Diosgenin inhibits proliferation and migration of ovarian cancer cells and induce apoptosis via upregulation of PTEN.

Diosgenin, a natural steroidal sapogenin, has recently attracted a high amount of attention, as an effective anticancer agent in ovarian cancer. However, diosgenin mediated anticancer impacts are still not completely understood. Thus, the present study evaluated the effect of diosgenin on the proliferation, apoptosis, and metastasis of ovarian cancer cells. OVCAR-3 and SKOV-3 cells were treated with diosgenin, cellular viability was assessed by MTT assay and apoptosis was measured by ELISA and evaluated the protein expression levels of apoptotic markers through western blotting. Cell migration was examined by measuring the mRNA levels of genes involved in the cell invasion. The protein expression levels of main components of PI3K signaling were evaluated via western blotting. Diosgenin led to significant inhibition of cellular proliferation in a dose-dependent manner. It also induced apoptosis through upregulating pro-apoptotic markers and downregulating antiapoptotic mediators. In addition, OVCAR-3 cells exposure to diosgenin decreased cell migration and invasion. More importantly, diosgenin downregulated the expression levels of main proteins in PI3K signaling including PI3K, Akt, mTOR, and GSK3. Diosgenin inhibited the proliferation and migration of OVCAR-3 ovarian cancer cells and induced apoptosis, which may be mediated by targeting PI3K signaling.

From Flora to Pharmaceuticals: 100 new additions to angiosperms of Gafargaon subdistrict in Bangladesh and unraveling antidiabetic drug candidates targeting DPP4 through in silico approach.

Addition to the angiosperm flora provides essential insights into the biodiversity of a region, contributing to ecological understanding and conservation planning. Gafargaon subdistrict under Mymensingh district in Bangladesh represents a diverse population of angiosperms with a multifaceted ecosystem that demands re-evaluation of the existing angiosperm diversity of Gafargaon to update the status of angiosperm taxa and facilitate their conservation efforts. With this endeavor, a total of 100 angiosperm taxa belonging to 90 genera and 46 families were uncovered as additional occurrence in Gafargaon. The species in the area showcased a variety of life forms, including 63 herbs, 14 shrubs, 14 trees, and 9 climbers. Among the recorded taxa, Chamaecostus cuspidatus (Nees & Mart.) C.D. Specht & D.W. Stev. was selected for antidiabetic drug design endeavor based on citation frequency and ethnomedicinal evidence. A total of 41 phytochemicals of C. cuspidatus were screened virtually, targeting the Dipeptidyl peptidase 4 protein through structure-based drug design approach, which unveiled two lead compounds, such as Tigogenin (-9.0 kcal/mol) and Diosgenin (-8.5 kcal/mol). The lead candidates demonstrated favorable pharmacokinetic and pharmacodynamic properties with no major side effects. Molecular dynamics simulation revealed notable stability and structural compactness of the lead compounds. Principal component analysis and Gibbs free energy landscape further supported the results of molecular dynamics simulation. Molecular mechanics-based MM/GBSA approach unraveled higher free binding energies of Diosgenin (-47.36 kcal/mol) and Tigogenin (-46.70 kcal/mol) over Alogliptin (-46.32 kcal/mol). The outcome of the present investigation would enrich angiosperm flora of Gafargaon and shed light on the role of C. cuspidatus to develop novel antidiabetic therapeutics to combat diabetes.

Polyphyllin I enhances tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of human osteosarcoma cell growth downregulating the Wnt/ß-catenin pathway.

OBJECTIVE: To investigate the synergistic effects of polyphyllin I (PPI) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the growth of osteosarcoma cells through downregulating the Wnt/ß-catenin signaling pathway. METHODS: Cell viability, apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays. The morphology of cancer cells was observed with inverted phase contrast microscope. The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays. The expressions of poly (adenosine diphosphate-ribose) polymerase, C-Myc, Cyclin B1, cyclin-dependent kinases 1, N-cadherin, Vimentin, Active-ß-catenin, ß-catenin, p-glycogen synthase kinase 3ß (GSK-3ß) and GSK-3ß were determined by Western blotting assay. RESULTS: PPI sensitized TRAIL-induced decrease of viability, migration and invasion, as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells. The synergistic effect of PPI with TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/ß-catenin signaling pathway. CONCLUSION: The combination of PPI and TRAIL is potentially a novel treatment strategy of osteosarcoma.

Potential targets of diosgenin for the treatment of oral squamous cell carcinoma and their bioinformatics and transcriptional profiling analyses.

Diosgenin can inhibit the proliferation and cause apoptosis of various tumor cells, and its inhibitory effect on oral squamous cell carcinoma (OSCC) and its mechanism are still unclear. In this study, we predicted the targets of diosgenin for the treatment of OSCC through the database, then performed bioinformatics analysis of the targets, and further verified the effect of diosgenin on the activity of OSCC cell line HSC-3, the transcriptional profile of the targets and the molecular docking of the targets with diosgenin. The results revealed that there were 146 potential targets of diosgenin for OSCC treatment, which involved signaling pathways such as Ras, TNF, PI3K-AKT, HIF, NF-κB, and could regulate cellular activity through apoptosis, autophagy, proliferation and differentiation, inflammatory response, DNA repair, etc. Diosgenin significantly inhibited HSC-3 cell activity. The genes such as AKT1, MET1, SRC1, APP1, CCND1, MYC, PTGS2, AR, NFKB1, BIRC2, MDM2, BCL2L1, MMP2, may be important targets of its action, not only their expression was regulated by diosgenin but also their proteins had a high binding energy with diosgenin. These results suggest that diosgenin may have a therapeutic effect on OSCC through AKT1, MMP2 and other targets and multiple signaling pathways, which is of potential clinical value.

Paris polyphylla ethanol extract and polyphyllin I ameliorate adenomyosis by inhibiting epithelial-mesenchymal transition.

BACKGROUND: The active ingredients of the Chinese medical herb Paris polyphylla, P. polyphylla ethanol extract (PPE) and polyphyllin I (PPI), potentially inhibit epithelial-mesenchymal transition (EMT) in tumors. However, the roles of these ingredients in inhibiting EMT in adenomyosis (AM) remain to be explored. PURPOSE: The primary goal of the study was to uncover the underlying molecular processes through which PPE and PPI suppress EMT in AM, alongside assessing the safety profiles of these substances. METHODS: To assess the suppressive impact of PPE on adenomyosis-derived cells (AMDCs), we employed Transwell and wound healing assays. The polyphyllins (PPI, PPII, PPVII) contained in PPE were characterized using high-performance liquid chromatography (HPLC). Then, bioinformatics techniques were performed to pinpoint potential PPI targets that could be effective in treating AM. Immunoblotting was used to verify the key proteins and pathways identified via bioinformatics. Furthermore, we examined the efficacy of PPE and PPI in treating Institute of Cancer Research (ICR) mice with AM by observing the morphological and pathological features of the uterus and performing immunohistochemistry. In addition, we assessed safety by evaluating liver, kidney and spleen pathologic features and serum test results. RESULTS: Three major polyphyllins of PPE were revealed by HPLC, and PPI had the highest concentration. In vitro experiments indicated that PPE and PPI effectively prevent AMDCs invasion and migration. Bioinformatics revealed that the primary targets E-cadherin, N-cadherin and TGFß1, as well as the EMT biological process, were enriched in PPI-treated AM. Immunoblotting assays corroborated the hypothesis that PPE and PPI suppress the TGFß1/Smad2/3 pathway in AMDCs to prevent EMT from progressing. Additionally, in vivo studies showed that PPE (3 mg/kg and 6 mg/kg) and PPI (3 mg/kg and 6 mg/kg), successfully suppressed the EMT process through targeting the TGFß1/Smad2/3 signaling pathway. Besides, it was observed that lower doses of PPE (3 mg/kg) and PPI (3 mg/kg) exerted minimal effects on the liver, kidneys, and spleen. CONCLUSIONS: PPE and PPI efficiently impede the development of EMT by inhibiting the TGFß1/Smad2/3 pathway, revealing an alternative pathway for the pharmacological treatment of AM.

Hepatogenic photosensitization in lambs supplemented with different levels of extruded urea in Brachiaria spp. pastures in the Brazilian Cerrado: Case report.

The aim was to report cases and risk factors for hepatogenous photosensitization in lambs kept on Brachiaria spp. pastures and supplemented with levels of extruded urea (EU). The herd consisted of 69 Texel crossbred lambs with known parentage (fathers and mothers adapted to the consumption of forage of the genus Brachiaria), randomly divided into 5 groups and distributed in individual paddocks for each group. The animals were supplemented with increasing levels of EU (Amireia® 200S): 0, 6, 12, 18, and 24 g of EU per 100 kg-1 of body weight (BW). The concentration of protodioscin was estimated in the mixed pastures of Brachiaria spp. (cv. Marandu and cv. Basilisk), structural components (leaf, stem, and dead material), samples of each cultivar, and in the months of December (2018), February, and April (2019). The animals were examined daily, and when behavioral changes were identified, they underwent clinical examinations and anamnesis. Weighing was performed every 14 days, followed by necropsy and serum biochemical analysis, including gamma-glutamyltransferase (GGT). The highest concentrations of protodioscin (p < 0.0001) were found in the pastures used by animals supplemented without extruded urea (7.07 ± 0.56), in the Basilisk cultivar (11.35 ± 0.06), in the leaf blade components (2.08 ± 0.05), and thatch (2.20 ± 0.00), and in the month of April (7.34 ± 0.29) (the month with the lowest rainfall), respectively. Fourteen (20.29%) cases of photosensitization were observed in lambs, of which six recovered, and eight died. Serum GGT levels ranged from 42.2 to 225 IU/L; however, in animals that died, values ranged from 209.4 to 225 IU/L. The use of levels 12 g and 18 g per 100 kg-1 of body weight of extruded urea may contribute to the lower occurrence of photosensitization, as the animals selected pastures with lower protodioscin content, presenting a smaller number of cases.

Stabilization of all-natural water-in-oil high internal phase pickering emulsion by using diosgenin/soybean phosphatidylethanolamine complex: Characterization and application in 3D printing.

This study aimed to prepare an all-natural water-in-oil high internal phase Pickering emulsion (W/O-HIPPE) using diosgenin/soybean phosphatidylethanolamine complex (DGSP) and investigate the 3D printing performance. Results suggested that the self-assembly of diosgenin crystal was modified by SP in DGSP (diosgenin-SP ratios at 3:1 and 1:1), revealing a variation from large-size outward radiating needle-like to small-size granular-like shape, which facilitated closely packing at the interface. Hydrophilicity of DGSP was also increased (contact angle varying from 133.3 o to 106.4 o), ensuring more adequate interfacial adsorption to reduce interfacial tension more largely (6.5 mN/m). Thus, the W/O-HIPPE made by DGSP with diosgenin-SP = 1:1, exhibited smaller droplets and better freeze/thawing stability. The W/O-HIPPE was also measured improved rheological properties for 3D printing: satisfied shear-thinning behavior, higher recovery and self-supporting (viscoelasticity and deformation resistance). Consequently, the W/O-HIPPE allowed for printing more delicate patterns. This work provided guidance to prepare W/O-HIPPE for 3D printing.

Isolation of endophytes from Dioscorea nipponica Makino for stimulating diosgenin production and plant growth.

KEY MESSAGE: Both bacterial and fungal endophytes exhibited one or more plant growth-promoting (PGP) traits. Among these strains, the Paenibacillus peoriae SYbr421 strain demonstrated the greatest activity in the direct biotransformation of tuber powder from D. nipponica into diosgenin. Endophytes play crucial roles in shaping active metabolites within plants, significantly influencing both the quality and yield of host plants. Dioscorea nipponica Makino accumulates abundant steroidal saponins, which can be hydrolyzed to produce diosgenin. However, our understanding of the associated endophytes and their contributions to plant growth and diosgenin production is limited. The present study aimed to assess the PGP ability and potential of diosgenin biotransformation by endophytes isolates associated with D. nipponica for the efficient improvement of plant growth and development of a clean and effective approach for producing the valuable drug diosgenin. Eighteen bacterial endophytes were classified into six genera through sequencing and phylogenetic analysis of the 16S rDNA gene. Similarly, 12 fungal endophytes were categorized into 5 genera based on sequencing and phylogenetic analysis of the ITS rDNA gene. Pure culture experiments revealed that 30 isolated endophytic strains exhibited one or more PGP traits, such as nitrogen fixation, phosphate solubilization, siderophore synthesis, and IAA production. One strain of endophytic bacteria, P. peoriae SYbr421, effectively directly biotransformed the saponin components in D. nipponica. Moreover, a high yield of diosgenin (3.50%) was obtained at an inoculum size of 4% after 6 days of fermentation. Thus, SYbr421 could be used for a cleaner and more eco-friendly diosgenin production process. In addition, based on the assessment of growth-promoting isolates and seed germination results, the strains SYbr421, SYfr1321, and SYfl221 were selected for greenhouse experiments. The results revealed that the inoculation of these promising isolates significantly increased the plant height and fresh weight of the leaves and roots compared to the control plants. These findings underscore the importance of preparing PGP bioinoculants from selected isolates as an additional option for sustainable diosgenin production.

Dioscin alleviates aplastic anemia through regulatory T cells promotion.

Objectives: Aplastic anemia (AA) is one of the immune-mediated bone marrow failure disorders caused by multiple factors, including the inability of CD4 + CD25 + regulatory T cells (Tregs) to negatively regulate cytotoxic T lymphocytes (CTLs). Dioscin is a natural steroid saponin that has a similar structure to steroid hormones. The purpose of this study is to look into the effect of Dioscin on the functions of CD4 + CD25+ Tregs in the AA mouse model and explore its underlying mechanism.Methods: To begin with, bone marrow failure was induced through total body irradiation and allogeneic lymphocyte infusion using male Balb/c mice. After 14 consecutive days of Dioscin orally administrated, the AA mouse model was tested for complete blood counts, HE Staining of the femur, Foxp3, IL-10 and TGF-ß. Then CD4 + CD25+ Tregs were isolated from splenic lymphocytes of the AA mouse model, Tregs and the biomarkers and cytokines of Tregs were measured after 24 h of Dioscin intervention treatment in vitro.Results: Dioscin promotes the expression of Foxp3, IL-10, IL-35 and TGF-ß, indicating its Tregs-promoting properties. Mechanistically, the administration of Dioscin resulted in the alteration of CD152, CD357, Perforin and CD73 on the surface of Tregs, and restored the expression of Foxp3.Conclusion: Dioscin markedly attenuated bone marrow failure, and promoted Tregs differentiation, suggesting the maintenance of theimmune balance effect of Dioscin. Dioscin attenuates pancytopenia and bone marrow failure via its Tregs promotion properties.

Effect of the NFIB rs28379954 T>C polymorphism on CYP2D6-catalyzed metabolism of solanidine.

Cytochrome P450 2D6 (CYP2D6) is important for metabolism of 20%-25% of all clinically used drugs. Many known genetic variants contribute to the large interindividual variability in CYP2D6 metabolism, but much is still unexplained. We recently described that nuclear factor 1B (NFIB) regulates hepatic CYP2D6 expression with the minor allele of NFIB rs28379954 T>C significantly increasing CYP2D6-mediated risperidone metabolism. In this study, we investigated the effect of NFIB T>C on metabolism of solanidine, a dietary CYP2D6 substrate. Analyses of solanidine and metabolites (M414, M416, and M444) were performed by ultra-high performance liquid chromatography-high-resolution mass spectrometry in a cohort of 463 CYP2D6-genotyped patients of which with 58 (12.5%) carried NFIB TC (n = 56) or CC (n = 2). Increased metabolism of solanidine was found in CYP2D6 normal metabolizers (NMs; n = 258, 55.7%) carrying the NFIB C variant (n = 27, 5.8%) with 2.83- and 3.38-fold higher M416-to-solanidine (p = 0.039) and M444-to-solanidine (p = 0.046) ratios, respectively, whereas this effect was not significant among intermediate metabolizers (n = 166, 35.9%) (p ≥ 0.09). Importantly, no effect of the NFIB polymorphism on solanidine metabolism was seen in TC or CC carriers lacking CYP2D6 activity (poor metabolizers, n = 30, 6.5%, p ≥ 0.74). Furthermore, the NFIB polymorphism significantly explained variability in solanidine metabolism (M414 p = 0.013, M416 p = 0.020, and M416 and M444 p = 0.009) in multiple linear regression models for each metabolic ratio in the entire population, correcting for covariates (including CYP2D6 genotypes). Thus, the study confirms the effect of NFIB in regulating CYP2D6 activity, suggesting an about 200% increase in CYP2D6-mediated clearance in NMs being NFIB CT or CC carriers, comprising around 6% of Europeans.

Diosgenyl glucosamine conjugates increase pro-apoptotic and selective activities in cancer cell lines.

BACKGROUND INFORMATION: Antiproliferative and apoptotic activities have been attributed to the phytosteroid diosgenin ((25R)-spirost-5-en-3ß-ol; 1). It is known that combining glucose with two rhamnoses (the chacotrioside framework) linked to diosgenin increases its apoptotic activity. However, the effects of diosgenin glucosamine glycosides on different cancer cell types and cell death have not been entirely explored. RESULTS: This study reports the antiproliferative, cytotoxic, and apoptotic activities of diosgenin and its glycosylated derivative ((25R)-spirost-5-en-3ß-yl ß-D-glucopyranoside; 2). It also explores the effects of two diosgenin glucosamine derivates, diosgenin 2-acetamido-2-deoxy-ß-D-glucopyranoside (3), and diosgenin 2-amino-2-deoxy-ß-D-glucopyranoside hydrochloride (4), on different cancer cell lines. We found that all the compounds affected proliferative activity with minimal toxicity. In addition, all cancer cell lines showed morphological and biochemical characteristics corresponding to an apoptotic process. Apoptotic cell death was higher in all cell lines treated with compounds 2, 3 and 4 than in those treated with diosgenin. Moreover, compounds 3 and 4 induced apoptosis better than compounds 1 and 2. These results suggest that combining glucosamine with modified glucosamine attached to diosgenin has a greater apoptotic effect than diosgenin or its glycosylated derivative (compound 2). Furthermore, diosgenin and the abovementioned glycosides had a selective effect on tumour cells since the proliferative capacity of human lymphocytes, keratinocytes (HaCaT) and epithelial cells (CCD841) was not significantly affected. CONCLUSIONS: Altogether, these results demonstrate that diosgenin glucosamine compounds exert an antiproliferative effect on cancer cell lines and induce apoptotic effects more efficiently than diosgenin alone without affecting non-tumour cells. SIGNIFICANCE: This study evidences the pro-apoptotic and selective activities of diosgenyl glucosamine compounds in cancer cell lines.

Diosgenin as a substitute for cholesterol alleviates NAFLD by affecting CYP7A1 and NPC1L1-related pathway.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) rapidly becomes the leading cause of end-stage liver disease or liver transplantation. Nowadays, there has no approved drug for NAFLD treatment. Diosgenin as the structural analogue of cholesterol attenuates hypercholesterolemia by inhibiting cholesterol metabolism, which is an important pathogenesis in NAFLD progression. However, there has been no few report concerning its effects on NAFLD so far. METHODS: Using a high-fat diet & 10% fructose-feeding mice, we evaluated the anti-NAFLD effects of diosgenin. Transcriptome sequencing, LC/MS analysis, molecular docking simulation, molecular dynamics simulations and Luci fluorescent reporter gene analysis were used to evaluate pathways related to cholesterol metabolism. RESULTS: Diosgenin treatment ameliorated hepatic dysfunction and inhibited NAFLD formation including lipid accumulation, inflammation aggregation and fibrosis formation through regulating cholesterol metabolism. For the first time, diosgenin was structurally similar to cholesterol, down-regulated expression of CYP7A1 and regulated cholesterol metabolism in the liver (p < 0.01) and further affecting bile acids like CDCA, CA and TCA in the liver and feces. Besides, diosgenin decreased expression of NPC1L1 and suppressed cholesterol transport (p < 0.05). Molecular docking and molecular dynamics further proved that diosgenin was more strongly bound to CYP7A1. Luci fluorescent reporter gene analysis revealed that diosgenin concentration-dependently inhibited the enzymes activity of CYP7A1. CONCLUSION: Our findings demonstrated that diosgenin was identified as a specific regulator of cholesterol metabolism, which pave way for the design of novel clinical therapeutic strategies.

Solanidine is a sensitive and specific dietary biomarker for CYP2D6 activity.

BACKGROUND: Individual assessment of CYP enzyme activities can be challenging. Recently, the potato alkaloid solanidine was suggested as a biomarker for CYP2D6 activity. Here, we aimed to characterize the sensitivity and specificity of solanidine as a CYP2D6 biomarker among Finnish volunteers with known CYP2D6 genotypes. RESULTS: Using non-targeted metabolomics analysis, we identified 9152 metabolite features in the fasting plasma samples of 356 healthy volunteers. Machine learning models suggested strong association between CYP2D6 genotype-based phenotype classes with a metabolite feature identified as solanidine. Plasma solanidine concentration was 1887% higher in genetically poor CYP2D6 metabolizers (gPM) (n = 9; 95% confidence interval 755%, 4515%; P = 1.88 × 10-11), 74% higher in intermediate CYP2D6 metabolizers (gIM) (n = 89; 27%, 138%; P = 6.40 × 10-4), and 35% lower in ultrarapid CYP2D6 metabolizers (gUM) (n = 20; 64%, - 17%; P = 0.151) than in genetically normal CYP2D6 metabolizers (gNM; n = 196). The solanidine metabolites m/z 444 and 430 to solanidine concentration ratios showed even stronger associations with CYP2D6 phenotypes. Furthermore, the areas under the receiver operating characteristic and precision-recall curves for these metabolic ratios showed equal or better performances for identifying the gPM, gIM, and gUM phenotype groups than the other metabolites, their ratios to solanidine, or solanidine alone. In vitro studies with human recombinant CYP enzymes showed that solanidine was metabolized mainly by CYP2D6, with a minor contribution from CYP3A4/5. In human liver microsomes, the CYP2D6 inhibitor paroxetine nearly completely (95%) inhibited the metabolism of solanidine. In a genome-wide association study, several variants near the CYP2D6 gene associated with plasma solanidine metabolite ratios. CONCLUSIONS: These results are in line with earlier studies and further indicate that solanidine and its metabolites are sensitive and specific biomarkers for measuring CYP2D6 activity. Since potato consumption is common worldwide, this biomarker could be useful for evaluating CYP2D6-mediated drug-drug interactions and to improve prediction of CYP2D6 activity in addition to genotyping.

Dioscin Alleviates Periodontitis by Inhibiting NLRP3 Inflammasome Activation via Regulation of K+ Homeostasis and Mitochondrial Function.

Gingival inflammation and alveolar bone loss are characteristic manifestations of periodontitis. Interleukin (IL)-1ß, the maturation of which is mainly regulated by NOD-like receptor protein (NLRP) 3 inflammasome, not only amplifies the inflammatory response but also triggers osteoclastogenesis, thereby accelerating the progression of periodontitis. Dioscin, a natural steroid saponin, has been shown to inhibit NLRP3 inflammasome. Nevertheless, research on the effectiveness of Dioscin for the management of periodontitis remains scarce. In this study, Dioscin was found to dramatically reduce the integral components of NLRP3 inflammasome, ultimately limiting IL-1ß secretion. Notably, the inhibitory impact of Dioscin on NLRP3 inflammasome might be exerted by curbing the generation of mitochondrial (mt) reactive oxygen species (ROS) and oxidized (ox) mtDNA, which were mediated by inhibition of K+ efflux. Furthermore, Dioscin effectively alleviated periodontitis in mice. Overall, the results established that Dioscin could alleviate periodontitis by inhibiting NLRP3 inflammasome via modulation of the K+ efflux-mtROS-ox-mtDNA pathway, holding the potential to treat periodontitis and other NLRP3-driven inflammatory diseases.

Microbial metabolism of diosgenin by a novel isolated Mycolicibacterium sp. HK-90: A promising biosynthetic platform to produce 19-carbon and 21-carbon steroids.

Green manufacture of steroid precursors from diosgenin by microbial replacing multistep chemical synthesis has been elusive. It is currently limited by the lack of strain and degradation mechanisms. Here, we demonstrated the feasibility of this process using a novel strain Mycolicibacterium sp. HK-90 with efficiency in diosgenin degradation. Diosgenin degradation by strain HK-90 involves the selective removal of 5,6-spiroketal structure, followed by the oxygenolytic cleavage of steroid nuclei. Bioinformatic analyses revealed the presence of two complete steroid catabolic gene clusters, SCG-1 and SCG-2, in the genome of strain HK-90. SCG-1 cluster was found to be involved in classic phytosterols or cholesterol catabolic pathway through the deletion of key kstD1 gene, which promoted the mutant m-∆kstD1 converting phytosterols to intermediate 9α-hydroxyandrostenedione (9-OHAD). Most impressively, global transcriptomics and characterization of key genes suggested SCG-2 as a potential gene cluster encoding diosgenin degradation. The gene inactivation of kstD2 in SCG-2 resulted in the conversion of diosgenin to 9-OHAD and 9,16-dihydroxy-pregn-4-ene-3,20-dione (9,16-(OH)2 -PG) in mutant m-ΔkstD2. Moreover, the engineered strain mHust-ΔkstD1,2,3 with a triple deletion of kstDs was constructed, which can stably accumulate 9-OHAD by metabolizing phytosterols, and accumulate 9-OHAD and 9,16-(OH)2 -PG from diosgenin. Diosgenin catabolism in strain mHust-ΔkstD1,2,3 was revealed as a progression through diosgenone, 9,16-(OH)2 -PG, and 9-OHAD to 9α-hydroxytestosterone (9-OHTS). So far, this work is the first report on genetically engineered strain metabolizing diosgenin to produce 21-carbon and 19-carbon steroids. This study presents a promising biosynthetic platform for the green production of steroid precursors, and provide insights into the complex biochemical mechanism of diosgenin catabolism.

Diosgenin biosynthesis investigation in medicinal herb (Tribulus terrestris) by transcriptome analysis.

Next-generation sequencing (NGS) has revolutionized the analysis of specific genes, pathways, and their regulation in various species. Tribulus terrestris L., an annual medicinal herb of Zygophyllaceae family, has gained significant attention due to its diverse medicinal properties, including anti-inflammatory, antimicrobial, and anti-cancer effects. Diosgenin, a steroidal saponin, is the major bioactive compound responsible for the medicinal importance of T. terrestris. However, there is a paucity of information regarding the genes involved in the diosgenin biosynthetic pathway in T. terrestris. To address this gap, this study aimed to identify candidate genes associated with diosgenin biosynthesis through whole transcriptome profiling. A total of ∼7.9 GB of data, comprising 482 million reads, was obtained and assembled into 148,871 unigenes. Subsequently, functional annotations were assigned to 50 % of the unigenes using sequence similarity searches against the NCBI non-redundant (NR), Uniprot, KEGG, Pfam, GO, and COG databases, primarily based on Gene Ontology and KEGG-KAAS pathways. The majority of unigenes associated with the biosynthesis of the steroidal diosgenin backbone exhibited up-regulation in the fruit, leaf, and root tissues, except the SQE gene in root. The differential expression of selected genes was further validated through quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the study identified 21,026 unigenes related to transcription factors and 15,551 unigenes containing simple sequence repeats (SSR). Notably, di-nucleotide SSR motifs exhibited a high repeat frequency. These findings greatly enhance our understanding of the diosgenin biosynthesis pathway and provide a basis for future research in molecular investigation and metabolic engineering, specifically for boosting diosgenin content.

The amelioration of salt stress-induced damage in fenugreek through the application of cold plasma and melatonin.

Nowadays, it is increasingly crucial to combine innovative approaches with established methods to enhance plant tolerance and maximize the production of beneficial compounds. With this aim in view, a study was carried out to investigate how different melatonin concentrations (0, 30, and 60 ppm), cold plasma treatment (at 3000 and 4000 V), and varying exposure durations (0, 1, 2, and 4 min) affect the physiological and biochemical attributes of fenugreek plants, as well as the levels of diosgenin under salinity stress. This study revealed that the application of 3000 V cold plasma for 2 min with 60 ppm melatonin by establishing cellular redox homeostasis in salinity-treated fenugreek plants, effectively prevented the destruction of pigments and reduced the electrolyte leakage index of malondialdehyde content. The utilization of these two elicitors has the potential to trigger multiple pathways, including the enzymatic and non-enzymatic antioxidants biosynthesis, and abscisic acid-dependent pathways. This activation results in an enhanced production of abscisic acid, auxin, and endogenous melatonin, along with the regulation of signal transduction pathways. Surprisingly, applying these two treatments increased the expression of SQS, CAS, SSR, BGL, SEP, SMT, and diosgenin content by 13, 22.5, 21.6, 19, 15.4, 12, and 6 times respectively. The findings highlight the intricate interplay between these treatments and the positive impact of their combined application, opening up avenues for further research and practical applications in improving plant tolerance to environmental stresses.

A new multi-template molecularly imprinted polymer for separation and purification of dioscin, protodioscin, and diosgenin from purple yam.

Saponin is an essential natural compound in purple yams with high nutritional and medicinal value. In this work, a multitemplate molecule-imprinted polymer (MMIP) was synthesized with dioscin, protodioscin, and diosgenin templates. The MMIPs were characterized with scanning electron microscopy, thermogravimetric analysis, Brunauer-Emmett-Teller (BET) adsorption, and Fourier transform infrared spectroscopy. The efficacy of the MMIPs was assessed with static, dynamic, selective adsorption, desorption, and reusability experiments. The three saponins were selectively extracted and determined by MMIP-high-performance liquid chromatography. The polymer morphology was regular and spherical. The amount of the MMIP adsorbed was 74.825 mg/g, and the imprinting factor was 2.1. The MMIP adsorbed the three saponins from purple yam extract, with recovery rates of 95.5-103.43 % and desorption rates of 85 %-98 %. In addition, the MMIPs were reused at least six times. These results demonstrated that the MMIPs efficiently and selectively extracted dioscin, protodioscin, and diosgenin from food matrices at high rates.

Diosgenin biosynthesis pathway and its regulation in Dioscorea cirrhosa L.

Dioscorea cirrhosa L. (D. cirrhosa) tuber is a traditional medicinal plant that is abundant in various pharmacological substances. Although diosgenin is commonly found in many Dioscoreaceae plants, its presence in D. cirrhosa remained uncertain. To address this, HPLC-MS/MS analysis was conducted and 13 diosgenin metabolites were identified in D. cirrhosa tuber. Furthermore, we utilized transcriptome data to identify 21 key enzymes and 43 unigenes that are involved in diosgenin biosynthesis, leading to a proposed pathway for diosgenin biosynthesis in D. cirrhosa. A total of 3,365 unigenes belonging to 82 transcription factor (TF) families were annotated, including MYB, AP2/ERF, bZIP, bHLH, WRKY, NAC, C2H2, C3H, SNF2 and Aux/IAA. Correlation analysis revealed that 22 TFs are strongly associated with diosgenin biosynthesis genes (-r2- > 0.9, P < 0.05). Moreover, our analysis of the CYP450 gene family identified 206 CYP450 genes (CYP450s), with 40 being potential CYP450s. Gene phylogenetic analysis revealed that these CYP450s were associated with sterol C-22 hydroxylase, sterol-14-demethylase and amyrin oxidase in diosgenin biosynthesis. Our findings lay a foundation for future genetic engineering studies aimed at improving the biosynthesis of diosgenin compounds in plants.

Mathematical models for predicting protodioscin in tropical forage grasses.

Protodioscin is a saponin present in grasses that can lead to losses in animal production. Our hypothesis was that mathematical models can accurately and precisely predict the protodioscin concentration in tropical grasses. We evaluated the ability of four mathematical models to describe the protodioscin concentration in Brachiaria and Panicum cultivars with different regrowth periods. Six cultivars of Panicum: Aruana, Massai, Mombaça, Tanzânia, Tamani, and Zuri; and five of Brachiaria-grass: Marandu, Paiaguás, Piatã, Xaraés and Basilisk. Protodioscin concentration evaluations were carried out at 51, 84, 110, and 111 days of age. Linear, Quadratic, Exponential, and Logarithmic models were evaluated, and the adequacy of the models was verified. The models were compared for accuracy and precision by pairwise mean squared error analysis and the delta Akaike information criterion. The models did not differ from each other in terms of accuracy and precision. The exponential model showed a high ability to explain the observed variability between protodioscin concentration and plant age for Brachiaria grasses. Panicum grasses have constant protodioscin concentration. Mathematical models are capable of predicting the protodioscin concentration in grasses of the genus Brachiaria based on plant age. We recommend Exponential model to predict the concentration of protodioscin in Brachiaria grasses.