Characterization of a glycosyltransferase from Paris polyphylla for application in biosynthesis of rare ophiopogonins and ginsenosides.

Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6'-O-glucosyltransferase PpUGT7 (PpUGT91AH7) was functionally characterized from Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6' position of diosgenin-3-O-rhamnosyl-(1 â†’ 2)-glucoside (1), pennogenin-3-O-rhamnosyl-(1 â†’ 2)-glucoside (2), and diosgenin-3-O-glucoside (5). The KM and Kcat values of PpUGT7 towards the substrate 2 were 8.4 µM and 2 × 10-3 s-1, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor 2 and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-O-glucosyl-(1 â†’ 6)-glucoside (10). Firstly, two monoglycosides 9 and 11 were generated using a known sterol 3-O-ß-glucosyltransferase PpUGT80A40 with ruscogenin (7) and 20(S)-protopanaxadiol (8) as substrates, which were further glycosylated to the corresponding diglycosides 10 and 12 under the catalysis of PpUGT7. In addition, compounds 7-11 were found to show inhibitory effects on the secretion of TNF-α and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in P. polyphylla var. yunnanensis, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.

Exploring the potential of Paris polyphylla var. yunnanensis pollen manipulation in modifying seed dormancy.

Paris polyphylla var. yunnanensis, a well-known Chinese medicinal herb, shows a unique physiological trait characterized by the cyclic opening and closing of its anthers after pollen maturation. The aim of this study was to explore the implications of this phenomenon on breeding. RNA sequencing coupled with methylation sequencing was used to scrutinize and compare gene expression profiles and methylation alterations in pollen and seeds during anther opening and closing, along with cold exposure. Genes enriched within Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were examined to identify gene clusters susceptible to temperature-related methylation changes in both pollen and seeds. Four pollen treatment models, namely, normal control, "pollen protected from low temperatures," "pollen from just-opened anther," and "pollen from close-blocked anther," were used to produce corresponding seeds via artificial pollination. Subsequently, qRT-PCR was used to validate modifications in the expression patterns of marker genes in pollinated seeds under diverse treatment scenarios. Genes exhibiting significant differences in expression between anthers and normal tissues, along with gene regions linked to methylation variations attributed to low-temperature-treated pollen and seeds, were identified through transcriptomic analysis. Convergence was observed in three signaling pathways: oxidative phosphorylation (ko00190), plant hormone signal transduction (Ko04075), and zeatin biosynthesis (ko00908). Notably, gene clusters prone to temperature-induced methylation changes, such as NADH-ubiquinone oxidoreductase chain 5, plasma membrane ATPase 4, cytochrome c oxidase subunit 2, cis-zeatin O-glucosyltransferase, ABSCISIC ACID-INSENSITIVE 5-like protein 4, and indole-3-acetic acid-amido synthetase (IAAS), were identified. Evaluation using various pollen pollination models revealed altered expression patterns of five dormancy-regulating marker genes: IAAS, sucrose synthase (SUS), gibberellin 2-oxidase (GA2ox), ABA INSENSITIVE 2 (ABI2), and auxin-repressed protein (ARP), in seeds pollinated with pollen from close-blocked anthers, cold-protected pollen, and pollen from freshly opened anthers. The close-blocked anther treatment led to significantly upregulated expression of IAAS, SUS, GA2ox, and ABI2, whereas ARP expression decreased markedly, indicating a propensity toward prolonged seed dormancy. Conversely, in the low-temperature-protected anther model, SUS, ARP, GA2ox, and IAAS exhibited reduced expression levels, whereas the expression of ABI2 was upregulated, overall facilitating seed germination.

Response Surface Optimization for Water-Assisted Extraction of Two Saponins from Paris polyphylla var. yunnanensis Leaves.

The process of extracting polyphyllin II and polyphyllin VII by water-assisted extraction was established and optimized in this study. Response surface methodology was used to establish a prediction model to optimize the extraction conditions. Based on the one-way test, the Box-Behnken design with three factors and three levels was used for the experimental program, and the composition analysis was carried out by high-performance liquid chromatography (HPLC). The optimal extraction conditions for polyphyllin II and polyphyllin VII were as follows: extraction time of 57 and 21 min, extraction temperature of 36 and 32 °C, solid-to-liquid ratio of 1:10 and 1:5 g/mL, respectively, and the yields of polyphyllin II and polyphyllin VII were 1.895 and 5.010%, which was similar to the predicted value of 1.835 and 4.979%. The results of the ANOVA showed that the model fit was good, and the Box-Behnken response surface method could optimize the water-assisted extraction of saponins from the leaves of Paris polyphylla var. yunnanensis. This study provides a theoretical basis for the application of polyphyllin II and polyphyllin VII in pharmaceutical production.

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.

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.

Analysis of the heavy metal contents' effect on steroidal saponins and the anti-breast cancer activity of Paris polyphylla var. yunnanensis.

Background: P. polyphylla var. yunnanensis, as a near-threatened and ethnic medicine in China, used to be a key ingredient in traditional Chinese medicine in treatment of traumatic injuries, sore throat, snakebites, and convulsions for thousands of years. However, there were no reports on the inverse relationship between the contents of heavy metals and saponins and its anti-breast cancer pharmacological activity in P. polyphylla var. yunnanensis. Methods: The present study aimed to reveal the characteristics of heavy metal contents and saponins and its anti-breast cancer pharmacological activity and their interrelationships in P. polyphylla var. yunnanensis from different production areas. The contents of heavy metal and steroidal saponins in P. polyphylla var. yunnanensis were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and the high-performance liquid chromatography technique, respectively. The Pearson correlation was used to study the correlation between saponins and heavy metals. 4T1 mouse mammary tumor cells were selected and cultivated for antitumor studies in vitro. Cell Counting Kit-8 (CCK-8) assay, Hoechst staining, and flow cytometry analysis were used for the examination of the proliferation and apoptosis of 4T1 tumor cells. Mouse breast cancer 4T1 cells were subcutaneously injected into BALB/c mice to construct a tumor model to explore the in vivo inhibitory effect on breast cancer. TUNEL assay and immunohistochemistry were used for the examination of the effect of P. polyphylla var. yunnanensis from different origins on cancer cell proliferation and apoptosis induction in 4T1 tumor mice. Results: Heavy metal contents were highly correlated with the content of steroidal saponins. The overall content of 10 metals in the three producing origins was of the order C3 >C2 >C1. The total content of eight steroidal saponins in the extracts of P. polyphylla var. yunnanensis from three different origins was C1 >C2 >C3. The Pearson correlation study showed that in all of the heavy metals, the contents of Cd and Ba were positively correlated with the main steroidal saponins in P. polyphylla var. yunnanensis, while Al, Cr, Cu, Fe, Zn, As, Hg, and Pb showed a negative correlation. In vitro experiments showed that the extracts of P. polyphylla var. yunnanensis from three origins could inhibit the proliferation and induce cell apoptosis of 4T1 cells in a concentration- and time-dependent manner, especially in the C1 origin. In vivo experiments showed that the extract of P. polyphylla var. yunnanensis from the three origins could inhibit the growth of tumors and induce the apoptosis of tumor cells. In the three origins, C1 origin had the lowest total heavy metal level but the highest total steroidal saponin level. Therefore, it showed a better effect in reducing the expression of the human epidermal growth factor receptor 2 (HER2) and Kiel 67 (Ki67) and increasing the expression of p53 in tumor tissues compared to the other origins. In conclusion, in the three origins, C1 origin exhibits antitumor pharmacological effects in vivo and in vitro which are better than those in the other origins. Conclusion: In this study, we found that with the increase of the heavy metal content, the content of steroid saponins and anti-breast cancer activity decreased. The results showed that the high content of the total heavy metals may not be conducive to the accumulation of steroidal saponins in P. polyphylla var. yunnanensis and lead to the low anti-breast cancer activity. The results of this study suggest that the content of heavy metals should be controlled in the artificial cultivation process of P. polyphylla var. yunnanensis.

First report of Lily virus X infecting Paris polyphylla var. yunnanensis in Yunnnan, China.

Lily virus X (LVX) is a positive-sense ssRNA virus belonging to the genus Potexvirus in the family Alphaflexiviridae. LVX is known to infect plants of the genera Lilium and Tricyrtis in the family Liliacea. LVX was first reported in an asymptomatic lily (Lilium formosanum) from England (Stone, 1980), but has been shown to infect plants in the Netherlands (Chen et al. 2005), the United States (Jordan et al. 2008) and Japan (Nijo et al. 2018). To date, the complete genomes of two LVX isolates from the Netherlands and Japan have been reported. Paris polyphylla var. yunnanensis, known as Dianchonglou in China, is a perennial plant of the family Melanthiaceae (formerly belonging to the family Trillium). In China, its rhizome is commonly used as an antispasmodic agent for stroke and cancer treatment (Chang et al. 2017). From 2019 to 2022, leaf mottle and shrinkage which are typical symptoms of viral infections were observed on the leaves of P. polyphylla var. yunnanensis plants in Dianchonglou fields in Qujing, Yunnan. Disease incidence ranged from 19% to 45% across 5 fields (90 plants per field) in Qujing. To identify the possible viral pathogen(s) associated with the disease, the mirVanaTM miRNA isolation Kit was used to extract total RNA was from a mixed sample pool of 5 symptomatic leaf samples collected from the 5 fields. RNA sequencing library was constructed using TruSeqTM RNA sample preparation kit. Sequencing on the Illumina HiSeqTM 2500 platform (Illumina, USA) with 125-bp paired-end reads yielded 23,077,786 raw reads. 22,534,100 clean reads were obtained by removing reads of low quality and poly-N using Trimmomatic software (Bolger et al. 2014). By utilizing the paired-end splicing method in Trinity software (Grabherr et al. 2011) the the raw reads were De novo assembled into 184,596 contigs, of which 303 were related to viruses, including Paris mosaic necrosis virus (PMNV), Pear alphapartitivirus (PAPV), Dahlia mosaic virus (DMV), and Lily virus X (LVX). BLASTn analysis revealed that 12 contigs (lengths ranging from 344 nt to 5,981 nt, query cover 6% to 99%) were most similar (57.32% to 91.67% nt identities) to the genome sequences of LVX, suggesting a possible infection of LVX in the plants. To confirm the result, a full-length genomic sequence of LVX was obtained by reverse transcription polymerase chain reaction (RT-PCR) using specific primers designed based on the sequence of the assembled contigs. The PCR products were cloned into pGEM-T vector (Promega Corporation, USA) and sequenced using the Sanger method (Sangon Biotech, Shanghai, China). The obtained full-length genomic sequence of the LVX isolate (LVX-PP, accession number OM100017) was 5,981 nt in length. BLASTp analysis demonstrated that the putative Rep and CP of LVX-PP shared 76.27% to 81.05% and 80.81% to 81.82% aa sequence similarities with that of other LVX isolates, respectively. Maximum-likelihood phylogenetic trees inferred from the Rep and CP aa sequences showed that LVX-PP clustered closely with LVX isolates. The leaf samples were further analyzed using a lily virus X (LVX) ELISA kit (DEIAPV181, Creative Diagnostics, U.S.A.). Healthy P. polyphylla var. yunnanensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for all five symptomatic plants (OD = 1.259 ± 0.007) relative to the negative (OD = 0.099) and blank (OD = 0.073) controls. These results indicate that LVX can infect P. polyphylla var. yunnanensis. To our knowledge, this is the first report that LVX has been detected in P. polyphylla var. yunnannensis. This study will serve as an important reference for the study of the host range of LVX. Further studies will be required to determine how LVX spreads between P. polyphylla var. yunnannensis and other host plants.

Unraveling the serial glycosylation in the biosynthesis of steroidal saponins in the medicinal plant Paris polyphylla and their antifungal action.

Sugar-sugar glycosyltransferases play important roles in constructing complex and bioactive saponins. Here, we characterized a series of UDP-glycosyltransferases responsible for biosynthesizing the branched sugar chain of bioactive steroidal saponins from a widely known medicinal plant Paris polyphylla var. yunnanensis. Among them, a 2'-O-rhamnosyltransferase and three 6'-O-glucosyltrasferases catalyzed a cascade of glycosylation to produce steroidal diglycosides and triglycosides, respectively. These UDP-glycosyltransferases showed astonishing substrate promiscuity, resulting in the generation of a panel of 24 terpenoid glycosides including 15 previously undescribed compounds. A mutant library containing 44 variants was constructed based on the identification of critical residues by molecular docking simulations and protein model alignments, and a mutant UGT91AH1Y187A with increased catalytic efficiency was obtained. The steroidal saponins exhibited remarkable antifungal activity against four widespread strains of human pathogenic fungi attributed to ergosterol-dependent damage of fungal cell membranes, and 2'-O-rhamnosylation appeared to correlate with strong antifungal effects. The findings elucidated the biosynthetic machinery for their production of steroidal saponins and revealed their potential as new antifungal agents.

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.

Cytotoxic and pro-apoptotic effects of botanical drugs derived from the indigenous cultivated medicinal plant Paris polyphylla var. yunnanensis.

Background: Cancer is one of the top two leading causes of death worldwide. Ethnobotanical research, it is one of methods, which is able to discover effective anticancer drugs based on "prototype" of indigenous people's historical experiences and practices. The rhizomes of Paris polyphylla var. yunnanensis (Franch.) Hand.-Mazz. have been used as botanical drugs to treat cancer by Yi, Bai, Dai, and Naxi ethnic groups in Yunnan, China, where this species is widely cultivated in a large scale in Yunnan. Materials and methods: To identify the substances of anticancer activities based on indigenous medicine knowledge, chromatography was performed to separate saponins from the rhizomes of P. polyphylla var. yunnanensis, followed by spectroscopy to determine the structure of six isolated saponins. The cytotoxicity of five extracts and six pure compounds were evaluated by MTS method. Quantitative determination of total saponins of P. polyphylla var. yunnanensis was analyzed by HPLC. Cell cycle assay, apoptosis assay, and mitochondrial membrane potential were used to evaluate the pro-apoptotic activity in vitro. Results: Five extracts and six pure saponins showed significant inhibitory cytotoxic activities of three human liver cancer cell lines (SMMC-7721, HepG2, and SK-HEP-1) and one non-small-cell lung cancer cell line (A549). The contents of Paris saponins I, II, and VII were 6.96% in the rhizomes of P. polyphylla var. yunnanensis, much higher than Chinese Pharmacopoeia standards (0.6%). Six saponins induced significant apoptosis and cell cycle arrest in three human cancer cell lines (A549, SMMC-7721, and HepG2), which was associated with the loss of mitochondrial membrane potential. Conclusion: The result of this study support that cultivated P. polyphylla var. yunnanensis could be a substitute for wild resource as an anticancer medicine based on indigenous medicine knowledge.

Effects of Organophosphate-Degrading Bacteria on the Plant Biomass, Active Medicinal Components, and Soil Phosphorus Levels of Paris polyphylla var. yunnanensis.

Paris polyphylla var. yunnanensis, a medicinal plant that originated in Yunnan (China), has been over-harvested in the wild population, resulting in its artificial cultivation. Given the negative environmental impacts of the excessive use of phosphorus (P) fertilization, the application of organophosphate-degrading bacteria (OPDB) is a sustainable approach for improving the P use efficiency in Paris polyphylla var. yunnanensis production. The present work aimed to analyze the effects of three organic phosphate-solubilizing bacteria of Bacillus on the yield and quality of P. polyphylla var. yunnanensis and the P concentrations in the soil. All the inoculation treatments distinctly increased the rhizome biomass, steroidal, and total saponin concentrations of the rhizomes and the Olsen-P and organic P in the soil. The highest growth rate of rhizomes biomass, steroidal saponins, available phosphorus, and total phosphorus content was seen in the S7 group, which was inoculated with all three OPDB strains, showing increases of 134.58%, 132.56%, 51.64%, and 17.19%, respectively. The highest total saponin content was found in the group inoculated with B. mycoides and B. wiedmannii, which increased by 33.68%. Moreover, the highest organic P content was seen in the group inoculated with B. wiedmannii and B. proteolyticus, which increased by 96.20%. In addition, the rhizome biomass was significantly positively correlated with the saponin concentration, together with the positive correlation between the Olsen-P and organic P and total P. It is concluded that inoculation with organophosphate-degrading bacteria improved the biomass and medicinal ingredients of the rhizome in P. polyphylla var. yunnanensis, coupled with increased soil P fertility, with a mixture of the three bacteria performing best.

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.

Separation and Purification of Two Saponins from Paris polyphylla var. yunnanensis by a Macroporous Resin.

An effective method for separating and purifying critical saponins (polyphyllin II and polyphyllin VII) from a Paris polyphylla var. yunnanensis extract was developed in this study which was environmentally friendly and economical. Static adsorption kinetics, thermodynamics, and the dynamic adsorption-desorption of macroporous resins were investigated, and then the conditions of purification and separation were optimized by fitting with an adsorption thermodynamics equation and a kinetic equation. Effective NKA-9 resin from seven macroporous resins was screened out to separate and purify the two saponins. The static adsorption and dynamic adsorption were chemical and physical adsorption dual-processes on the NKA-9 resin. Under the optimum parameters, the contents of polyphyllin II and polyphyllin VII in the product were 17.3-fold and 28.6-fold those in plant extracts, respectively. The total yields of the two saponins were 93.16%. This research thus provides a theoretical foundation for the large-scale industrial production of the natural drugs polyphyllin II and polyphyllin VII.

[Research progress of steroidal saponins in Paris polyphylla var. yunnanensis and their microbial transformation].

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.

First Report of Tobacco Mosaic Virus Infection in Paris polyphylla var. yunnanensis in China.

Paris polyphylla var. yunnanensis is a perennial herb in the family Trilliacea. The plants have immense medicinal and economic importance (Chen et al., 2021). Large-scale artificial planting has led to the emergence of various viral diseases in Paris polyphylla var. yunnanensis, including paris virus 1 (ParV1), paris mosaic necrosis virus (PMNV), paris polyphylla virus X, and pepper mild mottle virus (PMMoV) (Chen et al., 2021; Chen et al., 2022). However, tobacco mosaic virus (TMV) had not been reported as a pathogen on this host. In September 2021, symptoms of leaf shrinking, withering and mottling, and the plants demonstrating dwarfing first observed on Paris polyphylla var. yunnanensis in Qujing Province, Yunnan, China (Suppl Figure 1A). Leaves with these characteristic symptoms were collected from 20 plants. Virus particles in the samples were observed by transmission electron microscopy (TEM) using negative staining (Zhang et al., 2016). These samples revealed the presence of rod-shaped virions, which were approximately 300 nm long with a diameter of approximately 18nm (Suppl Figure 1B). Based on particle morphology these were identified as a putative Tobamovirus. To further identify the exact virus, total RNA was obtained using an RNA-easy Isolation Reagent (TaKaRaBiotech, Dalian, China), cDNA synthesis was performed and RT-PCR assays allowed to amplify a fragment of the CP gene of TMV using specific primers (Suppl table 1). A 480 bp fragment (Suppl Figure 1C) was obtained and cloned into the pMD-18T vector (TaKaRa Biotech, Dalian, China) and sequenced. BLASTn- analysis revealed that the 20 amplicons were identical and shared coat sequence (100%) identity with the TMV isolates Mile-1 (acc. no. MK584554.1) and the diseased P. polyphylla was infected with TMV. The sequence was deposited in the GenBank database with the accession number OM366238 (CP). The sap from infected plants was used as inoculum for transmission of TMV to 10 healthy Nicotiana glutinosa and N. tabacum K326, respectively. 15 days post-inoculation, obvious symptoms of necrosis and chlortisis for viral infection were observed on inoculated and systemic leaves. The systemic leaves of 20 from two species plants were collected, and tested positive for TMV by RT-PCR with the specific primers (Suppl table 1). The sequences of the movement protein (MP) gene (807 bp, OM3662406) and RNA-dependent RNA polymerase (RdRp) gene (3351 bp, OM366242) of TMV were obtained by RT-PCR assays using MP-and RdRp-specific primers (Suppl Table 1). A disease incidence survey was conducted by our team in three Paris polyphylla var. yunnanensis fields in Qujing province and we observed a symptom incidence of 60% across all three fields. To confirm that the symptoms corresponded to TMV infection, leaf samples from 20 plants were collected from per field and all plants tested positive for TMV using RT-PCR assays. To the best of our knowledge, this is the first report of TMV infection in P. polyphylla var. yunnanensis in China. This report, in combination with another recent report of new viruses (Paris mitovirus 1, Paris virus 2) that infects the plants (Chen et al., 2022), points toward a need to intensively monitor the viruses in fields to protect the P. polyphylla var. yunnanensis industry.

Comparative proteomic analysis between mature and germinating seeds in Paris polyphylla var. yunnanensis.

The long dormancy period of Paris polyphylla var. yunnanensis seeds affects the supply of this scarce plant, which is used as an important traditional Chinese medicine. Mature seeds with a globular embryo and germinating seeds with developed embryo were used to explore the mechanisms of seed germination in this species. The protein profiles between the mature and germinating seeds were compared using the isobaric tags for relative and absolute quantification (iTRAQ) approach. Of the 4,488 proteins identified, a total of 1,305 differentially expressed proteins (DEPs) were detected. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these DEPs indicated that metabolic pathways and the biosynthesis of secondary metabolites were the two top pathways. Additionally, phytohormone quantification shows that the abscisic acid (ABA) level significantly decreased, whereas the GA3 level dramatically increased among nine endogenous gibberellins (GAs), resulting in a significant increase of the GA3/ABA ratio in germinating seeds. The biosynthesis pathways of carotenoid as a precursor for ABA production and GA were further analyzed, and showed that proteinic expressions of the candidate genes in the two pathways did not correlate with the transcriptional abundances. However, 9-cis-epoxycarotenoid dioxygenase (NCED), a rate limited enzyme for ABA biosynthesis, was significantly decreased in mRNA levels in germinating seeds. By contrast, gibberellin 20-oxidase (GA20ox), a key enzyme GA biosynthesis, exhibited the major increase in one copy and a slight decrease in three others at the protentional level in germinating seeds. Gibberellin 2-oxidase (GA2ox), an inactivate enzyme in bioactive GAs, has the tendency to down-regulate in mRNA or at the proteinic level in germinating seeds. Altogether, these results suggested that the analyses of ABA and GA levels, the GA3/ABA ratio, and the expressional patterns of their regulatory genes may provide a novel mechanistic understanding of how phytohormones regulate seed germination in P. polyphylla var. yunnanensis.

A Potential Biofertilizer-Siderophilic Bacteria Isolated From the Rhizosphere of Paris polyphylla var. yunnanensis.

The increasing demands for crop production have become a great challenge while people also realizing the significance of reductions in synthetic chemical fertilizer use. Plant growth-promoting rhizobacteria (PGPR) are proven biofertilizers for increasing crop yields by promoting plant growth via various direct or indirect mechanisms. Siderophilic bacteria, as an important type of PGPR, can secrete siderophores to chelate unusable Fe3+ in the soil for plant growth. Siderophilic bacteria have been shown to play vital roles in preventing diseases and enhancing the growth of plants. Paris polyphylla var. yunnanensis (PPVY) is an important traditional Chinese herb. However, reports about its siderophilic bacteria are still rare. This study firstly isolated siderophilic bacteria from the rhizosphere soil of PPVY, identified by morphological and physio-biochemical characteristics as well as 16S rRNA sequence analysis. The dominant genus in the rhizobacteria of PPVY was Bacillus. Among 22 isolates, 21 isolates produced siderophores. The relative amount of siderophores ranged from 4 to 41%. Most of the isolates produced hydroxamate siderophores and some produced catechol. Four isolates belonging to Enterobacter produced the catechol type, and none of them produced carboxylate siderophores. Intriguingly, 16 strains could produce substances that have inhibitory activity against Candida albicans only in an iron-limited medium (SA medium). The effects of different concentrations of Fe3+ and three types of synthetic chemical fertilizers on AS19 growth, siderophore production, and swimming motility were first evaluated from multiple aspects. The study also found that the cell-free supernatant (CFS) with high siderophore units (SUs) of AS19 strain could significantly promote the germination of pepper and maize seeds and the development of the shoots and leaves of Gynura divaricata (Linn.). The bacterial solution of AS19 strain could significantly promote the elongation of the roots of G. divaricata (Linn.). Due to its combined traits promoting plant growth and seed germination, the AS19 has the potential to become a bioinoculant. This study will broaden the application prospects of the siderophilic bacteria-AS19 as biofertilizers for future sustainable agriculture.

[Spatial distribution characteristics of metabolities in rhizome of Paris polyphylla var. yunnanensis: based on MALDI-MSI].

In this study, a method was established for in-situ visualization of metabolite distribution in the rhizome of Paris polyphylla var. yunnanensis. To be specific, through matrix-assisted laser desorption/ionization-mass spectrometry imaging(MALDI-MSI), the spatial locations of steroidal saponins, amino acids, organic acids, phytosterols, phytoecdysones, nucleosides, and esters in rhizome of the medicinal plant were directly analyzed, and six unknown compounds with differential distribution in rhizome tissues were identified. The specific procedure is as follows: preparation of rhizome tissue section, matrix screening and optimization, and MALDI-MSI analysis. The results showed that the steroidal saponins were mainly distributed in the central, amino acids in epidermis and cortex, low-molecular-weight organic acids in central epidermis, phytosterols in the epidermis and lateral cortex, the phytoecdysones in epidermis and cortex, nucleosides(uneven distribution) in epidermis and cortex, growth hormones around the epidermis and cortex, particularly outside the cortex, and esters in cortex with unobvious difference among different tissues. In this study, the spatial distribution of meta-bolites in the rhizome of P. polyphylla var. yunnanensis was characterized for the first time. The result can serve as a reference for identifying and extracting endogenous metabolites of P. polyphylla var. yunnanensis, exploring the synthesis and metabolism mechanisms of the metabolites, and evaluating the quality of medicinal materials.