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).

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.

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.

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.

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.

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。

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.

七叶一枝花种子提取物对3种种子萌发特性的影响研究

为探究七叶一枝花种子提取物对不同类型种子萌发特性的影响,本研究以七叶一枝花休眠种子的甲醇、水浸提液为处理液进行小麦、绿豆、白菜种子萌发试验。结果表明,甲醇、水浸提液处理均可显著抑制不同类型种子的发芽率,在0.08 g/mL甲醇浸提液处理下,3种农作物种子发芽率均为0,在0.08 g/mL水浸提液处理下,绿豆发芽率为58.89%,小麦为7.78%。因此,推断七叶一枝花种子中含有内源抑制物、ABA含量过高均可能是导致七叶一枝花种子休眠的内源因素。

云南重楼果皮中1个新的C_(21)甾体皂苷

目的:研究云南重楼果皮中的甾体皂苷类成分。方法:采用HPD-100大孔吸附树脂柱、正相硅胶柱、ODS柱、Sephadex LH-20葡聚糖凝胶柱和半制备型高效液相色谱等方法对云南重楼果皮70%乙醇渗漉提取物中的甾体皂苷类成分进行系统研究,通过现代波谱学技术结合参考文献对化合物结构进行鉴定。结果:从云南重楼果皮中分离得到8个甾体皂苷类化合物,分别鉴定为:17α-hydroperoxide pregn-5-en-20-one-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside(1)、重楼皂苷Ⅳ(2)、ypsilandroside L(3)、重楼皂苷Ⅲ(4)、pregna-5,16-dien-3β-ol-20-one-3β-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside(5)、pregna-5,16-dien-3β-ol-20-one-3β-O-α-L-rhamnopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→4)]-β-D-glucopyranoside(6)、pregna-5,16-dien-3β-ol-20-one-3β-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside(7)、aethioside A(8)。结论:其中,化合物1为一个新的C_(21)甾体皂苷类化合物,化合物3、5、6、8为首次从重楼属植物中分离得到。

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.

不同育苗基质及激素处理对华重楼种子出苗的影响研究

针对重楼育苗常遇到的出苗时间长、出苗率低和种子播种后腐烂严重的问题,对最佳育苗方式、育苗基质及在较短时间内获得较多的重楼种苗等方面进行研究,旨在为重楼栽培和育苗方面提供技术参考。研究了不同厚度稻草覆盖、土壤与草炭不同配比、不同浓度KNO_(3)与质量浓度赤霉素(GA_(3))溶液浸种重楼种子对重楼种子出苗的影响。结果表明:在重楼种子播种前的土壤表面覆盖厚度为4 cm的稻草后播种重楼,再在重楼种子表面覆盖厚度为4 cm的稻草,重楼种子的出苗率最高,达90.8%,与其他覆盖厚度的差异具有高度统计学意义,出苗时间为播种后的第20月;土壤与草炭质量比为1∶3时,重楼种子的出苗率为73.0%,与其他质量比的出苗率的差异具有高度统计学意义(P<0.01),出苗时间为播种后的第20月;用19 mmol/L KNO_(3)、600 mg/L GA_(3)溶液浸种重楼种子,种子胚根长出率最高达56.67%,与排第2位溶液组合(KNO_(3)浓度为16 mmol/L、GA_(3)质量浓度为600 mg/L)胚根长出率的差异具有高度统计学意义(P<0.01),出苗时间为浸种处理后的第8月。3种育苗方式比较表明:稻草覆盖厚度为4 cm、土壤与草炭质量比为1∶3的育苗基质利于提高重楼种子的出苗率,KNO_(3)浓度为19 mmol/L、GA_(3)的质量浓度为600 mg/L的溶液浸种重楼种子,可大大缩短重楼种子的育苗时间,幼苗获得率约50%。

浇施磷酸二氢钾对华重楼幼苗生长与光合特性的影响

为了培育优质华重楼[Paris polyphlla Smith var.chinensis(Franch.)Hara]种苗,开展了二年生一叶期盆栽幼苗不同浓度磷酸二氢钾追肥研究,并测定其光合参数和生长量。结果表明,追施磷酸二氢钾后,华重楼幼苗的株高、地茎粗、光合色素及光合效率与对照相比均有显著提高。当磷酸二氢钾浓度为0~300 mg/L时,幼苗的株高、生根数量、茎鲜重、叶鲜重、叶绿素a、总叶绿素含量及类胡萝卜含量均随磷酸二氢钾浓度升高而增加,而地茎粗和叶绿素b含量则在磷酸二氢钾浓度为200 mg/L时达到最大值。当磷酸二氢钾浓度超过300 mg/L时,其相应的指标随着磷酸二氢钾浓度升高呈下降趋势。当磷酸二氢钾浓度达300 mg/L时,幼苗叶片净光合速率最大,胞间CO_(2)浓度最低。

云南重楼果皮化学成分研究

目的:研究云南重楼Paris polyphylla Smith var.yunnanensis(Franch.)Hand._Mazz.果皮中的化学成分。方法:采用HPD-100大孔吸附树脂柱层析、硅胶柱层析、ODS柱层析、Sephadex LH-20葡聚糖凝胶柱层析和半制备型高效液相色谱等方法对云南重楼果皮70%乙醇渗漉提取物中的化学成分进行系统研究,通过NMR和HR-MS现代波谱学技术结合参考文献对化合物的结构进行鉴定。结果:从云南重楼果皮的70%乙醇渗漉提取物中分离得到16个化合物,分别鉴定为:山柰酚(1)、槲皮素(2)、异鼠李素(3)、山柰酚-3-O-β-D-半乳糖苷(4)、紫云英苷(5)、异鼠李素-3-O-β-D-葡萄糖苷(6)、异槲皮苷(7)、金丝桃苷(8)、kaempferol-3-O-β-D-galactopyranosyl-(l→6)-β-D-glucopyranoside(9)、isorhamnetin-3-O-β-D-galactopyranosyl-(1→6)-β-D-glucopyranoside(10)、异鼠李素-3-O-β-D-龙胆二糖苷(11)、20-羟基蜕皮素(12)、薯蓣皂苷(13)、diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl(1→2)]-β-D-glucopyranoside(14)、偏诺皂苷元(15)、4-butyl-5-ethyl-3,4-dihydropyran-2-one(16)。结论:其中,化合物4、7、9、10、16为首次从云南重楼中分离得到,化合物9、10、16为首次从重楼属植物中分离得到。

滇重楼的化学成分研究

目的研究滇重楼Paris polyphylla var.yunnanensis根茎中的化学成分。方法利用正相硅胶柱色谱、葡聚糖凝胶Sephadex LH-20、反相制备色谱等手段进行分离纯化,并通过1H-NMR、13C-NMR、ESI-MS等波谱技术进行结构鉴定。结果从醋酸乙酯层中分离得到了7个化合物,分别鉴定为豆甾醇-3-O-β-D-葡萄糖苷(Ⅰ)、1-O-(β-D-葡萄糖基)-(2S,3S,4E,8E)-2-[(2′R)-2′-羟基十六酰氨基]-4(E),8(E)-十八二烯-1,3-二醇(Ⅱ)、β-蜕皮激素(Ⅲ)、薯蓣皂苷元-3-O-α-L-呋喃阿拉伯糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Ⅳ)、薯蓣皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Ⅴ)、薯蓣皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Ⅵ)和偏诺皂苷元-3-O-α-L-呋喃阿拉伯糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-葡萄糖苷(Ⅶ)。结论化合物Ⅱ为首次从该属植物中分离得到。

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。

云南重楼种子萌发过程内源激素含量及酶活性变化研究

为探讨云南重楼种子萌发不同阶段的酶和内源激素的变化,本研究采用生理生化方法测定云南重楼种子萌发不同阶段超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、苹果酸脱氧酶(MDH)和6-磷酸葡萄糖脱氢酶(G-6-PDH)活性,以及吲哚乙酸(IAA)、赤霉素(GA_3)、脱落酸(ABA)、水杨酸(SA)、反玉米素(TZ)、茉莉酸(JA)6种内源激素的含量,并分析比较其差异。结果表明,云南重楼种子在即将出苗阶段的SOD、POD活性迅速升高,CAT、MDH和G-6-PDH活性降低;IAA、GA_3含量均在即将出苗阶段显著高于其他阶段,种子休眠解除后ABA含量迅速下降。胚根刚突破种皮阶段,其SA、TZ、JA含量均最高,SOD、POD、IAA、GA_3间呈极显著正相关(P<0.01),MDH与G-6-PDH呈极显著正相关(P<0.01),ABA与GA_3呈显著负相关(P<0.05),SA、TZ、JA间呈极显著正相关(P<0.01),IAA/ABA、GA_3/ABA、SA/ABA在种子休眠解除和出苗阶段均呈上升趋势。综上,SOD、POD、GA_3是种子出苗阶段的关键物质,TZ、SA和JA对种子休眠解除具有重要的影响,IAA/ABA、GA_3/ABA及SA/ABA增大对种子休眠解除和出苗均有促进作用。本研究结果为云南重楼种子繁殖提供了理论依据和实践指导。

AM真菌对滇重楼根际土壤微生物数量及酶活性的影响

目的:研究接种AM真菌后滇重楼根际土壤微生物和酶活性的变化规律,为人工栽培滇重楼提供技术基础。方法:通过室温盆栽接种试验与室内分析相结合的方法,观察灭菌土壤条件下接种28种丛枝菌根(AM)真菌对滇重楼根际土壤微生物和土壤酶活性的影响。结果:滇重楼与AM真菌之间存在一定的相互选择性。AM真菌诱导处理后,不同AM真菌对滇重楼根际土壤微生物的数量、微生物生物量碳和土壤酶活性的影响有所差异,AM真菌的施用降低了滇重楼根际土壤中可培养真菌的数量,而对可培养细菌、放线菌数量呈现出增加的趋势,并提高了根际土壤微生物多样性指数、微生物生物量碳含量以及蛋白酶、脲酶、磷酸酶、过氧化氢酶和蔗糖酶活性。结论:滇重楼人工栽培中施用AM真菌能促使滇重楼根际微生物区系从低肥力的"真菌型"向高肥力的"细菌型"转化的趋势,提高根际土壤微生物多样性和酶活性,有助于维持滇重楼根际微生态系统的稳定性与和谐性,证明了田间人工栽培滇重楼施加AM菌剂的有效性及可能性。