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.

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.

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.

[Diosgenin alleviates NAFLD induced by a high-fat diet in rats via mTOR/SREBP-1c/HSP60/MCAD/SCAD signaling pathway].

This study aims to observe the effects of diosgenin on the expression of mammalian target of rapamycin(mTOR), sterol regulatory element-binding protein-1c(SREBP-1c), heat shock protein 60(HSP60), medium-chain acyl-CoA dehydrogenase(MCAD), and short-chain acyl-CoA dehydrogenase(SCAD) in the liver tissue of the rat model of non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin in alleviating NAFLD. Forty male SD rats were randomized into five groups: a control group, a model group, low-(150 mg·kg~(-1)·d~(-1)) and high-dose(300 mg·kg~(-1)·d~(-1)) diosgenin groups, and a simvastatin(4 mg·kg~(-1)·d~(-1)) group. The rats in the control group were fed with a normal diet, while those in the other four groups were fed with a high-fat diet. After feeding for 8 weeks, the body weight of rats in the high-fat diet groups increased significantly. After that, the rats were administrated with the corresponding dose of diosgenin or simvastatin by gavage every day for 8 weeks. The levels of triglyceride(TG), total cholesterol(TC), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were determined by the biochemical method. The levels of TG and TC in the liver were measured by the enzyme method. Oil-red O staining was employed to detect the lipid accumulation, and hematoxylin-eosin(HE) staining to detect the pathological changes in the liver tissue. The mRNA and protein levels of mTOR, SREBP-1c, HSP60, MCAD, and SCAD in the liver tissue of rats were determined by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. Compared with the control group, the model group showed increased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lipid deposition in the liver, obvious hepatic steatosis, up-regulated mRNA and protein expression levels of mTOR and SREBP-1c, and down-regulated mRNA and protein expression levels of HSP60, MCAD, and SCAD. Compared with the model group, the rats in each treatment group showed obviously decreased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lessened lipid deposition in the liver, ameliorated hepatic steatosis, down-regulated mRNA and protein le-vels of mTOR and SREBP-1c, and up-regulated mRNA and protein levels of HSP60, MCAD, and SCAD. The high-dose diosgenin outperformed the low-dose diosgenin and simvastatin. Diosgenin may prevent and treat NAFLD by inhibiting the expression of mTOR and SREBP-1c and promoting the expression of HSP60, MCAD, and SCAD to reduce lipid synthesis, improving mitochondrial function, and promoting fatty acid ß oxidation in the liver.

Anticancer Activity of Diosgenin and Its Molecular Mechanism.

Diosgenin, a steroidal sapogenin, obtained from Trigonella foenum-graecum, Dioscorea, and Rhizoma polgonati, has shown high potential and interest in the treatment of various cancers such as oral squamous cell carcinoma, laryngeal cancer, esophageal cancer, liver cancer, gastric cancer, lung cancer, cervical cancer, prostate cancer, glioma, and leukemia. This article aims to provide an overview of the in vivo, in vitro, and clinical studies reporting the diosgenin's anticancer effects. Preclinical studies have shown promising effects of diosgenin on inhibiting tumor cell proliferation and growth, promoting apoptosis, inducing differentiation and autophagy, inhibiting tumor cell metastasis and invasion, blocking cell cycle, regulating immunity and improving gut microbiome. Clinical investigations have revealed clinical dosage and safety property of diosgenin. Furthermore, in order to improve the biological activity and bioavailability of diosgenin, this review focuses on the development of diosgenin nano drug carriers, combined drugs and the diosgenin derivatives. However, further designed trials are needed to unravel the diosgenin's deficiencies in clinical application.

Deciphering the network of cholesterol biosynthesis in Paris polyphylla laid a base for efficient diosgenin production in plant chassis.

Cholesterol serves as a key precursor for many high-value chemicals such as plant-derived steroidal saponins and steroidal alkaloids, but a plant chassis for effective biosynthesis of high levels of cholesterol has not been established. Plant chassis have significant advantages over microbial chassis in terms of membrane protein expression, precursor supply, product tolerance, and regionalization synthesis. Here, using Agrobacterium tumefaciens-mediated transient expression technology, Nicotiana benthamiana, and a step-by-step screening approach, we identified nine enzymes (SSR1-3, SMO1-3, CPI-5, CYP51G, SMO2-2, C14-R-2, 8,7SI-4, C5-SD1, and 7-DR1-1) from the medicinal plant Paris polyphylla and established detailed biosynthetic routes from cycloartenol to cholesterol. Specfically, we optimized HMGR, a key gene of the mevalonate pathway, and co-expressed it with the PpOSC1 gene to achieve a high level of cycloartenol (28.79 mg/g dry weight, which is a sufficient amount of precursor for cholesterol biosynthesis) synthesis in the leaves of N. benthamiana. Subsequently, using a one-by-one elimination method we found that six of these enzymes (SSR1-3, SMO1-3, CPI-5, CYP51G, SMO2-2, and C5-SD1) were crucial for cholesterol production in N. benthamiana, and we establihed a high-efficiency cholesterol synthesis system with a yield of 5.63 mg/g dry weight. Using this strategy, we also discovered the biosynthetic metabolic network responsible for the synthesis of a common aglycon of steroidal saponin, diosgenin, using cholesterol as a substrate, obtaining a yield of 2.12 mg/g dry weight in N. benthamiana. Our study provides an effective strategy to characterize the metabolic pathways of medicinal plants that lack a system for in vivo functional verification, and also lays a foundation for the synthesis of active steroid saponins in plant chassis.

Diosgenin attenuates non-alcoholic fatty liver disease in type 2 diabetes through regulating SIRT6-related fatty acid uptake.

BACKGROUND: More than 70% of patients with type 2 diabetes (T2DM) concomitantly suffer from Non-alcoholic fatty liver disease (NAFLD), and the coexistence and interaction of them increases the intractability of NAFLD. With the protective effect against hepatic steatosis and liver fibrosis, SIRT6 is becoming a notable target of NAFLD. Diosgenin, an active monomer from Chinese herbs, has been reported to protect against NAFLD. PURPOSE: This study aims to figure out the mechanism how diosgenin alleviate NAFLD in T2DM and the relationship with SIRT6. METHODS: In vivo studies used spontaneous diabetic db/db mice and divided them into two parts. The first part included four groups consisting of control (Con) group, model (Mod) group, low dose of diosgenin (DL) group and high dose of diosgenin (DH) group. The second part included four groups consisting of Con group, Mod group, DH+OSS (OSS_128167, inhibitor of SIRT6) group, MDL (MDL800, agonist of SIRT6) group. HepG2 cell line was selected in study in vitro, which was mainly composed of six groups including Con group, palmitic acid (PA) group, PA+DL group, PA+DH group, PA+DH+OSS group, PA+MDL group. OGTT, Biochemical biomarker (including TG, TC, AST, ALT), inflammatory biomarker (including IL-6 and TNF-α) were measured. HE, Oil Red O, and DHE staining were conducted. Immunohistochemistry, immunofluorescence, mRNA-seq, and qPCR were used to explore the mechanism. RESULTS: Results in the first part of study in vivo indicated that diosgenin protected against lipid accumulation, oxidative stress, cell injury, and light inflammatory of liver in db/db mice and regulated the expression of SIRT6 and fatty acid transporter including CD36, FATP2, FABP1. The effect of diosgenin could be reversed in DH+OSS group and the same effect was observed in MDL group in the second part of study in vivo. The same results were also noted in followed study in vitro. Diosgenin inhibited the fatty acids uptake and regulated the expression of SIRT6 and fatty acid transporter including CD36, FATP2, and FABP1 in PA-induced hepG2 cells, and which was reversed in DH+OSS group and resembled in MDL group. CONCLUSIONS: Diosgenin could attenuate non-alcoholic fatty liver disease in type 2 diabetes through regulating SIRT6-related fatty acid uptake.

Exploring the medicinally important secondary metabolites landscape through the lens of transcriptome data in fenugreek (Trigonella foenum graecum L.).

Fenugreek (Trigonella foenum-graecum L.) is a self-pollinated leguminous crop belonging to the Fabaceae family. It is a multipurpose crop used as herb, spice, vegetable and forage. It is a traditional medicinal plant in India attributed with several nutritional and medicinal properties including antidiabetic and anticancer. We have performed a combined transcriptome assembly from RNA sequencing data derived from leaf, stem and root tissues. Around 209,831 transcripts were deciphered from the assembly of 92% completeness and an N50 of 1382 bases. Whilst secondary metabolites of medicinal value, such as trigonelline, diosgenin, 4-hydroxyisoleucine and quercetin, are distributed in several tissues, we report transcripts that bear sequence signatures of enzymes involved in the biosynthesis of such metabolites and are highly expressed in leaves, stem and roots. One of the antidiabetic alkaloid, trigonelline and its biosynthesising enzyme, is highly abundant in leaves. These findings are of value to nutritional and the pharmaceutical industry.

Diosgenin protects against podocyte injury in early phase of diabetic nephropathy through regulating SIRT6.

BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes mellitus. DN is the main cause of end-stage renal disease (ESRD). SIRT6 becomes the important target of DN. Diosgenin (a monomer from Chinese herbs) is probable to bind to SIRT6. PURPOSE: Based on studies presented in the literature on kidney injuries plus screening for the binding effects of the drug to Sirt6, we aimed to carry out the study to assess the effects of diosgenin involved in improving podocyte damage in the early phase of DN.. METHODS: DN model was established in spontaneous diabetic db/db mice. Animal experiment was in two parts. The first part includes four groups consisting of control (Con) group, model (Mod) group, low dose of diosgenin (DL) group and high dose of diosgenin (DH) group. The second part includes four groups consisting of control group, model group, DH+OSS_128167 (OSS, inhibitor of SIRT6) group, MDL800 (agonist of SIRT6) group. MPC5 cell line was selected in cell experiment, which was mainly composed of six groups including Con group, palmitic acid (PA) group, PA+DL group, PA+DH group, PA+DH+OSS group, PA+MDL800 group. Some procedures such as transcriptomics, RT-qPCR and so on were used in the study to explore and verify the mechanism. RESULTS: The abnormal changes of mesangial matrix expansion, glomerular basement membrane (GBM) thickness, foot process (FP) width, urine albumin/creatinine (UACR), DESMIN, ADRP, NEPHRIN, PODOCIN, SIRT6 in Mod group were alleviated in DH group rather than DL group in the first part of animal experiment. The effect in DH group could be reversed in DH+OSS group and the same effect was observed in MDL800 group in the second part of animal experiment. The same results were also found in cell experiment. Protein level and mRNA expression of pyruvate dehydrogenase kinase 4 (PDK4) and Angiopoietin-like-4 (ANGPTL4) were increased in PA group, which could be alleviated in DH group, MDL800 group rather than DH+OSS group. CONCLUSIONS: Diosgenin could protect against podocyte injury in early phase of diabetic nephropathy by regulating SIRT6.

22R- but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis.

Diosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16-oxidations of cholesterol followed by a late C26-oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16-oxygenase(s) shows strict 22R-stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S-dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16-oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R-oxidation and DzCYP90G6 for the C16-oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots.

Genome-Wide Identification of CYP72A Gene Family and Expression Patterns Related to Jasmonic Acid Treatment and Steroidal Saponin Accumulation in Dioscorea zingiberensis.

Dioscorea zingiberensis is a medicinal herb containing a large amount of steroidal saponins, which are the major bioactive compounds and the primary storage form of diosgenin. The CYP72A gene family, belonging to cytochromes P450, exerts indispensable effects on the biosynthesis of numerous bioactive compounds. In this work, a total of 25 CYP72A genes were identified in D. zingiberensis and categorized into two groups according to the homology of protein sequences. The characteristics of their phylogenetic relationship, intron-exon organization, conserved motifs and cis-regulatory elements were performed by bioinformatics methods. The transcriptome data demonstrated that expression patterns of DzCYP72As varied by tissues. Moreover, qRT-PCR results displayed diverse expression profiles of DzCYP72As under different concentrations of jasmonic acid (JA). Likewise, eight metabolites in the biosynthesis pathway of steroidal saponins (four phytosterols, diosgenin, parvifloside, protodeltonin and dioscin) exhibited different contents under different concentrations of JA, and the content of total steroidal saponin was largest at the dose of 100 µmol/L of JA. The redundant analysis showed that 12 DzCYP72As had a strong correlation with specialized metabolites. Those genes were negatively correlated with stigmasterol and cholesterol but positively correlated with six other specialized metabolites. Among all DzCYP72As evaluated, DzCYP72A6, DzCYP72A16 and DzCYP72A17 contributed the most to the variation of specialized metabolites in the biosynthesis pathway of steroidal saponins. This study provides valuable information for further research on the biological functions related to steroidal saponin biosynthesis.

Tribulus terrestris and female reproductive system health: A comprehensive review.

BACKGROUND: Tribulus terrestris L. (T. terrestris) positive performance on the male sexual system has been confirmed, but little is known about its effects on the female reproductive system. PURPOSE: This review discussed in detail the beneficial impact of T. terrestris and its secondary metabolites on the female reproductive system. STUDY DESIGN AND METHODS: In this review, the scientific Databases of Science direct, Pubmed, Web of Science, Google, Google Scholar, Researchgate, EMBASE, Scientific Information (SID), and Elsevier were searched profoundly. Studies about the pharmacological activities of T. terrestris on the female reproductive system in each aspect of investigations: human, in vivo, and in vitro studies, in the period from 1998 to 2020 were admitted. Our study was not limited by the language of publications. RESULTS: 23 articles about the effects of T. terrestris on the female reproductive system were found. These studies approved the T. terrestris efficacy on improvements in histological features of the ovary and uterus of polycystic ovary syndrome patients as well as the well-working of normal ovaries, enhancements in the sexual desire of postmenopausal syndrome, improve ovarian and breast cancers. CONCLUSION: These studies showed that the positive effect of T. terrestris on the female reproductive system was due to the presence of a secondary metabolite called protodioscin; a steroidal saponin compound, as the dominant active component of this plant.

[Biosynthetic pathways of Polygonatum cyrtonema polysaccharide and diosgenin based on its transcriptomic data].

Polygonatum cyrtonema belongs to the plant family Liliaceae, and its dried rhizome is one of the sources of Chinese traditional medicine of Polygonati Rhizoma. It possesses the dual function as both medicine and food. Its main chemical components are polysaccharides and saponins. In order to understand the biosynthesis pathway of polysaccharides and diosgenin in P. cyrtonema, the corresponding transcriptomic data were obtained by extracting and sequencing the RNA of four parts of P. cyrtonema, namely, leaves, stems, rhizomes and roots. By adopting BGISEQ-500 sequencing platform, 42.03 Gb data were retrieved. Subsequently, the de novo assembly was carried out by Trinity software to obtain 137 233 transcripts, of which 68.13% of unigenes were annotated in seven databases including KEGG, GO, NR, NT, SwissProt, Pfam and KOG. Transcripts that may be involved in the biosynthesis of polysaccharides and diosgenin were analyzed by data mining. With help of qPCR, we validated expression data of four genes that were possibly involved in the biosynthesis of target metabolites. This experiment provides data for the study of biosynthetic pathways of P. cyrtonema secondary metabolites and the clarification of related structural gene functions.

Microbial transformation of diosgenin to diosgenone by Wickerhamomyces anomalus JQ-1 obtained from Naxi traditional Jiu Qu.

Diosgenone [(20S,22R,25R)-spirost-4-en-3-one, C27H40O3] has been considered as a potential therapeutic alternative remedy for malaria. An efficient and economical approach of microbial transformation with diosgenin to diosgenone by the yeast strain Wickerhamomyces anomalus JQ-1 from Naxi traditional Jiu Qu was developed in this study. Chromatographic analysis confirmed that 85% of 0.1 mM diosgenin was transformed to diosgenone within 72 h. This research demonstrates that diosgenin could be converted to diosgenone through two-step pathway including 3ß-hydroxyl oxidation and double bond isomerization rather than through one-step pathway, which prompted a further inference that the oxidation activity in W. anomalus JQ-1 has the same function with the Oppenauer-type oxidation which can convert diosgenin into diosgenone. Gaining specific functional strains from traditional fermented products will be a potential direction and ethnobotanical researches could provide helps with discovery and utilization of microbial resources.

Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.

Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketal natural products is unknown. Here, we report the diosgenin-biosynthetic pathways in himalayan paris (Paris polyphylla), a monocot medicinal plant with hemostatic and antibacterial properties, and fenugreek (Trigonella foenum-graecum), an eudicot culinary herb plant commonly used as a galactagogue. Both plants have independently recruited pairs of cytochromes P450 that catalyze oxidative 5,6-spiroketalization of cholesterol to produce diosgenin, with evolutionary progenitors traced to conserved phytohormone metabolism. This study paves the way for engineering the production of diosgenin and derived analogs in heterologous hosts.

NMR-based metabolomics approach to investigate the distribution characteristics of metabolites in Dioscorea opposita Thunb. cv. Tiegun.

Dioscorea opposita Thunb. cv. Tiegun (DTT), a type of homologous medicinal plant, is commonly used as food in daily life. However, there has always been confusion regarding removal of the peel, as the nutrient metabolite composition of the peel is unclear. Here, a nuclear magnetic resonance (NMR)-based metabolomics approach was used to determine the metabolite distribution in DTT exclude-peel and peel. Thirteen characteristic metabolites with statistical significance were identified and compared using multivariate, univariate and cluster analyses. The results demonstrated that the peel contained the higher levels of α-glucose, batatasin IV, batatasin I, asparagine, ß-glucose, protodioscin, threonine, protogracillin, dioscin, and ß-sitosteryl acetate, and the samples without the peel had the higher levels of leucine, glutamine and alanine. This study provided scientific data for understanding the distribution characteristics of metabolites in DTT samples, promoting reasonable consumption of DTT.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial herb native to China. The rhizome of D. zingiberensis has long been used as a traditional Chinese medicine to treat rheumatic arthritis. Dioscin is the major bioactive ingredient conferring the medicinal property described in Chinese pharmacopoeia. Several previous studies have suggested cholesterol as the intermediate to the biosynthesis of dioscin, however, the biosynthetic steps to dioscin after cholesterol remain unknown. In this study, a comprehensive D. zingiberensis transcriptome derived from its leaf and rhizome was constructed. Based on the annotation using various public databases, all possible enzymes in the biosynthetic steps to cholesterol were identified. In the late steps beyond cholesterol, cholesterol undergoes site-specific oxidation by cytochrome P450s (CYPs) and glycosylation by UDP-glycosyltransferases (UGTs) to yield dioscin. From the D. zingiberensis transcriptome, a total of 485 unigenes were annotated as CYPs and 195 unigenes with a sequence length above 1000 bp were annotated as UGTs. Transcriptomic comparison revealed 165 CYP annotated unigenes correlating to dioscin biosynthesis in the plant. Further phylogenetic analysis suggested that among those CYP candidates four of them would be the most likely candidates involved in the biosynthetic steps from cholesterol to dioscin. Additionally, from the UGT annotated unigenes, six of them were annotated as 3-O-UGTs and two of them were annotated as rhamnosyltransferases, which consisted of potential UGT candidates involved in dioscin biosynthesis. To further explore the function of the UGT candidates, two 3-O-UGT candidates, named Dz3GT1 and Dz3GT2, were cloned and functionally characterized. Both Dz3GT1 and Dz3GT2 were able to catalyze a C3-glucosylation activity on diosgenin. In conclusion, this study will facilitate our understanding of dioscin biosynthesis pathway and provides a basis for further mining the genes involved in dioscin biosynthesis.

Diosgenin, a Novel Aldose Reductase Inhibitor, Attenuates the Galactosemic Cataract in Rats.

OBJECTIVE: To seek efficient aldose reductase inhibitors (ARIs) with excellent in vitro and in vivo biological activities against rat galactosemic cataract. METHODS: The method was firstly optimized to screen strong ARIs from nonoriented synthetic compounds and natural extracts. Then, diosgenin was assessed on osmotic expansion of primarily cultured lens epithelial cells (LECs) induced by galactose (50 mM). Diosgenin was administered to galactosemic rats by oral (100 and 200 mg/kg) or direct drinking (0.1%) to evaluate its anticataract effects. RESULTS: Diosgenin was found as the strongest ARI with IC50 of 4.59 × 10-6 mol/L. Diosgenin (10 µM) evidently inhibited the formation of tiny vacuoles and upregulation of AR mRNA in LECs. In vivo, diosgenin delayed lens opacification, inhibited the increase of ratio of lens weight to body weight, and decreased AR activity, galactitol level, and AR mRNA expression, especially in the diosgenin drinking (0.1%) group. CONCLUSIONS: Diosgenin was an efficient ARI, which not only significantly decreased the LECs' osmotic expansion in vitro but also markedly delayed progression of rat galactosemic cataract in vivo. Thus, diosgenin rich food can be recommended to diabetic subjects as dietary management to postpone the occurrence of sugar cataract, and diosgenin deserves further investigation for chronic diabetic complications.

Dioscin relieves endotoxemia induced acute neuro-inflammation and protect neurogenesis via improving 5-HT metabolism.

Sepsis, in addition to causing fatality, is an independent risk factor for cognitive impairment among sepsis survivors. The pathologic mechanism of endotoxemia induced acute neuro-inflammation still has not been fully understood. For the first time, we found the disruption of neurotransmitters 5-HT, impaired neurogenesis and activation of astrocytes coupled with concomitant neuro-inflammation were the potential pathogenesis of endotoxemia induced acute neuro-inflammation in sepsis survivors. In addition, dioscin a natural steroidal saponin isolated from Chinese medicinal herbs, enhanced the serotonergic system and produced anti-depressant effect by enhancing 5-HT levels in hippocampus. What is more, this finding was verified by metabolic analyses of hippocampus, indicating 5-HT related metabolic pathway was involved in the pathogenesis of endotoxemia induced acute neuro-inflammation. Moreover, neuro-inflammation and neurogenesis within hippocampus were indexed using quantitative immunofluorescence analysis of GFAP DCX and Ki67, as well as real-time RT-PCR analysis of some gene expression levels in hippocampus. Our in vivo and in vitro studies show dioscin protects hippocampus from endotoxemia induced cascade neuro-inflammation through neurotransmitter 5-HT and HMGB-1/TLR4 signaling pathway, which accounts for the dioscin therapeutic effect in behavioral tests. Therefore, the current findings suggest that dioscin could be a potential approach for the therapy of endotoxemia induced acute neuro-inflammation.

Preparation and Evaluation of Diosgenin Nanocrystals to Improve Oral Bioavailability.

Diosgenin (DSG), a well-known steroid sapogenin derived from Dioscorea nipponica Makino and Dioscorea zingiberensis Wright, has a variety of bioactivities. However, it shows low oral bioavailability due to poor aqueous solubility and strong hydrophobicity. The present study aimed to develop DSG nanocrystals to increase the dissolution and then improve the oral bioavailability and biopharmaceutical properties of DSG. DSG nanocrystals were prepared by the media milling method using a combination of pluronic F127 and sodium dodecyl sulfate as surface stabilizers. The physicochemical properties of the optimal DSG nanocrystals were characterized using their particle size distribution, morphology, differential scanning calorimetry, powder X-ray diffraction, Fourier transform infrared spectroscopy data, and solubility and dissolution test results. Pharmacokinetic studies of the DSG coarse suspension and its nanocrystals were performed in rats. The particle size and polydispersity index of DSG nanocrystals were 229.0 ± 3.7 nm and 0.163 ± 0.064, respectively. DSG retained its original crystalline state during the manufacturing process, and its chemical structure was not compromised by the nanonizing process. The dissolution rate of the freeze-dried DSG nanocrystals was significantly improved in comparison with the original DSG. The pharmacokinetic studies showed that the AUC0-72h and C max of DSG nanocrystals increased markedly (p < 0.01) in comparison with the DSG coarse suspension by about 2.55- and 2.01-fold, respectively. The use of optimized nanocrystals is a good and efficient strategy for oral administration of DSG due to the increased dissolution rate and oral bioavailability of DSG nanocrystals.