Two New Highly Oxygenated Spirostanol Saponins from Paris polyphylla var.stenophylla

Phytochemical investigation of the rhizomes of Paris polyphylla var.stenophylla led to the isolation of two new highly oxygenated spirostanol saponins,named paristenosides A(1)and B(2),together with seven known compounds.Their structures were established mainly on the base of NMR spectroscopic techniques and mass spectrometry,as well as chemical methods.In addition,the cytotoxicity of the two new saponins was tested.Graphical Abstract Two new highly oxygenated spirostanol saponins,paristenosides A(1)and B(2),were isolated from the rhizomes of Paris polyphylla var.stenophylla.Their structures were established mainly based on NMR spectroscopic techniques and mass spectrometry,as well as chemical methods.

华重楼(Paris polyphylla Smith var. chinensis)无性繁殖试验研究

华重楼为我国一味珍贵的民族药,本文以华重楼根茎为材料开展其无性繁殖试验,研究植物生长调节剂6-BA不同浓度对其潜伏芽萌发、生长的影响。试验结果显示5mg/L的6-BA可促进华重楼切段的繁殖系数,提高其双芽率、多芽率及有根率。

Isolation of the Endophytic Fungi of Paris polyphylla var. yunnanensis and Their Effects on the Embryo Development of P. polyphylla var. yunnanensis Seeds

[Objective] This study aimed to isolate the endophytic fungi of Paris polyphylla var. yunnanensis and investigate their effects on the embryo development of P. polyphylla var. yunnanensis seeds. [Method] The endophytic fungi of P. polyphylla were isolated and identified morphologically, and their effects on the embryo development of P. polyphylla var. yunnanensis seeds were studied by using paraffin sectioning and microphotography. [Result] Nine endophytic fungi, i.e. P. polyphylla var. yunnanensis endophytic fungi PPYEF-1, PPYEF-2, PPYEF-3, PPYEF-4, PPYEF-5, PPYEF-6, PPYEF-7, PPYEF-8 and PPYEF-9 belonging to seven genera in five families, three orders were isolated from the rhizomes. Except PPYEF-4 (Cladosporium sp.), other fungi could promote the embryo development of the P. polyphylla var. yunnanensis seeds, mostly reaching the extremely significant or significant level. PPYEF-9 (Trichoderma sp.) resulted in the highest embryo length and embryo-emerging ratio. [Conclusion] This paper could provide a reference for the application of the endophytic fungi of P. polyphylla var. yunnanensis in the dormancy-breaking of P. polyphylla var. yunnanensis seeds.

Temporal transcriptomics reveal the molecular mechanism of dormancy and germination regulated by temperature in Paris polyphylla seed

The mature seed of Paris polyphylla var.chinensis(PPC)is morphophysiologically dormant and develops differently under warm and cold temperatures.To elucidate the molecular mechanisms underlying temperature-dependent regulation of PPC seed dormancy and germination,we investigated the dynamic changes in PPC seed transcript levels under warm and cold temperature stratifications(WS and CS,respectively)by time-resolved RNA sequencing,focusing on genes related to hormone metabolism and signaling and cell wall remodeling(CWRM)and encoding transcription factors/regulators(TFs/TRs).A total of 48765 and 47836 differentially expressed genes(DEGs)were associated with WS and CS,respectively.Of these,17581 and 16652 DEGs,respectively,unique to WS and CS,and 5386 were common to both temperature stratifications across nine analyzed growth stages.The expression of hormone metabolism and signaling,TF/TR,and CWRM genes were closely associated with temperature.More genes related to gibberellin(GA),cytokinin,auxin,and brassinosteroid biosynthetic were upregulated in WS as compared to CS seeds,while genes related to dormancy release and germination were downregulated in WS seeds.However,only GA and abscisic acid levels were altered in PPC seeds breaking morphophysiological dormancy(MPD).Overall,37 TF and five TR families were upregulated whereas 24 TF and 16 TR families were downregulated in WS as compared to CS seeds.Most CWRM families were highly expressed under WS as compared to CS,suggesting that they promote endosperm weakening and embryo growth of WS seeds and facilitate MPD release and germination.A coexpression analysis revealed positive correlations between TFs/TRs and DEGs involved in hormone metabolism and signaling and CWRM.These results provided a basis for investigating the interaction between the endosperm and underdeveloped embryo in the regulation of PPC seed germination and seedling emergence.

A novel phenylpropanoid glycosides and a new derivation of phenolic glycoside from Paris Polyphylla var. yunnanensis

A novel phenylpropanoid glycosides 1, named parispolyside E and a novel derivation of phenolic glycoside 2, named parispolyside G, as well as two known flavonoid glycosides were isolated from the rhizome of Paris polyphylla var. yunnanensis. Their structures were elucidaed by spectroscopic methods.

Levels of rhizome endophytic fungi fluctuate in Paris polyphylla var.yunnanensis as plants age

Paris polyphylla var. yunnanensis is an important medicinal plant with abundant saponins that are widely used in the pharmaceuticals industry. It is unclear why the levels of active ingredients increase as these plants age. We speculated that the concentrations of those components in the rhizomes are mediated by fungal endophytes. To test this hypothesis, we took both culture-dependent and-independent(metagenomics) approaches to analyze the communities of endophytic fungi that inhabit those rhizomes in plants of different age classes(four, six, and eight years old). In all, 147 isolates representing 18 fungal taxa were obtained from 270 segments(90 per age class). Based on morphological and genetic characteristics, Fusarium oxysporum(46.55% frequency of occurrence) was the predominant endophyte,followed by Leptodontidium sp.(8.66%) and Trichoderma viride(6.81%). Colonization of endophytic fungi was maximized in the eight-year-old rhizomes(33.33%) when compared with four-year-old(21.21%) and six-year-old(15.15%) rhizomes. Certain fungal species were present only at particular ages. For example,Alternaria sp., Cylindrocarpon sp., Chaetomium sp., Paraphaeosphaeria sporulosa, Pyrenochaeta sp., Penicillium swiecickii, T. viride, and Truncatella angustata were found only in the oldest plants. Analysis of(metagenomics) community DNA extracted from different-aged samples revealed that, at the class level,the majority of fungi had the highest sequence similarity to members of Sordariomycetes, followed by Eurotiomycetes and Saccharomycetes. These results were mostly in accord with those we obtained using culture methods. Fungal diversity and richness also changed over time. Our investigation is the first to show that the diversity of fungi in rhizomes of P. polyphylla var. yunnanensis is altered as plants age, and our findings provide a foundation for future examinations of useful compounds.

Mycorrhizas Affect Polyphyllin Accumulation of Paris polyphylla var.yunnanensis through Promoting PpSE Expression

Paris polyphylla var.yunnanensis is a traditional Chinese medicinal plant,in which polyphyllin as the main medicinal component is an important secondary metabolite with bioactivity.Arbuscular mycorrhizal fungi(AMF)have multiple positive effects on plants,while it is not clear whether AMF increase the content of medicinal components in medicinal plants.In this study,a total of nine AMF treatments were laid to analyze the mycorrhizal effect on polyphyllin accumulation and PpHMGR and PpSE expression of P.polyphylla var.yunnanensis.AMF increased the content of polyphyllin in the cultivated variety with low relation to the increase of inoculation intensity.Polyphyllin I,II,and VII were identified and partly improved by AMF inoculation,dependent on AMF treatments and culture environments.Similarly,the PpHMGR and PpSE expression was induced by mycorrhization,dependent on AMF species,whilst the induction was more obvious in PpSE than in PpHMGR after mycorrhization.It concluded that the symbiotic relationship between P.polyphylla var.yunnanensis and AMF increased polyphyllin content level in the plant,which was associated with the up-regulation of PpSE transcripts.

Comprehensive Evaluation on Impacts of Diseases on Quality of Paris polyphylla var. chinensis Based on Ultraviolet and Infrared Spectra

[Objectives] The research aimed to evaluate impacts of diseases on quality of Paris polyphylla var. chinensis. [Methods]Ultraviolet spectrophotometry was used to determine and contrast content of total saponins in root,stem and leaf from healthy and diseased plants. Infrared characteristic absorption peaks of healthy and diseased plants were found by infrared fingerprint method for comparative analysis.[Results]Total saponins content in root sample J4 of healthy plant reached 4. 89% and was the highest,while it was 0. 13% in stem sample J11 of healthy plant and was the lowest. Total saponins content in root sample B1 of diseased plant was 1. 68% and was the highest,while it was 0. 1% in stem sample B7 of diseased plant and was the lowest. In healthy and diseased plants,total saponins content in root was significantly higher than that in stem and leaf. Content of total saponins in root from healthy plant was significantly higher than that from diseased plant,and there was little difference in stem and leaf of healthy and diseased plants. Root sample Jg of healthy plant had obvious C-O characteristic vibration of polysaccharides and glycosides and absorption peak of O-H bending vibration in steroidal saponin near 1 160. 02 cm-1,while diseased plant had no obvious absorption peak. Near 861. 97 and 768. 07 cm-1,root sample Jg of healthy plant had significant C-C stretching vibration in sugar ring and characteristic absorption peak of characteristic vibration of steroidal saponin,while root sample Bg of diseased plant had no obvious characteristic peak. [Conclusions] Diseases significantly affected composition and content of total saponins and steroidal saponins from P. polyphylla var. chinensis,further affecting quality of P. polyphylla var. chinensis.

Determination of 10 Kinds of Inorganic Elements in Paris polyphylla Smith var. yunnanensis(Franch.) Hand.-Mazz.(and Polygerm Varieties)

[Objectives] To determine the content of 10 kinds of inorganic elements(Pb,Cu,As,Cd,Hg,Zn,Fe,Mn,Sr and Cr) in samples of Paris polyphylla Smith var. yunnanensis( Franch.) Hand.-Mazz.( and polygerm varieties) produced in different areas. [Methods] The wet digestion and technique of inductively coupled plasma optical emission spectrometry( ICP-OES) were used. [Results] Under the experimental conditions,elements were not related to each other,and several kinds of elements could be measured at the same time. The content of Pb exceeding the standard only in sample S4 of P. polyphylla Smith var. yunnanensis( and polygerm varieties),Hg was not detected in all samples,and the rest heavy metals did not exceed the limit. [Conclusions] This study was expected to provide a certain reference for the development and utilization of P. polyphylla Smith var. yunnanensis( and polygerm varieties).

Mycorrhizal Fungal Effects on Growth,Antioxidant Capacity,and Medicine Quality of Paris polyphylla var.yunnanensis

A field experiment was conducted to determine the effects of two commercial strains composed of mulple arbuscular mycorrhizal fungi(AMF)species on plant growth,antioxidant capacity,and medicine quality of Paris polyphylla var.yunnanensis in three subtropical soils from Wanzhou,Anshun and Baoshan in fields.The results showed that AMF inoculation enhanced the fungal colonization rate and activities of both succinate dehydrogenase and alkaline phosphatase,thereby,enhancing the mycorrhizal viability of P.polyphylla var.yunnanensis.The concentrations of photosynthetic pigments(chlorophyll a,b,a+b and carotenoids),soluble sugar,soluble protein and photosynthetic capacity were higher in AMF-inoculated plants than in non-AMF-treated plants in field.AMFtreated plants recorded higher activities of catalase,peroxidase and superoxide dismutase,and caused the reduction in malondialdehyde content,indicating lower oxidative damage,compared with non-AMF plants.Polyphyllin I,Polyphyllin II,Polyphyllin III,Polyphyllin IV and total polyphyllin contents were increased by AMF treatment.In conclusion,AMF improved the plant growth,antioxidant capacity and medicinal quality of P.polyphylla var.yunnanensis seedlings.Hereinto,AMF effects on the soil from Wanzhou was relatively greater than on other soils.

Identification of Paris polyphylla Smith var. yunnanensis(Franch.) Hand.-Mazz.(Polygerm Varieties) Produced in Gaoligong Mountain Areas

[Objectives] To make original plant and microscopic identification and study the traits of Paris polyphylla Smith var. yunnanensis( Franch.) Hand.-Mazz.( polygerm varieties) produced in Gaoligong Mountain areas,and provide experimental data for their identification and application. [Methods] The original plant powder was used,and paraffin sections and physicochemical properties were identified. [Results]P. polyphylla Smith var. yunnanensis( polygerm varieties) produced in Gaoligong Mountain areas showed obvious microscopic features and physicochemical properties. [Conclusions] This study was expected to provide certain reference for identification of original plant and medicine and formulation of quality standard for P. polyphylla Smith var. yunnanensis( polygerm varieties) produced in Gaoligong Mountain areas,and provided basis for their further research and development.

Epigenetic Factors Altered in Dehisced Anther Correlated to Seed Dormancy in <i>Paris polyphylla</i>var. Yunnanensis

Paris polyphylla var. yunnanensis (Franch.), one of the best-known medicinal plants in China, has a dehiscent anther which physiologically work in pollination, however, the dehiscent anther always closes in response to darkness every day, and watering or raining every time. To explore this frequently closing and its unkown physiology, next-generation sequencing was performed, and the transcriptome was de novo assembled. RNA-sequencing was carried out in 15 samples including seven openning samples, four closed samples owing to darkness or watering, and tissue samples (leaf, petal, calyx, and stigma) were used for control. We obtained 72.75 GB data, assembled into 79,815 unigenes. Differentially expressed unigenes (DEGs) between opened and closed anther samples were 6231 and the DEGs between anther and control samples were 2831. Comparation between the two DEGs by KEGG enrichment showed that “plant hormone signal transduction” pathway is the most significant pathway for DEGs from closing anther vs. opening anther, and expression model of DEGs in the pathway might elicit change in germination and seed dormancy. Further examination of the action of the signal pathway on physiology showed “chromatin binding” function was prominent in “DNA binding” function of annotated DEGs between opened and closed anthers, of the 215 “chromatin binding” unigenes, 120 were involved in epigenetic silencing, and 50 of the epigenetic unigenes were directly related to germination or seed dormancy, strongly correlating anther closing to epigenetic modification and seed dormancy. These results were verified that at least three auxins involved in seed dormancy showed same expression patterns occurred in abnormal closing anther and seed embryo in Paris polyphylla var. yunnanensis. In conclusion, the information from transcriptome point out that frequent abnormal closing of dehiscent anthers possibly transfer the impact on seed dormancy, and epigenetic modification happened in closing may be the cause.

Investigation on the Resources of Paris Polyphylla var.yunnanensis

In this study,the resource distribution,morphological characteristics,artificial cultivation and market circulation of Paris Polyphylla var.yunnanensis and its polygerm varieties in Yunnan were investigated by referring to the research literature,field investigation and our planting experience.It is found that P.polyphylla var.yunnanensis is widely distributed in Yunnan Province,realizing large-scale artificial cultivation,while the wild resources of polygerm varieties are almost on the verge of extinction and are in urgent need of protection.

Comparison on Morphology and Micromorphology of Paris polyphylla Sm. var. polyphylla with Different Year Numbers

[Objectives] The research aimed to identify Paris polyphylla with different growth year numbers and evaluate its resource quality.[Methods]Comparative research on P. polyphylla with different growth year numbers was conducted by using morphological identification and microscopic identification method. [Results] By contrasting original plant morphology,transverse sections of rhizome and fibrous root,powder,it displayed that microstructure of P. polyphylla had regularity difference in cell shape and size,duct quantity,trait and size,size and distribution of calcium oxalate cluster crystal. [Conclusions] The microstructure change of P. polyphylla was related to the number of its growth year,which could provide the reference for identifying the growth year number of P. polyphylla and evaluating its resource quality.

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 nainly 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 ICso values of 15.22±1.73,18.87±1.81 and 17.64±1.69μmol^(-1),respectively.

Parisaponin Ⅰ和Parisvietnaside A的NMR数据分析

从滇重楼中分离得到2个甾体皂苷,利用1H NMR、13C NMR、1H-1H COSY、HSQC、HMBC、1D和2D TOCSY等多种核磁共振方法鉴定其结构分别为(25R)-26-O-β-D-吡喃葡萄糖基-3β,22α,26-三羟基-呋甾-5-烯-3-O-α-L-吡喃鼠李糖基-(1→2)-[α-L-呋喃阿拉伯糖基-(1→4)]-β-D-吡喃葡萄糖苷(1,Parisaponin I)和(25R)-3β,5α,6β-三羟基-△7-螺甾烯-3-O-β-D-吡喃葡萄糖基-(1→3)-[α-L-吡喃鼠李糖基-(1→2)]-β-D-吡喃葡萄糖苷(2,paris-vietnaside A).对化合物1和2的1H NMR和13C NMR信号分别进行了归属和详细分析,并纠正了文献中的核磁数据归属错误.化合物2为首次从滇重楼中分离得到.

四川地区华重楼(Paris polyphylla var.chinensis)主要病害调查及病原菌鉴定

华重楼是重要的中药材,近年来,华重楼病害已成为制约四川华重楼产量和品质提高的重要因素之一。本研究对四川种植区华重楼病害进行了调查,共调查到主要危害华重楼病害有4种。通过组织分离法对相应的病原菌进行分离,并对病原菌进行致病性测定、形态学观察和分子生物学鉴定。试验结果显示,结合病原菌形态特征及r DNA-ITS、HSP60、G3PDH、RPB2、TEF、ACT、LSU、Alt琢1、TUB基因序列分析,并构建多基因分子系统发育树,鉴定出引起华重楼4种真菌病害病原菌,分别为华重楼叶枯病(Epicoccum sorghinu)、褐斑病(Alternaria tenuissima)、灰霉病(Botrytis cinerea)和黑斑病(Alternaria alternata)。目前,危害华重楼的最为严重的病害是灰霉病、褐斑病和叶枯病。华重楼主要病害的调查及病原菌种类的鉴定为华重楼病害的防治提供理论依据。

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 UGT91AH1^(Y187A)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.

云南重楼种子育苗技术研究

目的：缩短云南重楼种子萌发和出苗所需时间，提高其出苗率；提出在生产中切实可行的云南重楼种子有性繁殖育苗方法。方法：考察不同温度、不同播种方式（播种时间、覆土厚度、地膜覆盖）等因素对云南重楼种子萌发和出苗的影响。结果：云南重楼种子胚后熟和萌发的最适温度在18～20℃，在此温度下处理3～4个月以后，转至0～10℃低温处理2～4个月，可在翌年4月出苗；适宜的播种时间是4月份以前，育苗土覆盖厚度为1cm左右，播种后用黑色地膜进行覆盖，可提高出苗率。结论：上述试验结果可为云南重楼种子育苗提供操作参考。

一株产重楼皂甙内生细菌的分离与鉴定

从产自四川彭州的华重楼(Parispolyphyllavar·chinensisFranch)地下块茎中分离得到16株菌。经以薯蓣皂甙元为标准对照的薄层层析分析,表明其中编号为SNUS-1(AY842149)的菌株能分泌重楼皂甙或其类似化合物。16SrDNA序列(约1,500bp)系统发育分析表明:菌株SNUS-1与Pseudomonastolassii(AF255336)和Pseudomonastolassii(D84028)的遗传距离分别为0和0·003,在系统进化树上三者又严格聚为一族,结合形态及生理特征确定其为托氏假单孢菌(Pseudomonastolassii)。