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Tribulus terrestris L. (TT) became a very popular plant due to its effects in sexual disorders. The pharmacological actions seem to be related with its content in sterolic saponins. Protodioscin has been reported to be the main compound from TT products with anabolic effects. Because of the popularity of the plant, numerous herbal supplements are consumed around the world. Quantification of steroidal saponins through HPLC methods is sometimes difficult. Several methods have been proposed but not all are reproducible. The aim of this study was to validate a LC MS/MS method for quantification of protodioscin in herbal supplements. Two herbal supplements found on the Romanian market have been chosen for this study. The compounds were separated on a C18 column with ammonium acetate buffer and acetonitrile as mobile phase. The ionization was performed in electrospray negative mode and detection of protodioscin was made by monitoring the sum of ions m/z 737.41, m/z 739.42 and m/z 755.42. In one of the herbal supplements, protodioscin was under the limit of quantification. In conclusion a simple and rapid method is proposed for the quantification of protodioscin in herbal supplements.

Background/Aims: Protodioscin (PD) is a steroidal saponin with anti-cancer effects on a number of cancer cells, but the anti-tumor effects and mechanism of action of PD on human cervical cancer cells is unclear. Methods: We determined cell viability using the MTT assay. Cell death, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) generation, and endoplasmic reticulum (ER) stress were measured on a flow cytometry. Caspase activation, ER stress, and MMP-dependent apoptosis proteins in cervical cancer cells in response to PD were determined by Western blot analysis. The ability of ATF4 binding to ChIP promoter was measured using the ChIP assay. Results: We demonstrated that PD inhibits cell viability, causes a loss of mitochondrial function, and induces apoptosis, as evidenced by up-regulation of caspase-8, -3, -9, -PARP, and Bax activation, and down-regulation of Bcl-2 expression. PD was shown to induce ROS and the ER stress pathway, including GRP78, p-eIF-2α, ATF4, and CHOP. Pre-treatment with NAC, a ROS production inhibitor, significantly reduced ER stress and apoptosis-related proteins induced by PD. Transfection of GRP78/CHOP-siRNA effectively inhibited PD-induced ER stress-dependent apoptosis. Moreover, treatment with PD significantly increased p38 and JNK activation. Co-administration of a JNK inhibitor (SP600125) or p38 inhibitor (SB203580) abolished cell death and ER stress effects during PD treatment. In addition, PD induced the expression of nuclear ATF4 and CHOP, as well as the binding ability of ATF4 to the CHOP promoter. Conclusion: Our results suggest that PD is a promising therapeutic agent for the treatment of human cervical cancer.

Protodioscin (PD) is a natural saponin with anti-inflammatory, anticancer, and metabolic regulatory properties. Senolytic agents, which selectively eliminate senescent cells, are gaining attention for enhancing cancer therapy. This study evaluated the senolytic potential of PD in 3D HepG2 spheroids subjected to Methotrexate (MTX)-induced senescence. Spheroids were generated using the hanging drop method with a 1:1 mixture of HepG2 medium and HFF1-conditioned medium. Senescence was induced with 6 µM MTX, followed by 24-h PD treatment at 1.6, 4, and 8.14 µM (10%, 25%, and 50% of IC₅₀). Cell viability was assessed via MTT; ROS, NO, and TGF-β levels via ELISA; apoptosis via Annexin V/PI; and cell death via Calcein-AM. Expression of p16, p21, p27, p53, and p-Smad2/4 was measured by Western blot, and β-Galactosidase activity was used as a senescence marker. PD dose-dependently reduced spheroid viability and, combined with MTX, significantly enhanced apoptosis, cell death, ROS, NO, β-Gal activity, and expression of p16, p27, and p53, while downregulating p-Smad2/4 and TGF-β compared to MTX treatment alone. These findings suggest that PD potentiates MTX-induced senescence through modulation of cell cycle regulators and inhibition of the TGF-β/p-Smad2/4 pathway, highlighting its potential as a senolytic adjuvant in liver cancer therapy.

Background: Tribulus terrestris (Tt) is a popular herbal supplement marketed to enhance fitness performance, despite inconclusive evidence regarding its efficacy and safety. This study aimed to investigate the prevalence of TT use, awareness, and motivations for its use among recreational athletes in Italy, helping to address the lack of empirical data describing who actually uses Tt, for what purposes, and with what behavioral risks. Methods: A cross-sectional anonymous survey was administered between May and October 2024 across Italian gyms and fitness clubs using Microsoft Forms. A total of 696 individuals initiated the questionnaire; after removal of duplicate, incomplete and ineligible entries, 510 responses were analyzed. Two indicators of Tt consumption were assessed: ever use and current use, with the latter designated as the primary outcome. A multivariable logistic regression evaluated predictors of current Tt use, entering sex, age category (18–24, 25–34, 35–44, ≥45 years), and motivation for supplement consumption. Results: Current Tt use was reported by 7.8% of respondents, while 10.5% declared ever using a Tt-containing product. Motivation was the only independent predictor of Tt consumption (p = 0.012). Individuals reporting performance enhancement as their primary motivation were markedly more likely to currently use Tt, compared with those using supplements for other purposes (adjusted OR ≈ 18.5; p = 0.008). Neither sex (p = 0.918) nor age category (p = 0.519) significantly predicted Tt use. Admission of anabolic steroid use was infrequent but was linked to online purchasing from potentially unregulated sources. Conclusions: Tt consumption in fitness settings is driven predominantly by performance-oriented expectations rather than demographic characteristics. The observed discrepancy between consumer beliefs and scientific evidence suggests a pressing need for educational interventions and regulatory vigilance in sports nutrition. Public health policies should focus on improving label literacy, strengthening consumer protection, and countering misinformation within supplement marketing environments.

The purpose of the current study was to demonstrate the neuroprotective effect of protodioscin (Prot) in an in vitro model of ischemia/reperfusion (I/R) and investigate the underlying molecular mechanism. After PC12 cells were exposed to oxygen and glucose deprivation (OGD) reperfusion, PI staining by flow cytometry was used to quantify the rate of apoptosis. The levels of hypoxia-inducible factor 1-alpha (HIF-1α), Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were determined using commercially available kits. Intracellular reactive oxygen species (ROS) level was detected using the 20,70-dichlorodihy-drofluorescein diacetate (DCFH-DA) fluorescence assay. The expression levels of heat-shock proteins (HSP), PI3K, AKT, Nrf2, and miR-124 were tested by western blot or quantitative PCR. Prot significantly attenuated oxygen-glucose deprivation/reperfusion (OGD/R)-induced apoptotic death. Prot also reduced the oxidative stress as revealed by increasing the activities of SOD and GSH-Px, decreasing the levels of ROS and MDA. Moreover, mechanism investigations suggested that Prot prevented the decrease of HSP70, HSP32 (hemeoxygenase-1, HO-1), and PI3K protein expression, phosphorylation of AKT, and the accumulation of nuclear Nrf2. The level of miR-124 was decreased in PC12 cells, which was also effectively reversed by Prot treatment. Prot protected PC12 cells against OGD/R-induced injury through inhibiting oxidative stress and apoptosis, which could be associated with increasing HSP proteins expression via activating PI3K/AKT/Nrf2 pathway and miR-124 modulation.

Oral cancer is one of the most common cancers in the world. Approximately 90% of oral cancers are subtyped to oral squamous cell carcinoma (OSCC). Despite advances in diagnostic techniques and improvement in treatment modalities, the prognosis remains poor. Therefore, an effective chemotherapy mechanism that enhances tumor sensitivity to chemotherapeutics is urgently needed. Methyl protodioscin (MP) is a furostanol bisglycoside with a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. The aim of the present study was to determine the antitumor activity of MP on OSCC and its underlying mechanisms. Our results show that treatment of OSCC cells with MP potently inhibited cell viability. Moreover, MP leading to cell cycle arrest at G2/M phase, which subsequently activates caspase-3, -8, -9 and PARP to induce cell apoptosis. Meanwhile, we also demonstrate that MP induces a robust autophagy in OSCC cells. The results indicate cathepsin S (CTSS) is involved in MP-induced apoptosis and autophagy by modulation of p38 MAPK and JNK1/2 pathways. These findings may provide rationale to combine MP with CTSS blockade for the effective treatment of OSCC.

Asparagus, a vital economic contributor, is a well‐liked vegetable grown around the globe, and some secondary metabolites in its spear are beneficial to human health. Asparagus spears possess a significant quantity of nutrients and phytochemicals; however, the difference in these chemical compositions among various varieties has not been sufficiently studied. This work aimed to detect the chemical compositions of 30 varieties of asparagus and to assess them by principal component analysis (PCA). The results showed that the contents of these chemical compositions varied in varieties. Selenium (Se, 1.12–2.9 μg/100 g dry‐weight [DW]) was abundant in asparagus, with an average dry matter content of 8.25%. Free amino acids (5.60–9.98 g/100 g DW) and polyphenols (6.34–8.67 mg/g DW) were both present in high amounts, along with flavonoids (4.218–8.22 mg/g DW) and protodioscin (0.44–1.96 mg/g DW). Correlation analysis, PCA, and hierarchical cluster analysis were used to conduct a comprehensive evaluation of asparagus. Atlas, Appolo, Jinggang 111, Jingke 2, and WS‐1 were the top five varieties with comprehensive scores. This study provided valuable data for the breeding, quality improvement, processing, and utilization of asparagus varieties in the future. A comprehensive evaluation of 30 asparagus varieties in Shandong Province China was conducted. Asparagus show differences in components depending on their varieties, and their quality varies between stem colors. The top five varieties with comprehensive scores were Atlas, Appolo, Jinggang 111, Jingke 2, and WS‐1.

Background Idiopathic pulmonary fibrosis is a persistent disease of the lung interstitium for which there is no efficacious pharmacological therapy. Protodioscin, a steroidal saponin, possesses diverse pharmacological properties; however, its function in pulmonary fibrosis is yet to be established. Hence, in this investigation, it was attempted to figure out the anti-pulmonary fibrosis influences of protodioscin and its pharmacological properties related to oxidative stress. Methods A mouse lung fibrosis model was generated using tracheal injections of bleomycin, followed by intraperitoneal injection of different concentrations of protodioscin, and the levels of oxidative stress and fibrosis were detected in the lungs. Multiple fibroblasts were treated with TGF-β to induce their transition to myofibroblasts. It was attempted to quantify myofibroblast markers’ expression levels and reactive oxygen species levels as well as Nrf2 activation after co-incubation of TGF-β with fibroblasts and different concentrations of protodioscin. The influence of protodioscin on the expression and phosphorylation of p62, which is associated with Nrf2 activation, were detected, and p62 related genes were predicted by STRING database. The effects of Nrf2 inhibitor or silencing of the Nrf2, p62 and NBR1 genes, respectively, on the activation of Nrf2 by protodioscin were examined. The associations between p62, NBR1, and Keap1 in the activation of Nrf2 by protodioscin was demonstrated using a co-IP assay. Nrf2 inhibitor were used when protodioscin was treated in mice with pulmonary fibrosis and lung tissue fibrosis and oxidative stress levels were detected. Results In vivo, protodioscin decreased the levels of fibrosis markers and oxidative stress markers and activated Nrf2 in mice with pulmonary fibrosis, and these effects were inhibited by Nrf2 inhibitor. In vitro, protodioscin decreased the levels of myofibroblast markers and oxidative stress markers during myofibroblast transition and promoted Nrf2 downstream gene expression, with reversal of these effects after Nrf2, p62 and NBR1 genes were silenced or Nrf2 inhibitors were used, respectively. Protodioscin promoted the binding of NBR1 to p62 and Keap1, thereby reducing Keap1-Nrf2 binding. Conclusion The NBR1-p62-Nrf2 axis is targeted by protodioscin to reduce oxidative stress and inhibit pulmonary fibrosis. Graphical Abstract

Extraction of the leaves of Carica papaya (family Caricaceae) and Azadirachta indica (family Meliacea) were done using solvents with varying polarities (acetone, hexane and ethylacetate). The crude extracts were screened for phytoconstituents using the preliminary method and high-performance liquid chromatography (HPLC) for separation and quantification of the constituents. Susceptibility of three medically important microorganisms (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Candida alblicans) to the solvent extracts was tested at 100 mg/mL, 50 mg/mL and 25 mg/mL concentrations the disc diffusion technique. Tannins, saponins, alkaloids, steroids, flavonoids and anthraquinone were present in all the solvent extracts of A. indica and C. papaya. Protein was present in all the solvent extracts of A. indica but absent in all the solvent extracts of C. papaya. Terpenoid was only present in hexane extract of A. indica but absent in other solvent extracts of A. indica and C. papaya. Flavonoid was present in all but only absent in ethylacetate extract of A. indica. Glycoside was present in all but absent in hexane extract of A. indica. Coumarin was only present in acetone extracts of both plants and absent in other solvent extracts of the two plants. Extracts of Carica papaya and Azadirachta indica displayed varying inhibitory activities (between 5.00-15.00 mm) against the organisms at all the tested concentrations. Acetone extract of A. indica produced zones of inhibition ranging from 5.00-14.00 mm while acetone extract of C. papaya produces a range of 4.00-10.00 mm. Also, hexane extract of A. indica produced inhibition range of 7.00-10.00 mm whereas hexane extract of C. papaya produces a range of 5.00-15.00 mm. However, ethylacetate extract of A. indica produced inhibition range of 5.00-13.67 mm, while ethylacetate extract of C. papaya produce a range of 5.00-15.00 mm. Different compounds quantified as different peaks by HPLC in the different solvent extracts of Carica papaya are acacic acid, genistein, protodioscin, betulinic acid, phorbolester, creptolepinone, brusatol and alpha ionone while the fractions from the solvent extracts of Azadirachta indica are myricetin, azadirachtol, azadirachtin a, pentadecane, phytol, azadirachnol, quercetin, b caryophyllen, alpha ionone, ascaridole, trams.b.farnes. Results obtained in this work indicated that all the solvent extracts of Carica papaya and Azadirachta indica contained active phytoconstituents and the extracts displayed good potentials at preventing diseases associated with the microorganisms tested in this work.

Protective effect of protodioscin or methyl protodioscin against inflammation had been reported in various inflammation diseases. This study aimed to investigate the effect of protodioscin against Complete Freund’s adjuvant (CFA)-induced arthritis rats. Rats randomly divided into model groups were injected with CFA, companied with different dose of protodioscin (50, 100, and 200 mg/kg body weight). The histology, changes in biochemical parameters and inflammatory cytokines expression were detected for anti-inflammation effect evaluation of protodioscin. CFA treatment induced arthritic rats with swelling paw, ankle inflammation, and area of lymphocyte infiltration, upregulated inflammatory cytokines (IL-1β, TNF-α, cyclo-oxygenase 2, and IL-6 as well as prostaglandin E2), articular elastase, myeloperoxidase, lipid peroxidase and nitrite oxide levels, downregulated glutathione, catalase, and superoxide dismutase. In contrast, protodioscin ameliorated all the changes induced by CFA in rats, suggesting the anti-inflammatory effect of protodioscin. We concluded that protodioscin administration into CFA-induced arthritis rats protected against CFA-induced oxidative stress, neutrophil infiltration, and inflammation, suggesting the anti-inflammatory effect and the therapeutic potential of protodioscin for arthritis.