泽泻原三萜、降三萜和倍半萜的分离及其抗炎活性研究＊

目的 对泽泻中原三萜、降三萜和倍半萜化学成分分离和鉴定及其抗炎活性进行研究；方法 采用硅胶柱色谱、凝胶柱色谱、高速逆流色谱、中压制备色谱和半制备色谱法对泽泻中各类化合物进行分离，利用¹H和¹³C NMR对分离化合物进行结构鉴定；通过测定各单体对LPS（1 μg·mL^-1）诱导Caco-2细胞中NO生成的抑制作用评价其抗炎活性。结果 从泽泻中分离得到19个化合物，包括8个原三萜类成分：alisol A 23 acetate （1），alisol G （2），16-oxo-11-anhydroalisol A （3），16-oxo-11-anhydroalisol A 24 acetate （4），alisoma A （5），alisoma B （6），alismanol J （7），alismanol B （8）；1个降三萜类成分：17-nor-protostane （9）；9个倍半萜类成分：orientalol L （10），orientalol M （11），orientalol N （12），orientalol O （13），orientalol P （14），alismanoid A （15），heyneanones D （16），leptocladol B （17），chabrolidione B （18）；1个木脂素类成分：pnoresinol （19）。原三萜类成分均具有显著的抑制活性，化合物1-8的IC₅₀分别为2.1、0.9、7.3、10.0、11.0、16.0、13.5和10.5 μmol·mL^-1，降三萜及倍半萜活性较弱，化合物10、11、13、15、16和17的IC₅₀分别为24.0、26.2、30.6、15.7、17.2和15.2 μmol·mL^-1。结论 化合物16、17、18和19为该植物首次分离得到。首次报道了倍半萜类成分对LPS诱导Caco-2细胞中NO生成的抑制作用，其中alisol A 23 acetate和alisol G的抑制活性最强。     

枇杷叶中五环三萜酸类有效成分的提取研究

目的：进行枇杷叶中五环三萜酸类有效成分的提取方法研究。方法：称取干燥粉碎的枇杷叶5g,以乙醇为溶剂,水浴中回流、过滤,滤液用旋转蒸发仪浓缩得到浸膏,浸膏用乙醇溶解、稀释,经香草醛显色后,在548nm处测定紫外吸收。单因素实验考察了不同水浴温度（60℃、70℃、80℃、90℃）、提取时间（2h、3h、4h、5h）、料液比（1∶10、1∶15、1∶20、1∶25、1∶30）、乙醇浓度（60%、70%、80%、90％）的影响,并进行了L16（44）的正交实验。结果：以乌索酸为对照建立标准曲线,曲线方程为A=0.021C＋0.0249,相关系数r2=0.9995,线性范围为10～50μg/mL,线性关系良好。正交实验结果表明,乙醇体积为90%,提取温度为80℃,提取时间为5h,料液比为1∶20为最佳提取条件。在该条件下进行提取实验验证,结果表明枇杷叶中总五环三萜类成分的含量约为1.32%。结论：该方法简便易行,可用于枇杷叶中五环三萜酸类有效成分的提取。     

雪胆属植物的化学成分及生物活性研究进展

雪胆属植物中含有丰富的三萜皂苷类成分,并且因其具有显著的抗肿瘤和抗菌消炎等生物活性而越来越受到医药领域的关注。近年来,国内外学者对雪胆属植物化学成分、药理活性等方面进行了大量研究,并取得了很大的进展。综述近30年来雪胆属的植物来源、三萜皂苷结构、波谱特征以及生物活性的研究进展,为雪胆属植物的进一步开发利用提供依据。     

Pentacyclic Triterpenoids from Spikes of Prunella vulgaris L. Inhibit Glycogen Phosphorylase and Improve Insulin Sensitivity in 3T3‐L1 Adipocytes

Phytochemical investigation of methanol extract from the spikes of Prunella vulgaris L. led to the isolation of two new pentacyclic triterpenoid glycosides Vulgasides I (1) and II (2) along with 13 known compounds (3–15). Their structures were established on the basis of nuclear magnetic resonance (1D and 2D) and mass spectroscopic data analysis. All the isolated compounds were screened for glycogen phosphorylase inhibitory activity and also evaluated for their effect on insulin sensitivity in 3T3‐L1 adipocytes. Two new compounds (1, 2) did not demonstrate the glycogen phosphorylase inhibitory activity, but other compounds (3–11) exhibited varying degrees of glycogen phosphorylase inhibitory activity with IC₅₀ values in the range from 30.69 to 68.85 μM. Compounds 3, 6, 7, 11, and 13 demonstrated markedly increased insulin‐mediated glucose consumption in 3T3‐L1 adipocytes. Copyright © 2014 John Wiley & Sons, Ltd.     

珍稀药用植物珠子参的转录组测序及分析

药用植物珠子参的根茎在中国西南区域已有上千年的药用历史,三萜皂苷为珠子参最主要的活性成分。为了探索珠子参根茎中皂苷物质生物合成的分子基础,该文采用Illumina HiSeq 2000高通量测序获得珠子参根茎的转录组数据;使用Trinity和TGICL软件实现Unigene的de novo拼接;基于BLAST完成Unigene的蛋白功能注释、KOG功能注释、GO分类和KEGG代谢通路分析。最终获得了短读序15.6 Gb,通过de novo拼接注释得到Unigene 62 240个。研究发现,珠子参根茎部表达的19个Unigene与三萜碳环骨架合成相关;69个Unigene与介导三萜碳环骨架的氧化和羟基化等修饰的CYP450相关,18个Unigene与负责三萜碳环骨架的糖基化的糖基化转移酶相关。该研究发现的三萜皂苷合成相关候选基因对于阐明珠子参三萜皂苷合成方式研究提供了重要的基础。     

Ursolic acid induces neural regeneration after sciatic nerve injury

In this study,we aimed to explore the role of ursolic acid in the neural regeneration of the injured sciatic nerve.BALB/c mice were used to establish models of sciatic nerve injury through unilateral sciatic nerve complete transection and microscopic anastomosis at 0.5 cm below the ischial tuberosity.The successfully generated model mice were treated with 10,5,or 2.5 mg/kg ursolic acid via intraperitoneal injection.Enzyme-linked immunosorbent assay results showed that serum S100 protein expression level gradually increased at 1-4 weeks after sciatic nerve injury,and significantly decreased at 8 weeks.As such,ursolic acid has the capacity to significantly increase S100 protein expression levels.Real-time quantitative PCR showed that S100 mRNA expression in the L4-6 segments on the injury side was increased after ursolic acid treatment.In addition,the muscular mass index in the soleus muscle was also increased in mice treated with ursolic acid.Toluidine blue staining revealed that the quantity and average diameter of myelinated nerve fibers in the injured sciatic nerve were significantly increased after treatment with ursolic acid.10 and 5 mg/kg of ursolic acid produced stronger effects than 2.5 mg/kg of ursolic acid.Our findings indicate that ursolic acid can dose-dependently increase S100 expression and promote neural regeneration in BALB/c mice following sciatic nerve injury.     

Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum

The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated β-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.Triterpene saponins are secondary metabolites that exhibit a large structural diversity and wide range of biological activities in many plant species (1, 2). Saponins are glycosides of sapogenins, which are composed of 30 carbon atoms arranged in 4- or 5-ring structures that are “decorated” by functional groups. Saponins are synthesized by multiple glycosylations of the sapogenin building blocks that are produced by multiple cytochrome P450-dependent monooxygenase (P450) or oxidoreductase-mediated modifications of basic backbones, such as β-amyrin (oleanane type), α-amyrin (ursane type), lupeol, and dammarenediol. These backbones are generated by oxidosqualene cyclase (OSC)-mediated cyclization of 2,3-oxidosqualene, which is also an intermediate in the synthesis of sterols in eukaryotes (3, 4). Both saponins and sapogenins include biologically active compounds or serve as starter molecules for the generation of novel, potentially bioactive structures by synthetic modification (5–7).The genus Bupleurum consists of perennial herbs that are used in Asian traditional medicine, either alone or in combination with other ingredients, for the treatment of common colds, fever, and inflammatory disorders (8). Saikosaponins constitute the largest class of secondary metabolites in Bupleurum and can account for up to ∼7% of root dry weight. Their accumulation can be further stimulated by jasmonate treatment (9). More than 120 closely related oleanane- and ursane-type saikosaponins have been identified from this genus and the oxidations at various positions suggest the presence of multiple enzymes, mainly P450s, capable of catalyzing specific modifications on the amyrin backbones (8, 10). To date, no P450 or oxidoreductase involved in triterpene saponin biosynthesis has been identified from Bupleurum species.P450s that modify the β-amyrin backbone on C-11; C-12,13; C-16; C-22; C-23; C-28 or C-30 have been characterized from Glycyrrhiza uralensis, Avena strigosa, Medicago truncatula, Glycine max, Vitis vinifera, and Catharanthus roseus (11–18). Hydroxylases from Panax ginseng that oxidize the dammarenediol-II backbone on C-6, C-12, or C-28 (19–21), and a C-20 hydroxylase from Lotus japonicus (22) that modifies lupeol, have also been identified. To characterize these P450s, they have been ectopically expressed in yeast strains either producing β-amyrin or externally supplied with candidate substrates. Similarly, several OSCs have been produced and functionally analyzed in yeast. From these studies, it is clear that yeast cells cannot only be used for the characterization of novel enzymes, but possibly also as a heterologous host for the production of triterpene sapogenins (23). To date only two pilot studies have aimed at engineering of β-amyrin production in yeast (24, 25), but no efforts toward engineering of sustainable production of sapogenins or saponins in yeast have been reported.Here, we identified and characterized CYP716Y1, a P450 from Bupleurum falcatum that corresponds to a C-16α oxidase, designated according to Nelson’s nomenclature (http://drnelson.uthsc.edu/cytochromeP450.html). By designing triterpene-hyperproducing starter strains, optimizing culturing conditions for triterpene synthesis, and using the CYP716Y1 gene in a combinatorial synthetic biology program, we established a platform that allows us to produce and sequester triterpene sapogenins in culture medium and to reconstitute a full saponin synthetic pathway in yeast cells.     

环境因子对药用植物三萜皂苷合成影响的研究进展

三萜皂苷类化合物是一类具有多种生物及药理活性的植物次生代谢产物,除去遗传因素外,环境对植物体内三萜皂苷的生物合成产生较大的影响。总结三萜皂苷类化合物的生物合成途径及其关键酶,并综述了环境中光照、温度、水分、盐、土壤状况等因子对药用植物三萜皂苷合成的影响和可能的作用机制,以期为优化药用植物的栽培环境和提高药材质量提供参考。     

河南产尾叶香茶菜叶部位化学成分的分离与纯化

目的:分离纯化河南产尾叶香茶菜(Rabdosia excisa)叶部位的化学成分.方法:利用硅胶柱色谱法对尾叶香茶菜叶部位的化学成分进行分离和纯化,通过理化性质和波谱数据鉴定结构.结果:从尾叶香茶菜叶中分离鉴定了10个化合物:1α,6,11β,15β-四乙酰基-6,7-断裂-7,20-内酯-对映-贝壳杉-16-烯(I)...     

HPLC-ELSD同时测定铁破锣中3种三萜皂苷含量及体外抗氧化性研究

目的 建立同时测定铁破锣中3种三萜皂苷BeesiosideⅠ、Ⅱ、Ⅲ含量的方法,并研究铁破锣中三萜皂苷的体外抗氧化性。方法 运用反相高效液相色谱-蒸发光散射检测法（HPLC-ELSD）对铁破锣中三萜皂苷进行含量测定,并对所用含量测定方法进行方法学考察;同时,对铁破锣中三萜皂苷清除自由基的能力和总的抗氧化能力进行体外试验。结果 HPLC-ELSD方法可同时测定BeesiosideⅠ、Ⅱ、Ⅲ的含量,且该方法线性关系良好、精密度、重复性、稳定性均好,回收率高;铁破锣中三萜皂苷能够有效清除ABTS⁺、DPPH·和羟基自由基,并且具有很好的抗氧化能力。结论 HPLC-ELSD方法简便、快速、准确、重现性好,可作为铁破锣中三萜皂苷（BeesiosideⅠ、Ⅱ、Ⅲ）的含量测定及质量控制标准方法,体外试验表明铁破锣中三萜皂苷具有良好的自由基清除和抗氧化能力。