雷公藤甲素和雷公藤内酯酮热稳定性与分解动力学

研究雷公藤甲素和雷公藤内酯酮的热稳定性与分解动力学。利用热重—微商热重(TG-DTG)和差热—微商差热(DTA-DDTA)分析技术测得这两种活性成分在氮气气氛中不同升温速率(β)下的热分解曲线,结合使用Kissinger法、Ozawa法、Coats-Redfern法和Achar法进行动力学处理,根据热分解的表观活化能(Ea)和指前因子(A)计算推断这两种活性成分的贮存期。结果表明,除脱水峰外,雷公藤甲素和雷公藤内酯酮热分解行为相似,失重均分为两步:第一步分别从200℃到340℃,200℃到290℃;第二步分别从340℃到440℃,290℃到490℃。雷公藤甲素和雷公藤内酯酮的两步热分解严重重叠,总失重率分别为85.5%和64.4%。DTA-DDTA曲线分别出现232℃和252℃吸热峰以及298℃和280℃放热峰。雷公藤甲素的第一步热分解的机制是Chemical reaction控制,对应的函数为Reaction order方程。测得平均表观活化能Ea为136.66 k J/mol,指前因子ln A为26.47;雷公藤内酯酮的第一步热分解的机制是Chemical reaction控制,对应的函数为二级反应方程。测得平均表观活化能Ea为127.19 k J/mol,指前因子ln A为25.23。根据第一步热分解的Ea和A推断,在室温(25℃)下,雷公藤甲素和雷公藤内酯酮的理论贮存期均为4~5年。研究结果可为含雷公藤甲素和雷公藤内酯酮药品的质量控制和制剂工艺、雷公藤的炮制减毒以及热分解产物研究提供参考。     

异戊烯基焦磷酸转运对雷公藤甲素生物合成的影响

雷公藤甲素是一种具有显著抗炎、抗肿瘤和免疫抑制活性的天然产物,市场需求量大,临床应用前景广阔。文中以雷公藤悬浮细胞为实验材料,通过对不同培养时期(7 d、14 d)的细胞外源性添加D,L-甘油醛(DLG)以阻断异戊烯基焦磷酸(IPP)转运,分析诱导前后的细胞活性及生物量、雷公藤甲素累积量及其生物合成上游途径关键酶基因的变化规律,探究IPP转运在雷公藤甲素生物合成过程中的时空特点及其影响机制。通过实验结果可知:IPP转运参与雷公藤甲素的生物合成;在细胞培养早期,IPP转运主要经由质体(MEP途径)转运到细胞质(MVA途径)中,而细胞培养晚期则相反;阻断IPP转运,可反馈调节雷公藤甲素生物合成上游途径关键酶基因的表达,进而影响雷公藤甲素的累积。上述研究填补了雷公藤甲素生物合成过程中IPP转运特点和机制方面的空白,为雷公藤甲素合成生物学关键技术的开发奠定了理论基础,具有重要的现实意义。     

雷公藤的杀虫作用研究与应用

卫矛科植物雷公藤(Tripterygium wilfordli Hook)是我国著名的杀虫植物。文章对雷公藤的杀虫活性、主要有效成分、作用机理及田间药效4个方面的研究与应用进行综述。雷公藤对多种昆虫有毒杀、拒食、麻醉、生长发育抑制和种群抑制作用;雷公藤定碱、雷公藤次碱、雷公藤春碱、雷公藤吉碱、雷公藤嗪碱、雷公藤新碱和雷公藤甲素、雷公藤内酯酮是其主要杀虫活性成分;雷公藤生物碱作用于昆虫的神经系统和中肠肠壁细胞,Na+,K+-ATP酶可能是雷公藤甲素的靶标之一;雷公藤还具有较好的田间防效。最后,对雷公藤作为杀虫植物资源的开发利用前景和今后的研究方向作了展望。     

不同采收期雷公藤甲素测定及其动态积累

采用HPLC法测定不同采收期雷公藤根、茎、叶的主要活性成分雷公藤甲素含量,掌握雷公藤甲素的动态积累。结果表明,雷公藤甲素浓度在7.75～250 μg·mL^-1范围内线性关系良好(R²=0.9997),雷公藤加样回收率为99.21%,RSD值为1.578%,该方法回收率较好。雷公藤甲素的含量在不同生长期、不同部位存在一定差异,不同部位甲素含量由高到低依次为叶、根、茎,雷公藤叶片具有资源开发利用潜力。7～8月采收的雷公藤叶片甲素含量最高,11～12月雷公藤根部甲素含量达最高。雷公藤不同器官均具有较高的药用价值,研究结果为雷公藤人工采收、质量评价和资源开发提供一定的科学依据。     

雷公藤红素抗肿瘤作用及机制研究进展

雷公藤红素是我国传统中药雷公藤中的天然活性成分,具有抗类风湿、抗炎、抗肿瘤等多种生物学活性。近年来,雷公藤红素由于低毒、多靶点、广谱性等优势,在抗肿瘤治疗中备受关注。雷公藤红素可以通过调控PI3K/AKT、NF-κB、MAPK和STAT3等多种信号通路抑制肿瘤增殖、侵袭和转移,诱导肿瘤细胞凋亡。综述了雷公藤红素的抗肿瘤作用及机制,以期促进雷公藤红素的深入研究与应用。     

雷公藤内酯醇干预肾脏疾病的研究进展

雷公藤的提取物在中国被用于治疗肾小球肾炎已经有30年的历史。雷公藤提取物的主要活性成分雷公藤内酯醇具有免疫抑制和抗炎的特性,然而,毒性作用限制了其临床应用。深入研究雷公藤内酯醇的药理机制,将有助于其临床应用和降低毒副反应。对国内外关于雷公藤内酯醇对肾脏影响的研究成果进行了综述。     

雷公藤倍半萜生物碱的研究（Ⅳ）

从雷公藤（Tripterygium uilfordii Hook.f.）的根皮中分离到两个倍半萜生物碱Ⅰ和Ⅱ,根据理化性质和波谱数据特别是2D－NMR技术(^1H-^1H COSY、^1H－^13C COSY、NOESY、COLOC）数据分析,鉴定Ⅰ为wilforine,Ⅱ为一新的生物碱－－雷公藤植碱(wilfordsuine）。     

内生真菌和内生细菌接种对雷公藤生长和次生代谢产物积累的影响

为探究内生真菌与内生细菌对雷公藤(Tripterygium wilfordii)的生长和次生代谢产物积累的相互作用,用内生真菌NS33、NS6和内生细菌LG3、LY1单独或跨界联合接种雷公藤,对雷公藤的生长和雷公藤甲素、雷公藤红素合成进行了研究。结果表明,单独或混合培养的菌株具有分泌铁载体、吲哚乙酸(IAA)和溶磷能力,对种子萌发、芽伸长和根系活力有显著促进作用。接种菌株NS33、NS6、NS6-LG3和NS6-LY1均显著促进了雷公藤组培苗的生长。单独或联合接种菌株均能显著提高雷公藤组培苗雷公藤甲素和雷公藤红素的积累,其中NS33-LG3和LG3的作用最显著。菌株NS33与LG3能够协同促进IAA的分泌、小麦幼苗根系活力和雷公藤红素的积累;菌株NS6与LY1协同提高了雷公藤组培苗的高度、质量和雷公藤红素的积累。因此,内生真菌与内生细菌联合接种对雷公藤生长和次生代谢产物积累具有一定的协同效应,显示出实际应用潜力。     

茉莉酸甲酯处理雷公藤悬浮细胞的基因差异表达分析

以不同浓度茉莉酸甲酯(MeJA)为诱导子对雷公藤悬浮细胞进行处理,采用cDNA-AFLP技术对差异表达基因进行研究。结果表明,茉莉酸甲酯在50～400μmol/L浓度范围内对雷公藤悬浮细胞总碱的积累呈抑制作用。茉莉酸甲酯处理后,分析筛选出了19个雷公藤悬浮细胞内差异表达的基因。通过与NCBI蛋白质数据库比对,7个片段的功能得以预测,涉及植物细胞的信号转导、转录调控和能量代谢等。这些结果对今后利用生物技术手段提高雷公藤生物碱含量奠定了一定基础。     

基于网络药理学及分子对接探讨雷公藤干预结缔组织相关间质性肺病的分子机制

为探究雷公藤干预结缔组织相关间质性肺疾病(CTD-ILD)的分子机制。运用网络药理学的方法,通过TCMSP数据库挖掘雷公藤的主要化学成分及作用靶点,利用Genecards、OMIM、DrugBank数据库获取CTD-ILD相关靶点,利用String平台进行蛋白质相互作用分析,构建PPI网络并挖掘网络中潜在的蛋白质功能模块,然后采用Metascape进行GO及KEGG富集分析,利用Cytoscape3.8.0软件构建"雷公藤成分-CTD-ILD-信号通路"网络,最后通过AutoDock Vina进行分子对接。通过分析得到雷公藤干预CTD-ILD的靶点共80个,核心成分为山奈酚、雷公藤内酯醇、川陈皮素、β-谷甾醇等,核心靶点有PTGS2、JUN、MAPK8、RELA、SCN5A、TNF等。GO和KEGG富集分析显示,IL17信号通路、Toll样受体信号通路、TNF信号通路、HIF-1信号通路、FoxO信号通路、Cancer信号通路等为雷公藤干预CTD-ILD的主要通路,涉及炎症,氧化应激,细胞凋亡及癌症等多个生物过程。分子对接结果亦显示,分子亲和力小于-7 kcal/mol占总数的62.5%,其中5次化合物的预测值超过原配体。综上,本研究初步揭示了雷公藤通过多成分、多靶点、多通路作用机制干预CTD-ILD的分子机制,并经分子对接予以验证,为临床应用雷公藤治疗CTD-ILD提供了理论依据。     

Applications of ultrafast HPLC to process development of recombinant DNA-derived proteins.

The application of ultrafast HPLC to the development of recovery processes for proteins produced by recombinant DNA technology has been explored using wide-pore HPLC resins and instrumentation designed for rapid analysis. High-resolution analysis of complex samples was achieved with a total analysis time of less than 5 min from injection to injection. Fractions collected during preparative chromatography were analyzed by SDS gel electrophoresis and fast HPLC. Specific proteins in the fractions were detected and quantitated by fast HPLC providing real-time analysis for pooling. The technique was also applied to the formidable task of detecting and quantitating protein variants during the development of recovery processes. Several examples of post-translational variant detection are shown. Ultrafast HPLC is a new analytical tool that can be applied to the development of robust manufacturing processes producing therapeutic proteins essentially free of known impurities and variants.     

雷公藤粗提物毒力测定及甲素含量分析

以无水乙醇为溶剂从雷公藤根皮中提取有效成分,用高效液相色谱法分析雷公藤粗提物中甲索含量,并将雷公藤粗提物配成5％的乳油,用浸叶法和药膜法测定小菜蛾二龄幼虫、烟粉虱成虫以及无翅成蚜的室内毒力,并以几种杀虫剂作为对比。结果表明：该批雷公藤粗提物中甲素含量为0．0012％;5％雷公藤粗提物乳油对小菜蛾二龄幼虫、烟粉虱成虫以及无翅成蚜具有一定的生物活性,但与其他几种常用杀虫剂相比活性较低,还需对其进一步精制和提纯。     

High performance capillary electrophoresis.

The application of recombinant-DNA methods for the production of therapeutic proteins has, over the past decade, driven the development of new technology for the analysis and characterization of biological molecules. High performance capillary electrophoresis (HPCE) has generated enormous interest among biochemists, analytical chemists and chromatographers, and is emerging as an extremely high-resolution separation technique, that may rival high performance liquid chromatography (HPLC) in its efficiency and breadth of application.     

Simultaneous determination of four sesquiterpene alkaloids in Tripterygium wilfordii Hook. F. extracts by high-performance liquid chromatography

Wilfortrine, wilfordine, wilforgine and wilforine are four major bioactive sesquiterpene alkaloids in Tripterygium wilfordii Hook. F. The first analytical determination of the four major bioactive alkaloids is described. The four alkaloids are well-resolved within 15 min using the developed HPLC method. The identity of the analytes was confirmed by an HPLC-MS experiment, with all compounds being clearly assignable by atmospheric pressure chemical ionization (APCI) positive mode analysis. The method was validated for limit of qualification, linearity and inter-day variation of precision and accuracy. Seven T. wilfordii samples (extracts and commercial product) were successfully analysed.     

The Application of HPLC Methodology for the Analysis of Receptor Mediated Changes in Phosphoinositide Metabolism

Abstract

HPLC methodology has found wide application in analytical problems in biochemistry. To study the metabolism of phosphatidylinositol and its regulation by receptor mediated events, HPLC could be a valuable technique. It has been recently demonstrated that a variety of hormones and neurotransmittors act to stimulate hydrolysis of phosphoinositides by a phospholipase C. To monitor this reaction, we have analysed the formation of radiolabelled inositol phosphates from phosphoinositides. The present paper describes a rapid HPLC procedure, to separate inositol phosphates from myo-inositol, which could be used in pharmacological studies of receptors linked to phosphoinositide hydrolysis. The potential of the application of HPLC to the analysis of the phospholipids involved is discussed.     

Rapid analysis and purification of polymerase chain reaction products by high-performance liquid chromatography

This report describes the use of high-performance liquid chromatography (HPLC) for the rapid analysis and purification of the polymerase chain reaction products. Employing a new anion-exchange nonporous column, efficient separations of both DNA restriction fragments and amplified PCR products are achieved in 10 to 20 minutes and quantitated within +/- 10%. The performance of the HPLC technique is described in terms of resolution, reproductibility, sensitivity and micropreparative capability and compared to that of gel electrophoresis for this application.     

A novel method to convert triptolide into tripchlorolide in Tripterygium wilfordii

A new method has been established to convert triptolide (1) into tripchlorolide (2) directly in the Chinese herbal drug Tripterygium wilfordii Hook F. and the influences of reaction times and pH values have been investigated. It was found that 1 could be most efficiently converted into 2 using a hydrochloric acid-acetic acid system. An HPLC method was devised in order to monitor the conversion and ESI-MS was used to identify the synthetic product 2. The sensitivity of the assay was sufficient to monitor the conversion of the main active components in T. wilfordii.     

雷公藤解毒持效双向发酵工艺的建立

采用双向发酵的原理,运用"发酵过程动态比较法"研究了灵芝接种于雷公藤药性基质上发酵不同时间所得菌质的化学成分、急性毒性和免疫功能的变化,以其确定雷公藤解毒持效双向发酵的最佳发酵终点。研究结果表明:发酵第30天所得的菌质(G30)总二萜的含量最低,为0.57%;与雷公藤生药比较,G30的LD50最高,且发酵第30天所得菌质的体液免疫和细胞免疫抑制作用最强。综合对成分含量、毒性及药效的动态数据联系比较、分析,确定了雷公藤解毒持效双向发酵的发酵终点与适宜的发酵周期是菌丝长满瓶后的第30天。     

High-performance liquid chromatography in enzymatic analysis: Opening lecture

During the previous two decades, high-performance liquid chromatography (HPLC) has proven to be an extremely useful technique with which to study the activity of enzymes and this paper will explore some of these uses. The success of the method can be seen not only from the increase in the number of papers utilizing this technique but also from the insights gained from its use on cellular phenomena. Given this success, it is no wonder that HPLC has become the technique of choice for many biologists seeking a more quantitative understanding of biological processes. Based on past experience, there is every reason to expect that the application of HPLC to the assaying of enzymatic activities will usher in another era of fundamental discoveries in the biological sciences. HPLC is particularly well suited to the assay of one activity in the presence of other activities obviating the need for extensive and tedious purification of biological samples. This advantage makes this technique particularly well suited to those who wish to use enzymes as markers for cellular processes, as indicators of metabolic activity and as evidence of gene function. To date, well over 100 activities have been assayed by this method. The method is particularly suited to problem-solving especially in such cases as when the presence of competing reactions prevents the recovery of the expected reaction products. Of the many applications, examples will be given on the use of HPLC for (1) monitoring the activity of an enzyme in a cell-free system, (2) monitoring the flow of metabolites through a multienzyme system and (3) the detection and study of new enzymatic activities. Some generalizations about the use of HPLC methods for the analysis of enzymatic activities will be presented.     

Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae)

Plants in the genus Tripterygium, such as Tripterygium wilfordii Hook.f., have a long history of use in traditional Chinese medicine. In recent years there has been considerable interest in the use of Tripterygium extracts and of the main bioactive constituent, the diterpene triepoxide triptolide (1), to treat a variety of autoimmune and inflammation-related conditions. The main mode of action of the Tripterygium extracts and triptolide (1) is the inhibition of expression of proinflammatory genes such as those for interleukin-2 (IL-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2) and interferon-gamma (IFN-gamma). The efficacy and safety of certain types of Tripterygium extracts were confirmed in human clinical trials in the US and abroad. Over 300 compounds have been identified in the genus Tripterygium, and many of these have been evaluated for biological activity. The overall activity of the extract is based on the interaction between its components. Therefore, the safety and efficacy of the extract cannot be fully mimicked by any individual constituent. This review discusses the biochemical composition and biological and pharmacological activities of Tripterygium extracts, and their main bioactive components.