Cloning and Functional Characterization of a Squalene Synthase from Paris polyphylla var. yunnanensis

Paris polyphylla Smith var. yunnanensis (Franch.) Hand. – Mazz. is a precious traditional Chinese medicine, and steroidal saponins are its major bioactive constituents possessing extensive biological activities. Squalene synthase (SQS) catalyzes the first dedicated step converting two molecular of farnesyl diphosphate (FDP) into squalene, a key intermediate in the biosynthetic pathway of steroidal saponins. In this study, a squalene synthase gene (PpSQS1) was cloned and functionally characterized from P. polyphylla var. yunnanensis, representing the first identified SQS from the genus Paris. The open reading frame of PpSQS1 is 1239 bp, which encodes a protein of 412 amino acids showing high similarity to those of other plant SQSs. Expression of PpSQS1 in Escherichia coli resulted in production of soluble recombinant proteins. Gas chromatography-mass spectrometry analysis showed that the purified recombinant PpSQS1 protein could produce squalene using FDP as a substrate in the in vitro enzymatic assay. qRT-PCR analysis indicated that PpSQS1 was highly expressed in rhizomes, consistent with the dominant accumulation of steroidal saponins there, suggesting that PpSQS1 is likely involved in the biosynthesis of steroidal saponins in the plant. The findings lay a foundation for further investigation on the biosynthesis and regulation of steroidal saponins, and also provide an alternative gene for manipulation of steroid production using synthetic biology.     

Molecular-networking-guided discovery of species-specific markers for discriminating five medicinal Paris herbs

BackgroundParidis Rhizoma (PR) is a famous traditional herbal medicine. Apart from two officially recorded species, viz. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. (PPY) and P. polyphylla Smith var. chinensis (Franch.) Hara (PPC), there are still many other species used as folk medicine. It is necessary to understand the metabolic differences among Paris species.PurposeTo establish a strategy that can discover species-specific steroidal saponin markers to distinguish closely-related Paris herbs for quality and safety control.MethodsA new strategy of molecular-networking-guided discovery of species-specific markers was proposed. Firstly, the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was applied to obtain the MS and MS/MS data of all samples. Then, molecular networking (MN) was created using MS/MS data to prescreen the steroidal saponins for subsequent analysis. Next, the principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) models were established to discover potential markers. Finally, the verification, identification and distribution of chemical markers were performed.ResultsA total of 126 steroidal saponins were screened out from five species using MN. Five species were classified successfully by OPLS-DA model, and 18 species-specific markers were discovered combining the variable importance in the projection (VIP) value, P value (one-way ANOVA) and their relative abundance. These markers could predict the species of Paris herbs correctly.ConclusionThese results revealed that this new strategy could be an efficient way for chemical discrimination of medicinal herbs with close genetic relationship.     

峨眉山2种重楼属植物同域分化的分子遗传学及其形态学证据初探

对峨眉山2种重楼－小重楼（Paris polyphylla Smith var. minora S.F.Wang）、黒籽重楼（P. thibetica Franch）DNA进行了AFLP (扩增的限制性片段长度多态性)分析和形态上的差异分析。AFLP分析可知小重楼和黑籽重楼在DNA分子水平上表现出极为丰富的遗传多样性,每个种内个体间的相似性很大,但有明显种间的遗传差异。形态对比分析可知小重楼和黑籽重楼在花部性状上有明显的差异。AFLP和形态分析结果表明小重楼和黑籽重楼无论是在形态还是遗传上均有明显差异。它们之间已经有了明显的分化。     

重楼属植物遗传多样性的SCOT标记

为评估重楼属植物的基因资源开发前景,初步探讨了SCOT标记技术在重楼属植物遗传多样性研究上的应用。采用目标起始密码子多态性(Start codon targeted polymorphism,SCOT)技术,对重楼属9个种40份材料进行基因组DNA多态性分析。结果表明:四川地区重楼属植物具有丰富的遗传多样性,40份供试材料可聚为4类:狭叶重楼单独聚为第一类;球药隔重楼、卵叶重楼、金线重楼及滇重楼聚为第二类;黑籽重楼与七叶一枝花聚为第三类;五指莲与毛重楼聚为第四类。说明SCOT标记能够对重楼属植物进行准确的分子鉴定,为重楼属植物的种类鉴定和种间的分类地位提供分子生物学依据,同时也为深入探究重楼皂苷合成关键酶基因定位研究提供重要参考。     

Steroidal saponins with cytotoxic activity from the rhizomes of Paris polyphylla var. yunnanensis

Two previously unreported spirostanol saponins, dianchonglouosides A and B (1 and 2), along with 7 known steroidal saponins (3–9) were isolated from the rhizomes of Paris polyphylla var. yunnanensis. Their structures were elucidated by extensive spectroscopic analysis (MS, 1D, and 2D NMR), and chemical methods. The cytotoxic activities of the isolated compounds 1–9 were also evaluated against two human cancer cell lines (HEK293 and HepG2). The results showed that compound 7 had the strongest cytotoxic activity against the two cancer cell lines with the IC₅₀ values of 0.6 and 0.9 μM, respectively.     

Pollen morphology inParis and its related genera

Pollen grains ofParis, Medeola, andScoliopus have been examined with light, scanning electron, and transmission electron microscopies.P. quadrifolia L.,P. verticillata Bieb.,P. delavayi Franch. andP. polyphylla Smith have monosulcate pollen characterized by psilate, foveolate or reticulate exine sculpture. In contrast to the former species,P. japonica (Fr. et Sav.) Franch. andP. tetraphylla A. Gray have monosulcate pollen with gemmate (rarely rugulate) exine.Medeola has monosulcate pollen with reticulate exine that is distinct from that ofParis. Scoliopus has monosulcate pollen characterized by a peculiar reticulate exine pattern. The palynological evidence suggests thatParis andTrillium are closely related to each other, andMedeola andScoliopus should be separated fromParis andTrillium.     

生态因子对滇重楼花药开裂的影响

滇重楼为延龄草科重楼属植物,具有极高的药用价值,由于重楼传统药用部位生长缓慢、繁殖力低下,以及人们对野生重楼资源的过度采挖使其资源日趋枯竭。滇重楼的花药在整个花期中存在开裂－关闭的现象,花药的有效闭合应是保护花粉、延长花粉寿命、增强雄性适合度的一种适应机制。该研究以滇重楼为对象,通过设计正交实验和对比实验,观测其花药开裂过程中的光照强度、温度、湿度的变化,探究光照强度、温度、湿度等生态因子对滇重楼花药开裂的影响以及滇重楼花药开裂与生态因子变化的关系。结果表明：(1)在滇重楼花药开裂的过程中,光照强度增强、温度升高、相对湿度下降;(2)温度是影响滇重楼花药开裂时间的主导因子,升温促进花药开裂,降温促进花药关闭;(3)高湿度及黑暗推迟花药开裂,但并不能阻止花药开裂;(4)低温可使滇重楼花药持续关闭,而光照强度越高,花药持续关闭所需的温度越低。该研究有利于解释滇重楼花药白天开裂夜晚关闭的现象与环境因子的关系,对滇重楼的栽培育种提供理论指导。     

中国重楼属(延龄草科)一新变种——攀西直瓣重楼

描述了延龄草科一新变种--攀西直瓣重楼(Paris polyphylla Smith var.panxiensis J.L.Liu),并绘制了形态图.新变种以其叶片狭长、条形而与狭叶重楼(Paris polyphylla Smith var.stenophylla Franch.)和小重楼(Paris polyphylla Smith var.minor S.F.Wang)相近似,但又以花瓣线状圆柱形、直立、不弯曲、远短于萼片、向上逐渐增粗呈棒状、先端圆形或钝圆,花药药隔明显头状突起而有明显区别.     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

Structure characterization of haemostatic diosgenin glycosides from paris polyphylia

From Paris polyphylla var. chinensis Hare (Liliaceae), four diosgenin glycosides with haemostatic effects were isolated. The structure of the major component was elucidated by chemical and spectroscopic methods as 3-{[α-L-rhamnopyranosyl(1_Rha → 2_Glu)]-[α-L-arabinofuranosyl(1_Ara → 4_Glu)]-β D-glucopyranosyl}-25(R)-spirost-5-en-3β-ol. This saponin was found to be identical to three previously reported compounds to which other structures were originally assigned, namely the major component from P. polyphylla Smith, the major cytotoxic component of yunnan paiyao, and polyphyllin D from P. polyphylla grown in the Himalaya region.     

药用植物华重楼(黑药花科)叶绿体全基因组研究

为探究华重楼(Paris polyphylla var. chinensis)的叶绿体基因组特征,利用叶绿体系统发育基因组学方法,对华重楼与其它百合目植物的叶绿体全基因组进行了比较。结果表明,华重楼的叶绿体全基因组长158307 bp,由4个区组成,包括2个反向重复区(IRA和IRB,27473 bp)、1个小单拷贝区(SSC,18175 bp)和1个大单拷贝区(LSC,85187 bp)。其叶绿体基因组有115个基因,包括81个编码蛋白质基因、30个转运RNA基因和4 个核糖体RNA基因。11种百合目植物的叶绿体全基因组的基因组成和基因顺序相似。华重楼的cemA基因是假基因,其起始密码子后有多聚核苷酸poly(A)及CA双核苷酸重复序列,编码序列中出现多个终止密码子, 且与北重楼(Paris verticillata)的cemA编码序列中的终止密码子位置不同。因此,华重楼叶绿体基因组比较保守;cemA结构及假基因化现象可能具有重要的进化与系统发育信息,其编码序列中的终止密码子可以区分华重楼和北重楼。     

Liquid chromatography tandem mass spectrometry in study of the pharmacokinetics of six steroidal saponins in rats

A rapid, simple and sensitive high-performance liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous determination of six main steroidal saponins in Paris polyphylla in rat plasma. Ginsenoside Rg3 was selected as the internal standard (IS). Plasma samples were pretreated with protein precipitation, and the separation was achieved on a reverse phase Agilent poroshell120 EC-C18 column using a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. The triple quadruple mass spectrometer was set in negative electrospray ionization mode and multiple reaction monitoring (MRM) was used for six steroidal saponins quantification. The precursors to produce ion transitions monitored for polyphyllin I, polyphyllin II, polyphyllin VI, polyphyllin VII, dioscin, gracillin and IS were m/z 899.5 > 853.4, 1059.5 > 1013.5, 783.4 > 737.4, 1075.5 > 1029.5, 913.5 > 867.4, 929.5 > 883.4 and 819.5 > 783.4, respectively. The intra- and inter-day precisions (RSD%) were less than 13% and the average extraction recoveries ranged from 85% to 97.0% for each analyte. Six steroidal saponins were proved to be stable during sample storage, preparation and analytical procedures. The established method was employed for simultaneous quantification and successfully used for the first time for the pharmacokinetics evaluation of the six main compounds after intragastric administration of P. polyphylla extract in Sprague–Dawley rats.     

华重楼内生真菌聚多曲霉的促生与拮抗作用

【背景】华重楼(Paris polyphylla var. chinensis)是我国一种名贵稀缺中药材,有多种药效,由于过度采挖等原因,其野生资源现已极度匮乏。华重楼的人工栽培技术尚未成熟,生长缓慢、病害频繁发生是主要的制约因素。【目的】植物益生菌的开发是一种环保且有效的解决途径,符合生态种植的要求。【方法】通过常规方法分离鉴定内生菌,选取已报道具有促生抗病作用的菌株进行玉米种子发芽试验,无氮培养基定性检测固氮活性,平板拮抗试验检测抗菌性;高效液相色谱-质谱联用技术(Liquid Chromatography-Mass Spectrometry,LC-MS)检测其菌液的代谢成分;分光光度法检测华重楼幼苗丙二醛和叶绿素含量,LC-MS检测叶片水杨酸、茉莉酸、脱落酸、赤霉素、细胞分裂素、生长素的含量,测量华重楼地下部分生物量指标。【结果】分离得到一株菌jdqmzz-1,经rDNA ITS序列扩增比对、形态学观察、生理生化鉴定,确定为聚多曲霉(Aspergillus sydowii);其可以促进玉米种子发芽和华重楼生长,具有固氮、拮抗病原真菌尖孢镰刀菌(Fusarium oxysporum GSICC 60612)、病原细菌胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovorum subsp. carotovorum,Pcc ATCC15713)的作用,真菌拮抗指数为42.50%,其菌液含有种类较多的促生和抑菌杀虫物质,菌液处理过的华重楼幼苗,叶片内源赤霉素和生长素含量较对照分别提高了54.1倍和2.3倍,叶绿素含量达到209.88 mg/g鲜重,较对照增加了48.80%,丙二醛含量较对照降低了15.20%;平均根数、平均根长、平均百株重较对照也有显著性(P0.01)提高。【结论】分离出的内生真菌聚多曲霉jdqmzz-1能够有效地促进华重楼的生长。     

Steroidal saponins with antimicrobial activity from stems and leaves of Paris polyphylla var. yunnanensis

Rhizoma Paridis, the root of Paris polyphylla var. yunnanensis (Trilliaceae), is a Chinese traditional medicine, which resources become less and less. However, the aerial parts of this herb, which can regenerate every year, were discarded. In order to expand the resources, detailed chemical investigation on the stems and leaves of Paris polyphylla var. yunnanensis led to isolation of one sapogenin and 24 steroidal saponins (1-25), including 6 new glycosides, named chonglouosides SL-1-SL-6 (1-6). Their structures were elucidated on the basis of detailed analyses of their 1D and 2D NMR spectra and acid hydrolysis. Among them, compounds 3 and 4 are the first 23,27-dihydroxydiosgenin saponin having a sugar chain attached to C-23 or C-27, while compound 6 is the first 27-hydroxyruscogenin glycoside bearing 1, 27-di-O-sugar chains. The known compounds 10, 12, 14, 19, 20, 22, and 25 were isolated from the genus Paris for the first time. Antimicrobial testing activities of the selected compounds showed that compound 2, 3, 6, 8, 9, 11, 13, 17, 18, 21, and 24 were active against Propionibacterium acnes with MIC values of 62.5, 62.5, 3.9, 16.5, 17.2, 7.8, 39.0, 17.2, 31.3, 62.5, and 31.3μg/ml, respectively.     

Furostanol saponins from Paris polyphylla: Structures of polyphyllin G and H

The structure of two new saponins, polyphyllins G and H, isolated from the tubers of Paris polyphylla have been elucidated as 3-O-{α-l-rhamnopyranosyl (1→3) [α-l-arabinofuranosyl (1→4)]-β-d-glucopyranosyl}-26-O-[β-d-glucopyranosyl] (25R)-22α-hydroxy-furost-5-en-3β, 26-diol and its 22-methoxy derivative respectively.     

Pennogenin Tetraglycoside Induces Rat Myometrial Contraction and MLC20 Phosphorylation via PLC-IP3 and RhoA/Rho Kinase Signaling Pathways

Background

Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been widely used in China for the treatment of abnormal uterine bleeding. We previously studied the main active constituents of TSSPs and their structure-activity relationships with respect to rat myometrial contractions. Tg (pennogenin tetraglycoside) was identified as one of the active ingredients in TSSPs able to induce rat myometrial contractions. However, the mechanisms underlying the pharmacological actions on uterine activity have not been described clearly.

Methods

Here Tg was screened for effects on contractile activity in isolated uterine strips from estrogen-primed rats and on MLC20 phosphorylation and related signaling pathways in cultured rat myometrial cells as determined by Western blot. Intracellular calcium ([Ca²⁺]_i) was monitored under a confocal microscope using Fluo-4 AM-loaded myometrial cells.

Results

Tg dose-dependently stimulated rat myometrial contractions as well as MLC20 phosphorylation in vitro, which could be completely suppressed by an inhibitor of myosin light chain kinase (MLCK). Use of Ca²⁺ channel blockers and kinase inhibitors demonstrated that Tg-induced myometrial contractions are mediated by activation of the phospholipase C (PLC)-inositol triphosphate (IP3) signaling pathway, resulting in increased MLC20 phosphorylation. Furthermore, Y27632, a specific inhibitor of Rho kinase (ROK), notably suppressed Tg-stimulated myometrial contractions and decreased MLC20 phosphorylation.

Conclusions

These data provide evidence that rat myometrial contractility induced by Tg results from enhanced MLC20 phosphorylation, while both PLC-IP3 and RhoA/ROK signaling pathways mediate the process. These mechanisms may be responsible for the therapeutic effects of TSSPs on abnormal uterine bleeding.     

Steroidal saponins from the rhizomes of Paris delavayi

Two new steroidal saponins, padelaosides A (1) and B (2), along with two other known steroidal saponins (3 and 4) were isolated from the rhizomes of Paris delavayi. Their structures were elucidated by 1D and 2D NMR techniques, HRFTMS, physical data and chemical methods. The two different absolute configurations of fucose, assigned as l and d that were found on compounds 1 and 2, respectively, were simultaneously reported in a natural medicine for the first time.     

Effect of cultivation years on saponins in <Emphasis Type="Italic">Paris Polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis> using ultra-high liquid chromatography–tandem mass spectrometry and Fourier transform infrared spectroscopy

Paris Polyphylla var. yunnanensis (PPY) has been used in traditional medicine for the treatment of parotitis and abscess. Previous studies showed the quality of PPY was associated with cultivation years. In this study, we applied ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and Fourier transform infrared (FT-IR) spectroscopy to measure the variation of saponins due to cultivation years in cultivated PPY. A rapid UHPLC–MS/MS method was developed for simultaneous determination of six major steroidal saponins (polyphyllin I, II, V, VI, VII and gracillin). Principal component analysis (PCA) was selected for pattern recognition of FT-IR profiles. The concentration of six targeted steroid saponins varied with the increase of cultivation year and attained the maximum level at 8th year (22.65?±?1.65 mg g^?1). A dynamic change with the growth of PPY presented in the content of diosgenin-type saponins and pennogenin-type saponins. After the treatment of standard normal variate (SNV), the PCA result showed that 4th and 5th years’ samples were solely classified into two parts and the chemical information of other samples was quite similar. According to FT-IR and UPLC-MS/MS results, an optimal harvest time recommend at 7th or 8th year in the current stage. These results could support the previous view that the variations of metabolites were associated with cultivation years and provided chemical evidence for further research on the agricultural practice of PPY.     

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

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.     

Three new napthalene derivatives from the endophytic fungus Phomopsis fukushii

An endophytic Phomopsis fukushii was isolated from the rhizome of Paris polyphylla var. yunnanensis, Diaporthaceae family, and three new (1–3) and four known napthalene derivatives (4–7) were isolated from the fermentation products of this fungus. Their structures were elucidated by spectroscopic methods including extensive 1D- and 2D-NMR techniques. Compounds 1–3 were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity with vancomycin as positive control. The results showed that compounds 1–3 showed good inhibitions with MCI valves of 4, 4 and 6 mg/mL, respectively.