Chemotaxonomic study of the genus Paris based on steroidal saponins

The study of saponins from the genus Paris (Trilliaceae) has led to the isolation of over 70 steroidal saponins. Their distributions in different species are summarised and possible patterns in the modifications of the aglycone moiety, based on a biosynthetic pathway for steroidal saponins, were reviewed in this study. The chemotaxonomic value of these secondary metabolites has been evaluated, and it is suggested that Paris thibetica, Paris vietnamensis, Paris delavayi and Paris pseudothibetica, which contain more active saponins, could be an ideal substitution material for Paris polyphylla Smith var. chinensis, and Paris polyphylla Smith var. yunnanensis. Distribution of the same types of saponins amongst the Trilliaceae suggests a close relationship between the Trillium and Paris.     

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.     

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。     

产甾体皂甙华重楼内生菌的筛选与鉴定

从华重楼(Paris polyphyllavar.chinensisFranch)的地下块茎中分离筛选得到2株可能产生甾体皂甙的内生菌(SS01和SS02),薄层层析检测菌株SS01、SS02的发酵产物分别有3条和2条层析带与重楼总皂甙的层析带迁移率相当。形态和生理生化特征初步表明SS01和SS02分属于肠杆菌科(Enterobacteriaceae)和芽孢杆菌属(Bacillussp.)细菌。扩增、测序得到SS01和SS02的部分16S rDNA序列,GenBank接收号分别为AY842143和AY842144。用Blastn调出与菌株16SrDNA同源的序列,用Clustal W进行多重序列对比,用软件Phylip按Neighbor-Joining方法构建16SrDNA系统发育树。菌株SS01和SS02分别与Cedecea davisae DSM4568、Paenibacillus daejeonensis处于同一分支,相似性分别为98.9%和97.7%,将它们鉴定为Cedecea davisae SS01和Paenibacillus daejeonensis SS02。     

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。     

Discovery of chemical markers for identifying species,growth mode and production area of Astragali Radix by using ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry

BackgroundAstragali Radix (AR) is a well-known Chinese herbal medicine. The quality of AR can be affected by many factors such as species, growth mode and production area, but there are still no chemical markers to distinguish it.PurposeTo explore chemical markers for improving the quality assessment of AR and discover chemical markers for identifying species, growth mode and production area of AR.MethodsA highly sensitive, efficient and accurate method based on ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) for simultaneous quantitative determination of 14 major chemical components (five flavonoids and nine triterpene saponins) in 94 batches of AR from China, Republic of Korea and Germany was developed for the first time. To explore chemical markers and assess changes in the contents of 14 compounds in the 94 batches of AR samples from different regions, hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed.ResultsAstragaloside III was not only an important chemical marker for distinguishing two species of AR, i.e.: Astragalus mongholicus and A. membranaceus, but also a potential chemical marker for the classification of cultivated and semi-wild AR. In addition, in the batches of cultivated AR, the content of isoastragaloside II and cyclocephaloside II were greater in batches from the region of Shaanxi Province than that of other Provinces in China, but the content of calycosin-7-O-β-D-glucoside and astragaloside IV, which are the quality control markers of AR required by the Chinese Pharmacopoeia, were higher than that of other Provinces in China. In addition, the content of calycosin-7-O-β-D-glucoside, ononin, calycosin and astragaloside I could be used to identify samples of AR collected from China, Republic of Korea and Germany.ConclusionThis UHPLC-QQQ-MS/MS method could be applied to the quantitative evaluation of AR and could be an important and meaningful reference to develop chemical markers for quality control of AR.     

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.     

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.     

Partial mycoheterotrophy is common among chlorophyllous plants with Paris-type arbuscular mycorrhiza

Background and AimsAn arbuscular mycorrhiza is a mutualistic symbiosis with plants as carbon providers for fungi. However, achlorophyllous arbuscular mycorrhizal species are known to obtain carbon from fungi, i.e. they are mycoheterotrophic. These species all have the Paris type of arbuscular mycorrhiza. Recently, two chlorophyllous Paris-type species proved to be partially mycoheterotrophic. In this study, we explore the frequency of this condition and its association with Paris-type arbuscular mycorrhiza.MethodsWe searched for evidence of mycoheterotrophy in all currently published ¹³C, ²H and ¹⁵N stable isotope abundance patterns suited for calculations of enrichment factors, i.e. isotopic differences between neighbouring Paris- and Arum-type species. We found suitable data for 135 plant species classified into the two arbuscular mycorrhizal morphotypes.Key ResultsAbout half of the chlorophyllous Paris-type species tested were significantly enriched in ¹³C and often also enriched in ²H and ¹⁵N, compared with co-occurring Arum-type species. Based on a two-source linear mixing model, the carbon gain from the fungal source ranged between 7 and 93 % with ferns > horsetails > seed plants. The seed plants represented 13 families, many without a previous record of mycoheterotrophy. The ¹³C-enriched chlorophyllous Paris-type species were exclusively herbaceous perennials, with a majority of them thriving on shady forest ground.ConclusionsSignificant carbon acquisition from fungi appears quite common and widespread among Paris-type species, this arbuscular mycorrhizal morphotype probably being a pre-condition for developing varying degrees of mycoheterotrophy.     

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.     

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.     

悬钩子属部分类群的分类订正

根据野外调查和查阅标本的结果, 对悬钩子属部分类群做了修订。将红毛悬钩子(R.pinfaensis Lévl.et Vant.)并入椭圆悬钩子(R.ellipticus Smith), 栽秧泡(R.ellipticus Smithvar.obcordatus (Franch.)Focke)提升为种(R.obcordatus (Franch.)Focke);棱枝细瘦悬钩子(R.macilentus Camb.var.angulatus Delav.)并入细瘦悬钩子(R.macilentus Camb.);毛叶插田泡(R.coreanus Miq.var.tomentosus Card.)并入插田泡(R.coreanus Miq.);狭叶绢毛悬钩子(R.lineatus Reinw.var.angustifolius Hook.)并入绢毛悬钩子(R.lineatus Reinw.)。对针刺悬钩子(R.pungens Camb.)及其各变种做了订正。     

大萼球药隔重楼,湖南重楼属(黑药花科)一新变种北大核心CSCD

报道了湖南重楼属(Paris L.)一新变种:大萼球药隔重楼(Paris fargesii Franch.var.macrosepala H.X.Yin&W.X.Rao&L.Zhao),其与原变种球药隔重楼(P.fargesii Franch.var.fargesii)区别在于外轮花被片极为宽大,长6.5~11.0 cm,宽2.5~4.0 cm,叶-萼长度比常小于2.0(平均1.91),叶-萼宽度比普遍小于3.0(平均2.86),内轮花被片较短,2.0~5.3 cm,常平展于外轮花被片之上,药隔凸出部分呈扁平状,具明显裂痕,顶面观呈蝶形或双肾形。     

Molecular Phylogeny Analysis and Species Identification of Dendrobium (Orchidaceae) in China

Dendrobium plants are important commercial herbs in China, widely used in traditional medicine and ornamental horticulture. In this study, sequence-related amplified polymorphism (SRAP) markers were applied to molecular phylogeny analysis and species identification of 31 Chinese Dendrobium species. Fourteen SRAP primer pairs produced 727 loci, 97% of which (706) showed polymorphism. Average polymorphism information content of the SRAP pairs was 0.987 (0.982–0.991), showing that plenty of genetic diversity exists at the interspecies level of Chinese Dendrobium. The molecular phylogeny analysis (UPGMA) grouped the 31 Dendrobium species into six clusters. We obtained 18 species-specific markers, which can be used to identify 10 of the 31 species. Our results indicate the SRAP marker system is informative and would facilitate further application in germplasm appraisal, evolution, and genetic diversity studies in the genus Dendrobium.     

Thrombin-based discovery strategy of bioactive-chemical quality marker combination for pollen of Typha orientalis by metabolomics coupled with chemometrics

BackgroundIt is of utmost significance to choose the bioactive components as quality markers for ensuring the effectiveness of traditional Chinese medicine (TCM). Nonetheless, some markers are able to assess effectively the quality of TCM without considering the pharmacological mechanisms and intrinsic chemical complexities.ObjectiveThis underscores the need to discover new and efficient markers which can assess both quality and mechanism of action. Herein, a strategy of bioactive-chemical quality marker combination was proposed to improve the level of the quality control of TCM by metabolomics coupled with chemometrics.MethodsA four-step plan was followed. Firstly, acquisition of metabolic features and component characterization of different batches of pollen of Typha orientalis C.Presl were performed using UHPLC-Q-TOF/MS. Secondly, the direct inhibitory effects of pollen of T. orientalis on thrombin was assessed by using chromogenic substrate method together with HPLC. Thereafter, bioactive-chemical marker combination associated with anti-thrombin segregation was screened using supervised classifiers. Finally, quantitative assay and prediction-model of selected markers were established for guarantying the quality of pollen of T. orientalis.ResultsA total of 22 compounds were annotated based on comparison with previous work from pollen of T. orientalis by UHPLC-Q-TOF/MS. Citric acid and linolenic acid inhibited the thrombin activity with IC₅₀ values, 0.52 ± 0.02 and 0.51 ± 0.02 mg/mL, respectively. A bioactive-chemical marker combination including citric acid, linolenic acid, typhaneoside, and isorhamnetin-3-O-neohesperidoside were discovered and selected as quality markers for evaluation of pollen of T. orientalis according to their capacity for inhibiting thrombin.ConclusionThe thrombin-based discovery strategy of bioactive-chemical marker combination was a powerful tool for screening the quality markers for evaluation of pollen of T. orientalis.     

Saponins in Pollen

Phosphate buffered saline (PBS) extracts of pollen of 34 out of 95 angiosperm species (in 40 families) tested, lysed human and/or snake head fish (Channa striatus Bloch) erythrocytes during assay for lectins in pollen. The bitter taste of the pollen extracts of these 34 species, the formation of a stable foam on shaking and the ability to lyse erythrocytes, suggested the presence of saponins, which have not, so far, been reported from pollen. Thin Layer Chromatography (TLC) and Mass Spectrum (MS) of the erythrocyte-lysing extracts of pollen of the garden gladiolus (Gladiolus gandavensis Van Hout.) confirmed that the pollen contained both triterpenoidal and steroidal saponins. The implications for the presence of saponins in pollen inhaled from the atmosphere, in the diagnosis and management of pollen allergy are discussed.     

重楼属的四个新异名

将小重楼Paris polyphylla Smith var．minora S．F．Wang、长药隔重楼P.polyhylla Smith var．pseudothibetica H．Li处理为金线重楼P.delavayi Franch．的异名;将广东重楼P.polyphylla Smith var．kwantungensis（R．H．Miau）S．C．Chen＆S．Y.Liang处理为多叶重楼P.polyphylla Smith的异名;将文县重楼P.wenxianensis Z．X．Peng＆R．N．Zhao处理为黑籽重楼P.thibetica Franch．的异名。     

Design of molecular markers for identification of species of the genus Chironomus (Diptera,Chironomidae)

Accurate identification and differentiation of species of the genus Chironomus based on their morphological features is a difficult problem. Unambiguous species identification by means of molecular markers is possible at any stage of the life cycle. Polymerase chain reaction (PCR) with species-specific primers was used to develop molecular markers (amplicons) for identification of Chironomus piger, Ch. dorsalis, and Ch. pseudothummi. Nucleotide sequences of the internal transcribed spacer region (ITS) of the locus coding for ribosomal RNA were used to design species-specific primers for these target species. Each of the species-specific primer pairs yielded species-specific amplicons (molecular markers) only with the DNA of target species: Ch. piger, Ch. dorsalis, and Ch. pseudothummi. Test PCRs with the DNA of eighteen Chironomus species confirmed the specificity of the primers obtained. The molecular markers produced in PCR with the designed species-specific primers permit reliable identification of Ch. piger, Ch. dorsalis, and Ch. pseudothummi and their differentiation from other species of the genus Chironomus.     

Concise synthesis and antitumor activities of trisaccharide steroidal saponins

The naturally derived trisaccharide steroidal saponin 1 and structurally modified derivatives 2 and 3 bearing the same sarsasapogenin aglycon were synthesized concisely via a direct transglycosylation strategy. The antitumor activities of the synthetic trisaccharide saponins 1–3 and their corresponding α-isomers 1a–3a were preliminarily evaluated against human gastric adenocarcinoma cell (MKN-45) and human epithelial cervical cancer cell (HeLa) by CCK-8 assay.