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.

Two previously undescribed cholestanol saponins from the rhizomes of Paris fargesii var. petiolata.

Two previously undescribed cholestanol saponins, parpetiosides F - G (1-2), and six known analogs (3-8) were isolated from the rhizomes of Paris fargesii var. petiolata. Their structures were elucidated by extensive spectroscopic data analysis and chemical methods. Compound 1 was a rare 6/6/6/5/5 fused-rings cholestanol saponin with disaccharide moiety linked at C-26 of aglycone which was hardly seen in genus Paris. All of these compounds were discovered in this plant for the first time. In addition, the cytotoxicities of saponins (1-8) against three human cancer cell lines (U87, HepG2 and SGC-7901) were evaluated by CCK-8 method, and saponins 5-8 displayed certain cytotoxicities. The strong interactions between saponins 5-8 and SCUBE3, an oncogene for glioma cells, were displayed by molecular docking.

New steroidal saponins from the aerial parts of Paris polyphylla var. yunnanensis and their effects on blood coagulation.

Five new steroidal saponins, paripolins D-H (1-5), and 6 known compounds (6-11) were isolated from the aerial parts of Paris polyphylla var. yunnanensis. The structures of 1-5 were determined using spectroscopic analyses in conjunction with acid hydrolysis. It is for the first time to report the 12-hydroxysteroidal saponins from the genus Paris. The effect of all isolated compounds on blood coagulation was determined in vitro using the plasma recalcification time method. Compounds 1 and 2 showed potent procoagulant activity, and 5-11 exhibited significant anticoagulant activity.

Multimodal integrated strategy for the discovery and identification of antiplatelet aggregation Q-markers in Paris polyphylla var. yunnanensis.

To enhance the quality evaluation and control of traditional Chinese medicine (TCM) and ensure the safety and efficacy of clinical medication, it is imperative to establish a comprehensive quality assessment method aligned with TCM efficacy. This study uses a representative Chinese medicine with multi-origin and multi-efficacy, Paris polyphylla var. yunnanensis (PY), as an illustrative example. Surprisingly, despite the high fingerprint similarity among the 12 batches of PY samples collected from various regions in Yunnan, a notable variation in the composition and content of components was observed. The chromatographic analysis identified seven common peaks, namely, polyphyllin I, polyphyllin II, polyphyllin V, polyphyllin VI, polyphyllin VII, polyphyllin H, and polyphyllin D. In the bioactivity evaluation, an in vitro antiplatelet aggregation model induced by adenosine diphosphate was established, showcasing excellent stability. The maximum antiplatelet aggregation inhibition rate for all PY samples consistently remained stable at 73.1%-99.1%. However, the 50% inhibitory concentration (IC50 ) values exhibited a range from 1.615 to 18.200 mg/mL. This approach not only meets high-throughput screening requirements but also demonstrates remarkable discrimination. The results of chemical and bioactivity evaluations were analyzed using hierarchical cluster analysis and canonical correlation analysis. Polyphyllin I, polyphyllin II, polyphyllin VII, polyphyllin H, and polyphyllin D were identified as the Q-markers for antiplatelet aggregation in PY samples. Validation of the bioactivity for these monomer components aligned with the previously mentioned findings. Notably, this study established a spectrum-effect model for PY samples, enhancing the scientific robustness of the quality evaluation method. Furthermore, these findings offer valuable research insights for improving the quality assessment of other TCMs.

Paris polyphylla saponins II inhibits invasive, migration and epithelial-mesenchymal transition of melanoma cells through activation of autophagy.

Malignant melanoma is a kind of malignant tumor derived from normal epidermal melanocytes or original nevus cells. It has a high degree of malignancy, rapid progress, dangerous condition, and poor prognosis. In recent years, the innovation of traditional Chinese medicine has broadened the scope and effect of tumor treatment. It is a hotspot and breakthrough to find new anti-tumor invasion and migration drugs from natural plants or traditional Chinese medicine. This study explored the role of PPII in promoting autophagy to inhibit EMT of melanoma cells, the role of the PI3K/Akt signaling pathway in the invasion and migration of melanoma cells induced by PPII. We found that PPII effectively inhibited the proliferation, invasion and migration of melanoma B16 and B16F10 in vitro, and induced autophagy. We also established the xenograft tumor and metastatic tumor model of C57BL/6 mice with B16F10 cells. Results showed that PPII effectively inhibited the growth of transplanted tumors, induced autophagy and inhibited the expression level of EMT related protein; Metastasis experiment showed that PPII inhibited the invasion and migration of B16F10, the effect of inhibiting lung metastasis is the most significant. Further mechanism studies showed that the inhibition of PPII on melanoma invasion and migration is related to its induction of autophagy and then inhibition of EMT.

A new insight into material basis of rhizoma Paridis saponins in alleviating pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Paris polyphylla, as a traditional Chinese herbal medicine, was often used to relieve inflammation and pain. Rhizoma Paridis saponins (RPS) as the main active components of Paris polyphylla have excellent analgesic effects. AIM OF THE STUDY: Determine the analgesic material basis of RPS. MATERIALS AND METHODS: LC-MS/MS was used to analyze RPS, plasma after intravenous injection of RPS, and oral administration of RPS. H22 plantar pain model was established to explore the analgesic material basis of RPS. Moreover, correlation analysis, network pharmacology, RT-PCR and molecular docking were applied in this research. RESULTS: RPS had dose-dependently analgesic effects in acetic acid- and formalin-induced pain models. LC-MS/MS detection indicated that diosgenin as the metabolite of RPS mainly distributed in brain tissues. The addition of antibiotics increased the anti-tumor effect of RPS, but reduced its analgesic effect. Network pharmacology, RT-PCR and molecular docking showed that diosgenin exerted its analgesic effect through SRC and Rap1 signaling pathway. CONCLUSION: Diosgenin exhibited analgesic effects, while saponins had good anti-tumor effects in RPS. This discovery provided a better indication for the later application of RPS in anti-tumor and analgesic settings.

Comparative metabolome profiling of Paris polyphylla var. yunnanensis cultivars and Paris luquanensis and their biological activity in zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Paris polyphylla var. Yunnanensis (Franch.) Hand.-Mazz., a perennial medicinal herb commonly known as "Chonglou" in Chinese, is mainly effective against innominate toxin swelling, insect sting, snake bite, traumatic injuries and various inflammatory. It is also recorded with mild toxicity. The rare species Paris luquanensis H. Li has been also used as folk medicine in Yunnan province for the same effects. Compared with P. polyphylla var. Yunnanensis (35-100 cm in height), this species has variegated leaves, and grows slower and is therefore shorter (6-23 cm in height). There are a number of different cultivars based on the shape of the petal and the height of Paris plant. However, currently, investigations into the differences of the chemical profiling of these cultivars are lacking. AIM OF THE STUDY: This study aims to: (1) examine metabolites variations in Paris polyphylla var. Yunnanensis cultivars and Paris luquanensis; (2) investigate the different metabolite accumulation patterns between rhizomes and leaves and provide more useful information for the application of P. polyphylla var. Yunnanensis leaves; (3) compare in vivo effects on the recruitment of reactive oxygen species (ROS) and Neutrophils and toxic effects in zebrafish model between leaves and rhizomes of P. polyphylla var. Yunnanensis and P. luquanensis. MATERIALS AND METHODS: The change patterns of metabolites in the leaves and rhizomes of four P. polyphylla var. Yunnanensis cultivars and one P. luquanensis cultivar were analyzed using an UPLC-ESI-MS/MS system. The total phenolic acid, total flavonoid, total saponin components and in vitro antioxidant activities were determined by spectrophotometric methods. The in vivo toxicity and their effects on the recruitment of ROS and neutrophils in zebrafish model were performed. RESULTS: The widely targeted metabolomics method detected 695 metabolites in tested samples and classified as 15 known classes according to structures of the metabolites. By overall-comparing the SDMs discerned between leaves and rhizomes of each samples, 161 metabolites were substantially altered in all the cultivars. There are 62 and 64 SDMs showing constitutive differential accumulation between leaves and rhizomes of P. polyphylla var. Yunnanensis (samples A-D) and P. luquanensis (sample E), respectively. The levels of TSC, TPC and TFC decreased significantly in leaves as compared to rhizomes for all cultivars, with the exception of TPC in cultivar A, which is almost the same in leave and rhizome. The DPPH scavenging property and FRAP values of rhizomes are higher than those of leaves for all cultivars. However, there is no distinct different between leaves and rhizomes of different sample extracts for in vivo effects on the recruitment of ROS and neutrophils in zebrafish model. BL extracts showed high toxicity to the developing embryos. CONCLUSION: As far as we are concerned, this study analyzes the P. polyphylla var. Yunnanensis and P. luquanensis variegation from the perspective of the metabolites pattern for the first time. The results give a valuable insight into the specie metabolic profiling and in vivo anti-oxidant, anti-inflammatory and toxic effects of these Paris plants.

Identification, Phytochemistry and Biological Activities of Paris polyphylla on Hepatocellular Carcinoma.

<b>Background and Objective:</b> Liver cancer is the common cause of cancer death. <i>Paris polyphylla</i> is used as a traditional folk medicine in Vietnam to treat pneumonia, mastitis, bruises and fractures but no study was available regarding its ability to treat liver cancer or slow its growth. In this study, <i>Paris polyphylla</i> samples were identified and evaluated cytotoxic activity against the liver cancer cells. <b>Materials and Methods:</b> <i>Paris polyphylla</i> species were collected from various areas in Yen Bai, Vietnam, which were identified by comparative morphological method and DNA barcoding for the <i>18S, matK</i> genes and <i>ITS</i> region. <i>Paris polyphylla</i> samples were dried until constant weight, ground into a fine powder and extracted in various solvents. The bioactivity of these extracts were done by the MTT assay. <b>Results:</b> The sequences of <i>18S, matK</i> genes and <i>ITS</i> region were high similarity to sequences of <i>P. polyphylla</i> in the National Center for Biotechnology Information. The N-hexane and ethyl acetate fractions were produced from the methanol extract of <i>P. polyphylla</i>. The TLC results showed that there was a significant difference in the component of n-hexane and ethyl acetate fraction. The N-hexane fraction contains mainly low-polarity and non-polarity substances. While ethyl acetate fraction consists mainly of polar substances. In addition, ethyl acetate fraction was shown the strongest cytotoxic activity on the cancer cell lines HepG2 and Huh7 with the evaluation of IC₅₀ = 115.11±2.77 µg mL¹ and IC₅₀ = 148.11±1.78 µg mL¹. <b>Conclusion:</b> The extract of <i>Paris polyphylla</i> demonstrated strong potential to inhibit the growth of the liver cancer cell line. The ethyl acetate fraction has the highest ability for cytotoxicity on the liver and cell line at a concentration of 200 µg mL¹ through MTT.

Steroid and triterpenoid saponins from the rhizomes of Paris polyphylla var. stenophylla.

Five new saponins, including three steroid saponins, paristenoids A-C (1-3), and two triterpenoid saponins, paristenoids D-E (4-5), along with four known ones (6-9) were isolated from the rhizomes of Paris polyphylla var. stenophylla. The structures of the isolated compounds were identified mainly by detailed spectroscopic analysis, including extensive 1D and 2D NMR, MS, as well as chemical methods. Compound 3 is a new cyclocholestanol-type steroidal saponin with a rare 6/6/6/5/5 fused-rings cholestanol skeleton, and this skeleton has been first found from the genus Paris. The cytotoxicities of the isolated compounds against three human three glioma cell lines (U87MG, U251MG and SHG44) were evaluated, and compound 7 displayed certain inhibitory effect with IC50 values of 15.22 ± 1.73, 18.87 ± 1.81 and 17.64 ± 1.69 µmol·L-1, respectively.

[New steroidal saponins from aerial parts of Paris polyphylla var. chinensis].

The shortage of Paridis Rhizoma promotes comprehensive utilization and development research of waste aerial parts of the original plant. The chemical compositions of the aerial parts of Paris polyphylla var. chinensis were clarified based on the ultrahigh performance liquid chromatography tandem quadrupoles time of flight mass spectrometry(UPLC-QTOF-MS/MS) in the previous investigation, and a series of flavonoids and steroidal saponins were isolated. The present study continued the isolation and structure identification of the new potential compounds discovered based on UPLC-QTOF-MS/MS. By using silica gel, ODS, flash rapid preparation, and other column chromatography techniques, combined with prepared high performance liquid chromatography, five compounds were isolated from the 75% ethanol extract of the aerial parts of P. polyphylla var. chinensis, and their structures were identified by spectral data combined with chemical transformations, respectively, as(23S,25R)-23,27-dihydroxy-diosgenin-3-O-α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→3)]-ß-D-glucopyranoside(1),(25R)-26-O-ß-D-glucopyranosyl-furost-5-en-3ß,22α,26-triol-3-O-α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranoside(2),(25R)-27-O-ß-D-glucopyranosyl-5-en-3ß,27-dihydroxyspirost-3-O-α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranoside(3),(25R)-27-O-ß-D-glucopyranosyl-5-en-3ß,27-dihydroxyspirost-3-O-α-L-rhamnopyranosyl-(1→2)-[ß-D-glucopyranosyl-(1→3)]-ß-D-glucopyranoside(4), and aculeatiside A(5). Among them, compounds 1-4 were new ones, and compound 5 was isolated from P. polyphylla var. chinensis for the first time.

Population Genetic Analysis of Paris polyphylla var. yunnanensis Based on cpDNA Fragments.

Paris polyphylla var. yunnanensis is a well-known medicinal plant that is mainly distributed in Southwest China; however, its genetic diversity and biodiversity processes are poorly understood. In this study, the sequences of cpDNA trnL-trnF fragments of 15 wild populations and 17 cultivated populations of P. polyphylla var. yunnanensis were amplified, sequenced, and aligned to study the population genetics of this species. Genetic diversity was analyzed based on nucleotide diversity, haplotype diversity, Watterson diversity, population-level diversity, and species-level genetic diversity. Genetic structure and genetic differentiation were explored using haplotype distribution maps and genetic distance matrices. A total of 15 haplotypes were identified in the 32 populations of P. polyphylla var. yunnanensis. Five unique haplotypes were identified from the fourteen haplotypes of the cultivated populations, while only one unique haplotype was identified from the ten haplotypes of the wild populations. The haplotype richness and genetic diversity of the cultivated populations were higher than those of the wild populations (HT = 0.900 vs. 0.861). In addition, there were no statistically significant correlations between geographic distance and genetic distance in the cultivated populations (r = 0.16, p > 0.05), whereas there was a significant correlation between geographical distance and genetic structure in the wild populations (r = 0.32, p > 0.05), indicating that there was a geographical and genetic connection between the wild populations. There was only 2.5% genetic variation between the wild populations and cultivated populations, indicating no obvious genetic differentiation between the wild and cultivated populations. Overall, the genetic background of the cultivated populations was complex, and it was hypothesized that the unique haplotypes and higher diversity of the cultivated populations were caused by the mixed provenance of the cultivated populations.

Anatomical, Morphological, and Chemical Characterizations and Biological Activities of Gagea taurica Steven (Liliaceae): A New Record for the Turkish Flora.

Gagea genus, which is native to the Mediterranean and Black Sea regions, has attracted significant attention due to its biodiversity and potential health benefits. In this study, the biochemical composition and biological activities of methanol extracts from various parts of G. taurica were investigated, along with their anatomical and morphological characteristics. The best antimicrobial activity was found to be MeOH extracts of corm and leaf against several Candida strains with MIC=640 µg/mL. The highest level of phenolics together with significant results of antioxidant activities were observed in flowers extracts. The α-amylase inhibition assay results showed that the highest inhibition percentage was observed with acarbose (59 %), followed by leaf extract (43 %). Leaf exhibited the most effective inhibitory activity in AChE inhibition assay, whereas flower demonstrated the most significant inhibitory activity in BChE inhibition assay. Hesperidin was found as 1621.0001 ng/ml value in flower extract and 283.9339 ng/ml value leaf.

Optimum Reaction Conditions for the Synthesis of Selenized Ornithogalum caudatum Ait. (Liliaceae) Polysaccharides and Measurement of Their Antioxidant Activity In Vivo.

This study determined the optimum reaction conditions for synthesizing selenium-containing polysaccharides. Polysaccharide IIA (with the highest yield) from Ornithogalum caudatum Ait. (Liliaceae) (OCAPIIA) was extracted and purified. Then, three parameters were selected to optimize the synthesis of selenized OCAPIIA (Se-OCAPIIA) using the Box-Behnken design (BBD) and response surface methodology (RSM). The morphology of Se-OCAPIIA was analyzed by scanning electron microscopy (SEM). The characteristic peaks and the monosaccharide composition of Se-OCAPIIA were evaluated by Fourier-transform infrared spectroscopy and gas chromatography. A D-galactose-induced aging mouse model was established, and the in vivo antioxidant activity of Se-OCAPIIA was measured. The optimal conditions for the synthesis of Se-OCAPIIA were as follows: reaction temperature, 72.38 °C; Na2SeO3 to OCAPIIA mass ratio, 0.93 g/g; and reaction time, 8.05 h. The selenium content of Se-OCAPIIA obtained using the optimized process was 3.131 ± 0.090 mg/g, close to the maximum predicted value (3.152 mg/g). Se-OCAPIIA contained D-mannose, D-glucose, and D-galactose at a molar ratio of 1.00:0.34:0.88. SEM showed that Se-OCAPIIA was spherical and flocculated. Compared with OCAPIIA, Se-OCAPIIA exhibited two characteristic peaks at 833 and 610 cm-1 in the infrared spectrum. Se-OCAPIIA increased catalase, glutathione peroxidase, and superoxide dismutase activities and decreased MDA concentrations in the mouse liver. Moreover, Se-OCAPIIA treatment improved liver morphology, decreased the levels of IL-1ß and IL-6, and increased IL-10 concentration. In conclusion, the synthesis of Se-OCAPIIA is effective, simple, and feasible. Se-OCAPIIA demonstrated high antioxidant activity in vivo and is a promising antioxidant and therapeutic agent.

[Gene clone and functional identification of sterol glycosyltransferases from Paris polyphylla var. yunnanensis].

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 ß-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.

[Chemical constituents from Paris rugosa rhizomes and their antimicrobial activities].

Paris rugosa(Melanthiaceae) only grows in Yunnan province of China at present, and its chemical constituents have not been systematically studied. In this study, nine compounds, including one new compound pariposide G(1) and eight known compounds of cerin(2), stigmast-4-en-3-one(3), ß-ecdysone(4), ophiopogonin C'(5), methyl protogracillin(6), gracillin(7), parissaponin H(8), and parisyunnanoside G(9), were isolated and identified from the ethanol extract of P. rugosa rhizomes by column chromatography methods and semi-preparative high-performance liquid chromatography(HPLC). Compounds 1-9 were isolated from this plant for the first time. The antibacterial and antifungal activities of all the compounds were evaluated. The results showed that ophiopogonin C' had strong inhibitory effects on Candida albicans [MIC_(90)=(4.68±0.01) µmol·L~(-1)] and the fluconazole-resistant strain of C. albicans [MIC_(90)=(4.66±0.02) µmol·L~(-1)].

Steroidal sapogenins from the aerial parts of Paris polyphylla var. yunnanensis and activity evaluation.

Phytochemical investigation of an extract of the aerial parts of Paris polyphylla var. yunnanensis resulted in the identification of three new steroidal sapogenins, namely as paripolins A-C (1-3). With the aid of comprehensive spectroscopic techniques (NMR, IR, UV, MS), the structures of all isolated compounds were elucidated and subsequently screened for anti-inflammatory activity.

Dynamic variation of Paris polyphylla root-associated microbiome assembly with planting years.

MAIN CONCLUSION: P. polyphylla selectively enriches beneficial microorganisms to help their growth. Paris polyphylla (P. polyphylla) is an important perennial plant for Chinese traditional medicine. Uncovering the interaction between P. polyphylla and the related microorganisms would help to utilize and cultivate P. polyphylla. However, studies focusing on P. polyphylla and related microbes are scarce, especially on the assembly mechanisms and dynamics of the P. polyphylla microbiome. High-throughput sequencing of the 16S rRNA genes was implemented to investigate the diversity, community assembly process and molecular ecological network of the bacterial communities in three root compartments (bulk soil, rhizosphere, and root endosphere) across three years. Our results demonstrated that the composition and assembly process of the microbial community in different compartments varied greatly and were strongly affected by planting years. Bacterial diversity was reduced from bulk soils to rhizosphere soils to root endosphere and varied over time. Microorganisms benefit to plants was selectively enriched in P. polyphylla roots as was its core microbiome, including Pseudomonas, Rhizobium, Steroidobacter, Sphingobium and Agrobacterium. The network's complexity and the proportion of stochasticity in the community assembly process increased. Besides, nitrogen metabolism, carbon metabolism, phosphonate and phosphinate metabolism genes in bulk soils increased over time. These findings suggest that P. polyphylla exerts a selective effect to enrich the beneficial microorganisms and proves the sequential increasing selection pressure with P. polyphylla growth. Our work adds to the understanding of the dynamic processes of plant-associated microbial community assembly, guides the selection and application timing of P. polyphylla-associated microbial inoculants and is vital for sustainable agriculture.

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.

Cytotoxic steroidal glycosides from the rhizomes of Paris polyphylla var. yunnanensis.

Paris polyphylla var. yunnanensis (Franch.) Hand.-Mazz. (Melanthiaceae), an important specie of the genus Paris, has long been in a traditional Chinese medicine (TCM) for a long time. This study aimed to isolate and identify the structures of bioactive saponins from the rhizomes of P. polyphylla var. yunnanensis and evaluate their cytotoxicity against BxPC-3, HepG2, U373 and SGC-7901 carcinoma cell lines. Seven previously undescribed and seven known saponins were identified, and Paris saponins VII (PSVII) showed significant cytotoxicity against the BxPC-3 cell line with IC50 values of 3.59 µM. Furthermore, flow cytometry, transmission electron microscopy and western-bolt analysis revealed that PSVII inhibited the proliferation of BxPC-3 cells and might be involved in inducing apoptosis and pyroptosis by activating caspase-3, -7 and caspase-1, respectively.

A novel sterol glycosyltransferase catalyses steroidal sapogenin 3-O glucosylation from Paris polyphylla var. yunnanensis.

BACKGROUND: Paris polyphylla var. yunnanensis is an important medicinal plant, and the main active ingredient of the plant is polyphyllin, which is a steroid saponin with pharmacological activities. The central enzyme genes participating in the biosynthesis of polyphyllin are increasingly being uncovered; however, UGTs are rarely illustrated. METHODS AND RESULTS: In this study, we cloned a new sterol glycosyltransferase from Paris polyphylla var. yunnanensis and identified its catalytic function in vitro. PpUGT6 showed the ability to catalyse the C-3 glycosylation of pennogenin sapogenin of polyphyllin, and PpUGT6 showed catalytic promiscuity towards steroids at the C-17 position of testosterone and methyltestosterone and the triterpene at the C-3 position of glycyrrhetinic acid. Homology modelling of the PpUGT6 protein and virtual molecular docking of PpUGT6 with sugar acceptors and donors were performed, and we predicted the key residues interacting with ligands. CONCLUSIONS: Here, PpUGT6, a novel sterol glycosyltransferase related to the biosynthesis of polyphyllin from P. polyphylla, was characterized. PpUGT6 catalysed C-3 glycosylation to pennogenin sapogenin of polyphyllin, which is the first glycosylation step of the biosynthetic pathway of polyphyllins. Interestingly, PpUGT6 demonstrated glycodiversification to testosterone and methyltestosterone at C-17 and triterpene of glycyrrhetinic acid at the C-3 position. The virtual molecular docking of PpUGT6 protein with ligands predicted the key residues interacting with them. This work characterized a novel SGT glycosylating pennogenin sapogenin at C-3 of polyphyllin from P. polyphylla and provided a reference for further elucidation of the phytosterol glycosyltransferases in catalytic promiscuity and key residues interacting with substrates.