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.

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.

Structural basis of mammalian complex IV inhibition by steroids.

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme's catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

Dioscin Activates Endoplasmic Reticulum Unfolded Protein Response for Defense Against Pathogenic Bacteria in Caenorhabditis elegans via IRE-1/XBP-1 Pathway.

The unfolded protein response (UPR) is an evolutionarily conserved pathway that senses and responds to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen during bacterial infection. The IRE-1/XBP-1 pathway is a major branch of the UPRER that has been conserved from yeast to human. Dioscin, a steroidal saponin exhibits a broad spectrum of properties. However, whether dioscin influences the immune response and the underlying molecular mechanisms remain obscure. We find that dioscin increases resistance to Gram-negative pathogen Pseudomonas aeruginosa. Furthermore, dioscin also inhibits the growth of pathogenic bacteria. Meanwhile, dioscin enhances the resistance to pathogens by reducing bacterial burden in the intestine. Through genetic screening, we find that dioscin activates the UPRER to promote innate immunity via IRE-1/XBP-1 pathway. Intriguingly, dioscin requires the neural XBP-1 for immune response. Our findings suggest that dioscin may be a viable candidate for the treatment of infectious diseases.

Polyphyllin I alleviates neuroinflammation after cerebral ischemia-reperfusion injury via facilitating autophagy-mediated M2 microglial polarization.

Microglial activation and polarization play a central role in poststroke inflammation and neuronal damage. Modulating microglial polarization from pro-inflammatory to anti-inflammatory phenotype is a promising therapeutic strategy for the treatment of cerebral ischemia. Polyphyllin I (PPI), a steroidal saponin, shows multiple bioactivities in various diseases, but the potential function of PPI in cerebral ischemia is not elucidated yet. In our study, the influence of PPI on cerebral ischemia-reperfusion injury was evaluated. Mouse middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation and reoxygenation (OGD/R) model were constructed to mimic cerebral ischemia-reperfusion injury in vivo and in vitro. TTC staining, TUNEL staining, RT-qPCR, ELISA, flow cytometry, western blot, immunofluorescence, hanging wire test, rotarod test and foot-fault test, open-field test and Morris water maze test were performed in our study. We found that PPI alleviated cerebral ischemia-reperfusion injury and neuroinflammation, and improved functional recovery of mice after MCAO. PPI modulated microglial polarization towards anti-inflammatory M2 phenotype in MCAO mice in vivo and post OGD/R in vitro. Besides, PPI promoted autophagy via suppressing Akt/mTOR signaling in microglia, while inhibition of autophagy abrogated the effect of PPI on M2 microglial polarization after OGD/R. Furthermore, PPI facilitated autophagy-mediated ROS clearance to inhibit NLRP3 inflammasome activation in microglia, and NLRP3 inflammasome reactivation by nigericin abolished the effect of PPI on M2 microglia polarization. In conclusion, PPI alleviated post-stroke neuroinflammation and tissue damage via increasing autophagy-mediated M2 microglial polarization. Our data suggested that PPI had potential for ischemic stroke treatment.

Diosgenin improves post-myocardial infarction cardiac function via HAND2-induced angiogenesis.

While diosgenin has been demonstrated effective in various cardiovascular diseases, its specific impact on treating heart attacks remains unclear. Our research revealed that diosgenin significantly improved cardiac function in a myocardial infarction (MI) mouse model, reducing cardiac fibrosis and cell apoptosis while promoting angiogenesis. Mechanistically, diosgenin upregulated the Hand2 expression, promoting the proliferation and migration of endothelial cells under hypoxic conditions. Acting as a transcription factor, HAND2 activated the angiogenesis-related gene Aggf1. Conversely, silencing Hand2 inhibited the diosgenin-induced migration of hypoxic endothelial cells and angiogenesis. In summary, these findings provide new insights into the protective role of diosgenin in MI, validating its effect on angiogenic activity and providing a theoretical basis for clinical treatment strategies.

Identification of genetic variants controlling diosgenin content in Dioscorea zingiberensis tuber by genome-wide association study.

BACKGROUND: Diosgenin is an important steroidal precursor renowned for its diverse medicinal uses. It is predominantly sourced from Dioscorea species, particularly Dioscorea zingiberensis. Dioscorea zingiberensis has an ability to accumulate 2-16% diosgenin in its rhizomes. In this study, a diverse population of 180 D. zingiberensis accessions was used to evaluate the genomic regions associated with diosgenin biosynthesis by the genome wide association study approach (GWAS). RESULTS: The whole population was characterized for diosgenin contents from tubers by gas chromatography mass spectrometry. The individuals were genotyped by the genotyping-by-sequencing approach and 10,000 high-quality SNP markers were extracted for the GWAS. The highest significant marker-trait-association was observed as an SNP transversion (G to T) on chromosome 10, with 64% phenotypic variance explained. The SNP was located in the promoter region of CYP94D144 which is a member of P450 gene family involved in the independent biosynthesis of diosgenin from cholesterol. The transcription factor (TF) binding site enrichment analysis of the promoter region of CYP94D144 revealed NAC TF as a potential regulator. The results were further validated through expression profiling by qRT-PCR, and the comparison of high and low diosgenin producing hybrids obtained from a bi-parental population. CONCLUSIONS: This study not only enhanced the understanding of the genetic basis of diosgenin biosynthesis but also serves as a valuable reference for future genomic investigations on CYP94D144, with the aim of augmenting diosgenin production in yam tubers.

Fatty acid and nutrient profiles, diosgenin and trigonelline contents, mineral composition, and antioxidant activity of the seed of some Iranian Trigonella L. species.

BACKGROUND: Fenugreeks (Trigonella L. spp.), belonging to the legume family (Fabaceae), are well-known multipurpose crops that their materials are currently received much attention in the pharmaceutical and food industries for the production of healthy and functional foods all over the world. Iran is one of the main diversity origins of this valuable plant. Therefore, the aim of the present study was to explore vitamins, minerals, and fatty acids profile, proximate composition, content of diosgenin, trigonelline, phenolic acids, total carotenoids, saponins, phenols, flavonoids, and tannins, mucilage and bitterness value, and antioxidant activity of the seed of thirty populations belonging to the ten different Iranian Trigonella species. RESULTS: We accordingly identified notable differences in the nutrient and bioactive compounds of each population. The highest content (mg/100 g DW) of ascorbic acid (18.67 ± 0.85‒22.48 ± 0.60) and α-tocopherol (31.61 ± 0.15‒38.78 ± 0.67) were found in the populations of T. filipes and T. coerulescens, respectively. Maximum content of catechin was found in the populations of T. teheranica (52.67 ± 0.05‒63.50 ± 0.72 mg/l). Linoleic acid (> 39.11% ± 0.61%) and linolenic acid (> 48.78 ± 0.39%) were the main polyunsaturated fatty acids, with the majority in the populations of T. stellata (54.81 ± 1.39‒63.46 ± 1.21%). The populations of T. stellata were also rich in trigonelline (4.95 ± 0.03‒7.66 ± 0.16 mg/g DW) and diosgenin (9.06 ± 0.06‒11.03 ± 0.17 mg/g DW). CONCLUSIONS: The obtained data provides baseline information to expand the inventory of wild and cultivated Iranian Trigonella species for further exploitation of rich chemotypes in the new foods and specific applications.

Diosgenin restores memory function via SPARC-driven axonal growth from the hippocampus to the PFC in Alzheimer's disease model mice.

Central nervous system axons have minimal capacity to regenerate in adult brains, hindering memory recovery in Alzheimer's disease (AD). Although recent studies have shown that damaged axons sprouted in adult and AD mouse brains, long-distance axonal re-innervation to their targets has not been achieved. We selectively visualized axon-growing neurons in the neural circuit for memory formation, from the hippocampus to the prefrontal cortex, and showed that damaged axons successfully extended to their native projecting area in mouse models of AD (5XFAD) by administration of an axonal regenerative agent, diosgenin. In vivo transcriptome analysis detected the expression profile of axon-growing neurons directly isolated from the hippocampus of 5XFAD mice. Secreted protein acidic and rich in cysteine (SPARC) was the most expressed gene in axon-growing neurons. Neuron-specific overexpression of SPARC via adeno-associated virus serotype 9 delivery in the hippocampus recovered memory deficits and axonal projection to the prefrontal cortex in 5XFAD mice. DREADDs (Designer receptors exclusively activated by designer drugs) analyses revealed that SPARC overexpression-induced axonal growth in the 5XFAD mouse brain directly contributes to memory recovery. Elevated levels of SPARC on axonal membranes interact with extracellular rail-like collagen type I to promote axonal remodeling along their original tracings in primary cultured hippocampal neurons. These findings suggest that SPARC-driven axonal growth in the brain may be a promising therapeutic strategy for AD and other neurodegenerative diseases.

Polyphyllin I Sensitizes Cisplatin-Resistant Human Cervical Cancer Cells to Cisplatin Treatment.

Cervical cancer (CC) is a common gynecological malignancy, and improving cisplatin sensitivity has become a hot topic in CC chemotherapy research. Polyphyllin I (PPI), a potent bioactive compound found in Rhizoma Paridis, known for its anticancer properties, remains underexplored in CC resistance. In this study, we evaluated PPI's impact on cisplatin-resistant CC cells and elucidated its underlying mechanism. Our findings reveal that PPI enhances the sensitivity of cisplatin-resistant CC cells to the drug, promotes apoptosis, and inhibits cell migration. Mechanistically, PPI was found to regulate p53 expression and its target genes, and suppressing p53 expression reverses PPI's sensitizing effect in drug-resistant CC cells. In conclusion, PPI showed promise in sensitizing cisplatin-resistant human CC cells to cisplatin treatment, suggesting that it could serve as a potent adjunct therapy for cervical cancer, particularly for cases that have developed resistance to cisplatin, thereby providing a promising basis for further clinical investigation into PPI for enhancing the efficacy of existing chemotherapy regimens in resistant cervical cancer.

Evidence for solanidine as a dietary CYP2D6 biomarker: Significant correlation with risperidone metabolism.

AIMS: The extensive variability in cytochrome P450 2D6 (CYP2D6) metabolism is mainly caused by genetic polymorphisms. However, there is large, unexplained variability in CYP2D6 metabolism within CYP2D6 genotype subgroups. Solanidine, a dietary compound found in potatoes, is a promising phenotype biomarker predicting individual CYP2D6 metabolism. The aim of this study was to investigate the correlation between solanidine metabolism and the CYP2D6-mediated metabolism of risperidone in patients with known CYP2D6 genotypes. METHODS: The study included therapeutic drug monitoring (TDM) data from CYP2D6-genotyped patients treated with risperidone. Risperidone and 9-hydroxyrisperidone levels were determined during TDM, and reprocessing of the respective TDM full-scan high-resolution mass spectrometry files was applied for semi-quantitative measurements of solanidine and five metabolites (M402, M414, M416, M440 and M444). Spearman's tests determined the correlations between solanidine metabolic ratios (MRs) and the 9-hydroxyrisperidone-to-risperidone ratio. RESULTS: A total of 229 patients were included. Highly significant, positive correlationswere observed between all solanidine MRs and the 9-hydroxyrisperidone-to-risperidone ratio (ρ > 0.6, P < .0001). The strongest correlation was observed for the M444-to-solanidine MR in patients with functional CYP2D6 metabolism, i.e., genotype activity scores of 1 and 1.5 (ρ 0.72-0.77, P < .0001). CONCLUSION: The present study shows strong, positive correlations between solanidine metabolism and CYP2D6-mediated risperidone metabolism. The strong correlation within patients carrying CYP2D6 genotypes encoding functional CYP2D6 metabolism suggests that solanidine metabolism may predict individual CYP2D6 metabolism, and hence potentially improve personalized dosing of drugs metabolized by CYP2D6.

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.

Nano-enabled delivery of diosgenin and emodin ameliorates respirable silica dust-induced pulmonary fibrosis silicosis in rats.

Oxidative stress and inflammation play a fundamental role in the beginning and advancement of silicosis. Hence, questing active phytocompounds (APCs) with anti-oxidative and anti-inflammatory properties such as diosgenin (DG) and emodin (ED) can be a therapeutic intervention targeting silica-induced pulmonary inflammation and fibrosis. Hydrophobicity and low bioavailability are the barriers that restrict the therapeutic efficacy of DG and ED against pulmonary defects. Encapsulating these APCs in polymeric nanoparticles can overcome this limitation. The present study has thus explored the anti-inflammatory and anti-fibrotic effects of polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) individually loaded with DG (DGn) or ED (EDn) and in combine DG+ED [(DG+ED)n] in respirable silica dust (RSD)-induced pulmonary fibrosis silicosis rat model. Our study found that individual and combined NPs revealed physiochemical characteristics appropriate for IV administration with sustained-drug release purposes. Physiological evaluations of RSD-induced silicosis rats suggested that no treatment could improve the body weight. Still, they reduced the lung coefficient by maintaining lung moisture. Only (DG+ED)n significantly cleared free lung silica. All interventions were found to attribute the increased per cent cell viability in BALF, reduce cytotoxicity via minimizing LDH levels, and balance the oxidant-antioxidant status in silicotic rats. The expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6, MCP-1, and TGF-ß1) were efficiently down-regulated with NPs interventions compared to pure (DG+ED) treatment. All drug treatments significantly declined, the 8-HdG and HYP productions indicate that RSD-induced oxidative DNA damage and collagen deposition were successfully repaired. Moreover, histopathological investigations proposed that individual or combined drugs NPs interventions could decrease the fibrosis and alveolitis grades in RSD-induced silicosis rats. However, (DG+ED)n intervention significantly inhibited pulmonary fibrosis and alveolitis compared to pure (DG+ED) treatment. In conclusion, the RSD can induce oxidative stress and inflammation in rats, producing reactive oxygen species (ROS)-mediated cytotoxicity to pulmonary cells and leading to silicosis development. The IV administration of combined NP suppressed lung inflammation and collagen formation by maintaining oxidant-antioxidant status and effectively interrupting the fibrosis-silicosis progression. These results may be attributed to the improved bioavailability of DG and ED through their combined nano-encapsulation-mediated targeted drug delivery.

In vitro Evaluation of Fungal Susceptibility and Inhibition of Virulence by Diosgenin.

Fungal infections are a public health problem that mainly affects immunosuppressed people, Candida spp. have been responsible for most sources of contamination and invasive fungal infections described around the world. The need arises to find new therapeutic approaches to combat growing infections. Plants and natural products have been considered a valuable source for discovering new molecules with active ingredients. Diosgenin is a sapogenin found in the families of Leguminosae and Dioscoreaceae, it is obtained mainly from the dioscin saponin through the hydrolysis method, it is a phytochemical that has been highlighted in the treatment of various diseases, as well as in combating microbial resistance. The present study aimed to evaluate the susceptibility of fungal strains to diosgenin, as well as verify the association with the reference drug and evaluate the inhibition of the virulence factor through morphological changes in the yeast state to the filamentous form of hyphae and pseudohyphae in strains of Candida albicans, Candida tropicalis and Candida krusei using the broth microdilution method and microculture technique. Antifungal assays revealed that diosgenin was not able to inhibit the growth of the tested strains. However, it was able to inhibit the fungal dimorphism of the strains evaluated, however further studies are recommended to verify its effectiveness against other virulence factors.

Proposing anti-counterfeiting pharmacopoeia quality markers for Shenlingbaizhu granule based on UHPLC-Q-orbitrap-MS identification.

INTRODUCTION: Shenlingbaizhu granule, a Traditional Chinese Medicine prescription comprising Renshen, Gancao, and Shanyao, is widely consumed in China nowadays. OBJECTIVE: The study tries to propose pharmacopoeia quality markers (Q-markers) to prevent counterfeiting involving Renshen, Gancao, and Shanyao. METHODOLOGY: A novel strategy, that is, library-based ultra-high-performance liquid chromatography-quadrupole-orbitrap mass spectrometry, was used to analyse the lyophilised aqueous powder of Shenlingbaizhu granule. Subsequently, quantum chemistry calculation and UV-vis spectra scanning were also performed through theoretical or experimental approaches. RESULT: Thirty-two isomers have been strictly distinguished, especially positional isomeric isochlorogenic acid B versus isochlorogenic acid C, positional isomeric schaftoside versus isoschaftoside, positional isomeric ginsenoside Rg2 versus 20S-ginsenoside Rg3, and stereoisomeric 20S-ginsenoside Rg3 versus 20R-ginsenoside Rg3. Seventeen compounds were unexpectedly observed, particularly scoparone and pectolinarigenin, while a total of 76 bioactive compounds have been putatively identified in the study. The quantum chemistry calculation and UV-vis spectra scanning results revealed that glycyrrhizic acid, ginsenoside Re, ginsenoside Rb1, and diosgenin displayed different dipole moment values and maximum absorption wavelengths from each other. CONCLUSION: The study recommends glycyrrhizic acid, ginsenoside Re, ginsenoside Rb1, and diosgenin as four anti-counterfeiting Q-markers for the pharmacopoeia. The anti-counterfeiting Q-markers can be detected using conventional HPLC. The observation of 17 unexpected compounds updates our knowledge regarding the bioactives of Shenlingbaizhu granule.

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.

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.

Dietary diosgenin transcriptionally down-regulated intestinal NPC1L1 expression to prevent cholesterol gallstone formation in mice.

BACKGROUND: Cholesterol gallstone disease is a common disease. Reducing cholesterol burden is important to prevent/treat gallstone. In this study, we investigated the application of diosgenin (DG) to prevent the formation of gallstone in mice. METHODS: Adult male C57BL/6J mice were fed with the lithogenic diet (LD) only or LD supplemented with DG or ezetimibe for 8 weeks. Incidences of gallstone formation were documented. Intestine and liver tissues were collected to measure the lipid contents and expression of genes in cholesterol metabolism. Caco2 cells were treated with DG to monitor the regulation on cholesterol absorption and the transcriptional regulation of Npc1l1 gene. Changes of gut microbiota by DG was analyzed. Intraperitoneal injection of LPS on mice was performed to verify its effects on STAT3 activation and Npc1l1 expression in the small intestine. RESULTS: LD led to 100% formation of gallstones in mice. In comparison, dietary DG or ezetimibe supplementary completely prevents gallstones formation. DG inhibited intestinal cholesterol absorption in mice as well as in Caco2 cells by down-regulation of Npc1l1 expression. DG could directly inhibit phosphorylation of STAT3 and its transcriptional regulation of Npc1l1 expression. Furthermore, DG could modulate gut microbiota profiles and LPS mediated STAT3 activation and Npc1l1 expression. CONCLUSION: Our results demonstrated that dietary DG could inhibit intestinal cholesterol absorption through decreasing NPC1L1 expression to prevent cholesterol gallstone formation.

Prediction of CYP2D6 poor metabolizers by measurements of solanidine and metabolites-a study in 839 patients with known CYP2D6 genotype.

PURPOSE: Poor metabolizers (PMs) of the highly polymorphic enzyme CYP2D6 are usually at high risk of adverse effects during standard recommended dosing of CYP2D6-metabolized drugs. We studied if the metabolism of solanidine, a dietary compound found in potatoes, could serve as a biomarker predicting the CYP2D6 PM phenotype for precision dosing. METHODS: The study included 839 CYP2D6-genotyped patients who were randomized by a 4:1 ratio into test or validation cohorts. Full-scan high-resolution mass spectrometry data files of previously analyzed serum samples were reprocessed for identification and quantification of solanidine and seven metabolites. Metabolite-to-solanidine ratios (MRs) of the various solanidine metabolites were calculated prior to performing receiver operator characteristic (ROC) and multiple linear regression analyses on the test cohort. The MR thresholds obtained from the ROC analyses were tested for the prediction of CYP2D6 PMs in the validation cohort. RESULTS: In the test cohort, the M414-to-solanidine MR attained the highest sensitivity and specificity parameters from the ROC analyses (0.98 and 1.00) and highest explained variance from the linear models (R2 = 0.68). Below these thresholds, CYP2D6 PM predictions were tested in the validation cohort providing positive and negative predictive values of 100% for the MR of M414, while similar values for the other MRs ranged from 20.5 to 73.3% and 96.7 to 99.3%, respectively. CONCLUSION: The M414-to-solanidine MR is an excellent predictor of the CYP2D6 PM phenotype. By measuring solanidine and metabolites using liquid chromatography-mass spectrometry in patient serum samples, CYP2D6 PMs can easily be identified, hence facilitating the implementation of precision dosing in clinical practice.

Semi-Synthesis and Biological Evaluation of 25(R)-26-Acetoxy-3ß,5α-Dihydroxycholest-6-One.

Previously, we identified a series of steroids (1-6) that showed potent anti-virus activities against respiratory syncytial virus (RSV), with IC50 values ranging from 3.23 to 0.19 µM. In this work, we first semi-synthesized and characterized the single isomer of 5, 25(R)-26-acetoxy-3ß,5α-dihydroxycholest-6-one, named as (25R)-5, in seven steps from a commercially available compound diosgenin (7), with a total yield of 2.8%. Unfortunately, compound (25R)-5 and the intermediates only showed slight inhibitions against RSV replication at the concentration of 10 µM, but they possessed potent cytotoxicity activities against human bladder cancer 5637 (HTB-9) and hepatic cancer HepG2, with IC50 values ranging from 3.0 to 15.5 µM without any impression of normal liver cell proliferation at 20 µM. Among them, the target compound (25R)-5 possessed cytotoxicity activities against 5637 (HTB-9) and HepG2 with IC50 values of 4.8 µM and 15.5 µM, respectively. Further studies indicated that compound (25R)-5 inhibited cancer cell proliferation through inducing early and late-stage apoptosis. Collectively, we have semi-synthesized, characterized and biologically evaluated the 25R-isomer of compound 5; the biological results suggested that compound (25R)-5 could be a good lead for further anti-cancer studies, especially for anti-human liver cancer.