In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl β-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.
Ligands ; Glycosyltransferases/genetics* ; Sterols ; Phylogeny ; Ascomycota ; Liliaceae/chemistry* ; Melanthiaceae ; Diosgenin ; Sugars ; Glucose ; Uridine Diphosphate

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.
Male ; Humans ; Carcinoma, Squamous Cell/drug therapy* ; Diosgenin/metabolism* ; Mouth Neoplasms/drug therapy* ; Apoptosis ; Prostatic Neoplasms/drug therapy*

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 β oxidation in the liver.
Rats ; Male ; Animals ; Non-alcoholic Fatty Liver Disease/genetics* ; Sterol Regulatory Element Binding Protein 1/metabolism* ; Diet, High-Fat/adverse effects* ; Diosgenin/metabolism* ; Chaperonin 60/therapeutic use* ; Rats, Sprague-Dawley ; Liver ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Triglycerides ; RNA, Messenger/metabolism* ; Simvastatin/therapeutic use* ; Body Weight ; Lipid Metabolism ; Mammals/metabolism*

Steroidal saponins, important natural organic compounds in Paris polyphylla var. yunnanensis, have good biological activity. Structural modification of steroidal saponins by microbial transformation could produce a large number of products with novel structures and excellent bioactivity, which can provide functional compounds for the research and development of steroidal drugs. This study summarized the research progress in steroidal saponins and their microbial transformation in P. polyphylla var. yunnanensis. P. polyphylla var. yunnanensis contains 112 steroidal saponins, 8 of which are used as substrates in 35 transformation reactions by 25 microbial species, with the highest transformation rate of 95%. Diosgenin is the most frequently used substrate. Furthermore, the strains, culture medium, reaction conditions, transformation rate, transformation reaction characteristics, and biological activities of the transformed products were summarized. This review may provide reference for the further research on microbial transformation of steroidal saponins in P. polyphylla var. yunnanensis.
Diosgenin/analysis* ; Liliaceae/chemistry* ; Melanthiaceae/chemistry* ; Rhizome/chemistry* ; Saponins/analysis*

This study aims to investigate the molecular mechanism of polyphyllin Ⅰ(PPⅠ) inhibiting proliferation of human breast cancer cells. Human breast cancer BT474 and MDA-MB-436 cells were treated with different concentrations of PPⅠ, and then the effect of PPⅠ on cell proliferation was detected by MTT assay, trypan blue dye exclusion assay, real-time cell analysis, and clone forming assay, respectively. The apoptosis was detected by Annexin V-FITC/PI staining and then analyzed by flow cytometry. The change of mitochondrial membrane potential was detected by flow cytometry after fluorescent probe JC-1 staining. Western blot was used to detect protein expression and phosphorylation. Molecular docking was performed to detect the binding between PPⅠ and EGFR. The affinity between PPⅠ and EGFR was determined by drug affinity responsive target stability assay. The results indicated that PPⅠ inhibited the proliferation and colony formation of BT474 and MDA-MB-436 cells in a time-and concentration-dependent manner. The PPⅠ treatment group showed significantly increased apoptosis rate and significantly decreased mitochondrial membrane potential. PPⅠ down-regulated the expression of pro-caspase-3 protein, promoted the cleavage of PARP, and significantly reduced the phosphorylation levels of EGFR, Akt, and ERK. Molecular docking showed that PPⅠ bound to the extracellular domain of EGFR and formed hydrogen bond with Gln366 residue. Drug affinity responsive target stability assay confirmed that PPⅠ significantly prevented pronase from hydrolyzing EGFR, indicating that PPⅠ and EGFR have a direct binding effect. In conclusion, PPⅠ inhibited the proliferation and induced apoptosis of breast cancer cells by targeting EGFR to block its downstream signaling pathway. This study lays a foundation for the further development of PPⅠ-targeted drugs against breast cancer.
Apoptosis ; Breast Neoplasms/genetics* ; Cell Line, Tumor ; Cell Proliferation ; Diosgenin/analogs & derivatives* ; ErbB Receptors ; Female ; Humans ; Molecular Docking Simulation

As a naturally occurring steroid sapogenin, diosgenin acts as the precursor of hundreds of steroid medicines, and thereby has important medicinal value. Currently, industrial production of diosgenin relies primarily on chemical extraction from plant materials. Clearly, this strategy shows drawbacks of excessive reliance on plant materials and farmland as well as environment pollution. Due to development of metabolic engineering and synthetic biology, bio-production of diosgenin has garnered plenty of attention. Although the biosynthetic pathways of diosgenin have not been completely identified, in this review, we outline the identified biosynthetic pathways and key enzymes. In particular, we suggest heterologous biosynthesis of diosgenin in Saccharomyces cerevisiae. Overall, this review aims to provide valuable insights for future complete biosynthesis of diosgenin.
Biosynthetic Pathways/genetics* ; Diosgenin ; Metabolic Engineering

Diosgenin is widely distributed in many plants, such as Polygonatum sibiricum, Paris polyphylla, Dioscorea oppositifolia, Trigonella foenum-graecum, Costus speciosus, Tacca chantrieri, which has good anti-tumor activity and preferable effects on preventing atherosclerosis, protecting the heart, treating diabetes, etc. This review combed through the anti-tumor mechanisms of diosgenin encompassing lung, breast, gallbladder, liver, oral cavity, stomach, bladder, bone marrow, etc. Besides, it was discovered that diosgenin mainly exerts its effect by inhibiting tumor cell migration, suppressing tumor cell proliferation and growth, and inducing cell apoptosis. However, problems like low yield and bioavailability frequently exist in natural diosgenin. This review introduced methods such as structural modification, dosage form optimization and combination medication to improve the yield and anti-tumor activity of diosgenin. Via the summary of this paper, it is expected to provide theoretical basis for the rational exploitation and utilization of diosgenin.
Apoptosis ; Biological Products ; Cell Proliferation ; Diosgenin/pharmacology* ; Trigonella

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.
Biosynthetic Pathways ; Diosgenin/metabolism* ; Gene Expression Profiling ; Phytochemicals/biosynthesis* ; Polygonatum/metabolism* ; Polysaccharides/biosynthesis* ; Transcriptome

Polyphyllin D is a steroid saponin monomer in Polyphyllin, with antibacterial, analgesic, sedative, anti-tumor and other pharmacological effects, but is rarely reported in pancreatic cancer. This study detected apoptosis-relevant indicators, in order to explore the effect of polyphyllin D on the proliferation and apoptosis of human pancreatic cancer Panc-1 cells and relevant mechanisms of action. After pancreatic cancer Panc-1 cells were treated with polyphyllin D(0, 1, 2, 3, 4, 5 μg·μL~(-1)) for 24, 48 and 72 hours, CCK-8 method was used to detect the effect of polyphyllin D on the proliferation of pancreatic cancer Panc-1 cells. Flow cytometry was used to detect cell cycle and changes in mitochondrial membrane potential(MMP). The apoptosis was detected by Annexin V-FITC/PI staining, and Western blot was used to detect the protein expressions of cytochrome C(Cyto C), Bax, Bcl-2, cleaved caspase-3 and cleaved caspase-9. The results indicated that compared with the control group, polyphyllin D could inhibit the proliferative activity of Panc-1 cells in a time and concentration-dependent manner. Flow cytometry results showed that polyphyllin D could block the cells in S and G_2/M phase in a concentration manner, the MMP of the cells was significantly reduced, and the apoptosis rate increased with the concentration of polyphyllin D. Western blot results showed that polyphyllin D could concentration-dependently up-regulate the protein expression levels of Bax, Cyto C, cleaved caspase-3 and cleaved caspase-9, and down-regulate the protein expression level of Bcl-2. The above findings suggested that polyphyllin D could effectively inhibit the proliferation of Panc-1 cells, and its mechanism may be related to the blocking of cell growth cycle and the apoptosis induced by mitochondrial pathway.
Antineoplastic Agents, Phytogenic/pharmacology* ; Apoptosis ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Diosgenin/pharmacology* ; Humans ; Pancreatic Neoplasms/pathology* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Saponins/pharmacology* ; bcl-2-Associated X Protein/metabolism*

ObjectiveTo explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.

METHODSWe cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.

RESULTSCompared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).

CONCLUSIONSPPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.

Apoptosis ; Cell Proliferation ; drug effects ; DNA (Cytosine-5-)-Methyltransferase 1 ; metabolism ; Diosgenin ; analogs & derivatives ; pharmacology ; Flavonoids ; metabolism ; Humans ; MAP Kinase Signaling System ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; NF-kappa B ; metabolism ; PC-3 Cells ; Phosphorylation ; Prostatic Neoplasms, Castration-Resistant ; drug therapy ; metabolism ; pathology ; Signal Transduction ; Transcription Factor RelA ; metabolism