significant activity with IC50 values of 5.14-19.87 μM. The above results indicate that ultrasonic-assisted extraction technology can be used to obtain new withanolides more efficiently from T. anomalum, thereby enhancing the utilization rate of T. anomalum resources.

Withania somnifera (W. somnifera) has a long history of safety in the amelioration of neuro-active ailments. The current study aims to explore Withania somnifera phyto-active principle anti-microbial, ant-neuropathic, and anti-inflammatory activities, and to modify these activities utilizing nano-cubosomes exploiting their mechanisms of action. Bio-guided fractionation technique was utilized, to identify the most phyto-active compound, using LC-MS-NMR online technique and biological models of diabetes, neuropathy, and inflammation. In-vitro antibacterial activity was also monitored. The HbA1c, in-vivo antioxidant (serum-catalase, TBARS, and GSH), serum insulin, and pro-inflammatory serum cytokines (TNF alpha, IL-six, and IL-ten) levels have been assessed to establish the anti-neuropathic and anti-inflammatory mechanisms. The nano-cubosomal formulations (CUB 1-3) were utilized to improve the W. somnifera most active compound efficacy. W. somnifera has shown ten major peaks; coagulin Q (10.2 %), dihydrowithanolide A (2.4 %), dihydrowithaferin D (1.8 %), physagulin D (7.6 %), withanoside V (2.3 %), withanolide A (WDA, 10.3 %), withafrin A (4.9 %), withaferin D (7.7 %), withanone 9 (9.9 %), withanolide D (4.8 %). The bio-guided fractionation technique utilizing LC-MS-NMR technique has proved that withanolide A (WDA) is the most phyto-active compound in W. somnifera. The latter has shown better results than WDA, which might be due to other effective compounds in Ws. However, CUB 3 (WDA nano-cubosomes dispersion) has shown more prominent anti-diabetic, anti-neuropathic, anti-inflammatory, and anti-bacterial potentials than Ws and WDA. Thus, CUB 3 modified WDA activity, and improved its efficacy. The normalization of HbA1c levels, increased insulin secretagogue potential, and the amelioration of the oxidative-stress may be the underlying Ws, WDA, and CUB 3 antidiabetic neuropathy mechanism. Moreover, the Ws, WDA, and CUB 1-3 anti-inflammatory mechanism might be due to the amelioration of the pro-inflammatory serum cytokines (decreasing TNF alpha and IL-six levels and increasing IL-ten). Thus, CUB 3 might be a powerful tool in augmenting Withania somnifera activity as an oral drug-delivery system and improving its efficacy against neuropathy and inflammation.

OBJECTIVE: Phytotherapy employs phytoconstituents/phytomedicines derived from plants for treating and preventing illnesses. Withania somnifera is known in the Indian Ayurveda Pharmacopoeia for its medicinal applications and pharmacological properties. In this study, we examined the biological activity spectrum of Withania somnifera methanolic extract of stem (WSME), which is valued as a \"Rasayana\" due to its extensive range of medicinal uses.
METHODS: WSME was subjected to TPC and TFC quantification and bioactive components were characterized using LC-MS. Its antioxidant potential was gauged by DPPH and H2O2 radical scavenging assays, while antibacterial efficacy was assessed against S. aureus and E. coli using disc diffusion assay. In vitro anticancer activity was evaluated against human breast cancer MDA-MB-231 cells while toxicity was evaluated against normal Vero cells using MTT assay.
RESULTS: WSME, rich in Withaferin A, showed TPC of 4.73 ± 0.15 mg GAE/g and TFC of 94.94 ± 6.15 mg QE/g dry weight of extract. It exhibited significant antioxidant activity (43.28 and 66.8 % inhibition at 1,000 μg/mL using DPPH and H2O2 assays, respectively) and mild antibacterial effects against S. aureus (300-500 mg/mL). WSME induced cell death in MDA-MB-231 cells and significantly inhibited their growth (IC50: 66 μg/mL, P value<0.05) without affecting normal Vero cells in the studied range of 25-400 μg/mL (IC50: 6.09 mg/mL, P value>0.05).
CONCLUSIONS: The present study lends support to further testing of WSME against other cancer cell lines and animal models of cancer. These preclinical studies would provide further validation to its prospective use as an adjunct in human breast cancer therapy.

Characterization of botanical extracts by mass spectrometry-based metabolomics analysis helps in determining the phytochemical composition that underlies their bioactivity and potential health benefits, while also supporting reproducibility of effects in clinical trials. The quantification of seven withanolides in Withania somnifera using three mass-spectrometry methods was evaluated using Deming regression. Two high-resolution time-of-flight mass spectrometry methods were used, one operating in data-dependent acquisition mode and the other in parallel-reaction-monitoring mode with an inclusion list. The two high-resolution time-of-flight mass spectrometry methods were compared to a multiple-reaction-monitoring method. We evaluated in-source fragmentation of steroidal glycosides and optimized the methods accordingly. A novel software approach to integrating parallel-reaction-monitoring data acquired with an inclusion list was developed. Combining and comparing quantitative results allowed for quantitative specificity, good precision, and adjustment of instrument source conditions for optimal quantification by multiple-reaction-monitoring mass spectrometry, an analytical method that is widely accessible in analytical and phytochemical laboratories.

Withanolides are a group of naturally occurring plant-based small molecules known for their wide range of host cellular functions. The anticancer potential of withanolides has been explored in varying cancer cell lines in vitro. Based on our prior studies, among the tested withanolides, withametelin (WM) has shown significant cytotoxicity with the highest efficacy on HCT-116 colon cancer cells (IC50 0.719 ± 0.12μM). Treatment with WM reduced the TGF-β driven proliferation, colony-forming ability, migration, and invasiveness of HCT-116 cells in vitro. WM also downregulated the expression of mesenchymal markers such as N-CADHERIN, SNAIL, and SLUG in HCT-116 cells. At the molecular level, WM inhibited TGF-β induced phosphorylation of SMAD2/3 and reduced the expression of an immune checkpoint inhibitor programmed-death ligand-1 (PD-L1). Our study highlights the possible anticancer mechanisms of WM involving modulation of the TGF-β pathway and associated target gene expression, suggesting its potential utility in cancer therapy.

BACKGROUND: Epilepsy is among the most frequent severe brain diseases, with few treatment options available. Neuronal ferroptosis is an important pathogenic mechanism in epilepsy. As a result, addressing ferroptosis appears to be a promising treatment approach for epilepsy. Withaferin A (WFA) is a C28 steroidal lactone that has a broad range of neuroprotective properties. Nonetheless, the antiepileptic action of WFA and the intrinsic mechanism by which it inhibits ferroptosis following epilepsy remain unknown.
OBJECTIVE: This study aimed at investigating to the antiepileptic potential of WFA in epilepsy, as well as to propose a potential therapeutic approach for epilepsy therapy.
METHODS: We conducted extensive research to examine the impacts of WFA on epilepsy and ferroptosis, using the kainic acid (KA)-treated primary astrocyte as an in vitro model and KA-induced temporal lobe epilepsy mice as an in vivo model. To analyze the neuroprotective effects of WFA on epileptic mice, electroencephalogram (EEG) recording, Nissl staining, and neurological function assessments such as the Morris water maze (MWM) test, Y-maze test, Elevated-plus maze (O-maze) test, and Open field test were used. Furthermore, the mechanism behind the neuroprotective effect of WFA in epilepsy was investigated using the transcriptomics analysis and verified on epileptic patient and epileptic mouse samples using Western blotting (WB) and immunofluorescence (IF) staining. In addition, WB, IF staining and specific antagonists/agonists were used to investigate astrocyte polarization and the regulatory signaling pathways involved. More critically, ferroptosis was assessed utilizing lipocalin-2 (LCN2) overexpression cell lines, siRNA knockdown, JC-1 staining, WB, IF staining, flow cytometry, electron microscopy (TEM), and ferroptosis-related GSH and MDA indicators.
RESULTS: In this study, we observed that WFA treatment reduced the number of recurrent seizures and time in seizure, and the loss of neurons in the hippocampal area in in epileptic mice, and even improved cognitive and anxiety impairment after epilepsy in a dose depend. Furthermore, WFA treatment was proven to enhance to the transformation of post-epileptic astrocytes from neurotoxic-type A1 to A2 astrocytes in both in vivo and in vitro experiments by inhibiting the phosphoinositide 3-kinase /AKT signaling pathway. At last, transcriptomics analysis in combination with functional experimental validation, it was discovered that WFA promoted astrocyte polarity transformation and then LCN2 in astrocytes, which inhibited neuronal ferroptosis to exert neuroprotective effects after epilepsy. In addition, we discovered significant astrocytic LCN2 expression in human TLE patient hippocampal samples.
CONCLUSIONS: Taken together, for the first, our findings suggest that WFA has neuroprotective benefits in epilepsy by modulating astrocyte polarization, and that LCN2 may be a novel potential target for the prevention and treatment of ferroptosis after epilepsy.

Physalis alkekengi L. is a valuable medicinal plant from the Solanaceae family and has multiple therapeutic applications. This study aimed to develop an optimized protocol for callogenesis in P. alkekengi to obtain friable calluses with high biomass. The effect of different concentrations of picloram, casein hydrolysate (CH), basal media (Murashige and Skoog (MS) and Gamborg (B5)), and static magnetic field (SMF) were investigated on the callus induction and growth, signaling molecules, and enzymatic and non-enzymatic antioxidants. Results showed that CH (200 mgL-1) and SMF4 mT for 90 min increased callus induction and fresh weight in P. alkekengi, while different concentrations of picloram reduced callogenesis. Hypocotyl explants showed various callogenesis and metabolic responses depending on the basal medium type. The 2B5 medium supplied with CH 200 (mgL-1) induced friable and cream calluses with high biomass (0.62 g) compared to the MS medium (control). The maximum activity of superoxide dismutase and catalase activities was identified in the 2B5 medium and peroxidase in the 2MS medium. The highest total phenolic (129.44 µg g-1DW) content and phenylalanine-ammonia lyase activity were obtained in the 2MS medium, and total withanolides (49.86 µg g-1DW) and DPPH radical scavenging activity were observed in the 2B5 medium. The 2MS medium boosted the hydrogen peroxide and nitric oxide levels, while their contents alleviated in the 2B5 medium, although these parameters were higher than the control. The findings of this study suggest that an effective protocol for successful callogenesis in P. alkekengi and the nutrient composition of culture medium by affecting the level of signaling molecules can control the antioxidant defense system and callus growth.

The Solanaceae family and the Withania genus specifically are rich sources of medicinal plants. Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS/MS) revealed a predominance of withanolides from an organic extract of Withania obtusifolia. A constructed molecular network uncovered the presence of potentially novel withanolides. A series of withanolides were then isolated and structurally characterized from the extract including two new withanolides (withafolia A and withafolia B) and seven previously reported metabolites. Of the isolated compounds, cytotoxicity of withanolide J, physaperuvin G, and a commercial STAT3 inhibitor (S3I-201) were assessed against a human leukemia HL-60 cell line resulting in IC50 values of 26, 29, and 120 μM, respectively. In silico molecular docking simulations indicate that withanolide J and physaperuvin G can bind as an inhibitor in the active site of STAT3 with docking scores comparable to the selective STAT3 inhibitor, S3I-201.

Vimentin has been reported to play diverse roles in cell processes such as spreading, migration, cell-matrix adhesion, and fibrotic transformation. Here, we assess how vimentin impacts cell spreading, morphology, and myofibroblast transformation of human corneal fibroblasts. Overall, although knockout (KO) of vimentin did not dramatically impact corneal fibroblast spreading and mechanical activity (traction force), cell elongation in response to PDGF was reduced in vimentin KO cells as compared to controls. Blocking vimentin polymerization using Withaferin had even more pronounced effects on cell spreading and also inhibited cell-induced matrix contraction. Furthermore, although absence of vimentin did not completely block TGFβ-induced myofibroblast transformation, the degree of transformation and amount of αSMA protein expression was reduced. Proteomics showed that vimentin KO cells cultured in TGFβ had a similar pattern of protein expression as controls. One exception included periostin, an ECM protein associated with wound healing and fibrosis in other cell types, which was highly expressed only in Vim KO cells. We also demonstrate for the first time that LRRC15, a protein previously associated with myofibroblast transformation of cancer-associated fibroblasts, is also expressed by corneal myofibroblasts. Interestingly, proteins associated with LRRC15 in other cell types, such as collagen, fibronectin, β1 integrin and α11 integrin, were also upregulated. Overall, our data show that vimentin impacts both corneal fibroblast spreading and myofibroblast transformation. We also identified novel proteins that may regulate corneal myofibroblast transformation in the presence and/or absence of vimentin.

Silver (Ag) is a non-essential heavy metal with substantial environmental toxicity but an excellent promotor for plant organogenesis. It is used as an elicitor for secondary metabolite production and for in planta synthesis of metal nanoparticles (MNPs). In the present study, the Ag accumulation and reduction capability of in vitro shoots of Withania somnifera and the toxicity and elicitation effect of Ag on in vitro shoots were explored. In vitro shoot cultures of W. somnifera were treated with different concentrations of silver nitrate for a specific treatment period. Growth index, withaferin A, elemental and electron microscopy analyses were done on silver-treated in vitro shoots of W. somnifera. 1 mM silver nitrate treatment for 12 days period was found to give increased growth index (1.425 ± 0.05c) and withaferin A (2.568 ± 0.08e mg g-1) content. The concentration of bioaccumulated Ag in 1 mM silver nitrate treated in vitro shoot was found to be 50.8 ppm. The presence of nano-Ag was also found in the leaves of 1 mM silver nitrate-treated in vitro shoots. In summary, this is the first report portraying the bioaccumulation and in planta reduction capability of the in vitro shoot system of W. somnifera, which makes it a potential medicinal plant of commercial value for silver contaminated soils.