不同产地蒺藜核糖体DNA内转录间隔区序列分析

目的通过测定核糖体DNA内转录间隔区(Ribosomal DNA internal transcribed spacer,rDNA-ITS)基因序列,确定不同产地的蒺藜在种质遗传上是否存在差异。方法利用PCR产物直接测序,测定不同产地蒺藜的rDNA-ITS基因序列。结果测得ITS碱基序列742 bp,其中ITS1全部序列267 bp,5.8 S全部序列167bp,ITS2全部序列209 bp。6个产地的蒺藜样品的ITS的碱基序列完全相同。结论不同地区的蒺藜在种质上没有发生变异。     

基于5S-rRNA 基因间区碱基序列鉴定繁缕

目的建立快速准确鉴定民间药繁缕(Stellariamedia)的方法。方法首次利用DNA分子鉴定技术对繁缕及混淆品牛繁缕(Myosotonaquaticum)进行了5SrRNA基因间区的PCR扩增和测序。结果DNA序列和限制性酶切谱可以用于鉴定繁缕及牛繁缕。同时对石生繁缕(S.vestita)、长叶繁缕(S.longifolia)及垂梗繁缕(S.radians)进行了5SrRNA基因间区的PCR扩增和测序,认为上述5种植物各自的DNA序列和限制性酶切谱可用于繁缕属分子系统学研究。结论该方法可用于药用植物繁缕的鉴定及繁缕属分子系统学研究。     

香港特有止泻中药材红党参的化学和分子生物学鉴定

目的鉴定香港特有止泻中药材红党参的基源植物和表面红色物质。方法将红党参的5SrRNA基因间隔区序列和HPLC色谱指纹谱与正品党参5SrRNA基因间隔区序列和HPLC色谱指纹谱进行比较研究;将红党参表面红色物质的X-衍射光谱与中药赤石脂X-衍射光谱进行比较研究。结果红党参的HPLC色谱指纹谱和党参药材具有相同的特征色谱峰;红党参的5SrRNA基因间隔区序列与党参正品之一的素花党参（Codonopsis pilosula var.modesta）的5SrRNA基因间隔区序列最为相似;红色物质的X-衍射光谱与中药赤石脂X-衍射光谱完全相同。结论红党参的基源是素花党参（Codonopsis pilosula var.modesta）的根,红党参表面的红色物质是传统中药赤石脂（Halloysitum Rubrum）。红党参是素花党参根用中药赤石脂炮制而成,科学名可定为Radix Codonopsis Praeparata Halloysita Rubra。     

儿童近端小肠粘膜相关菌群分布研究

目的研究儿童近端小肠粘膜相关菌群种类和分布。方法取儿童近端小肠粘膜活检组织,提取总DNA,经PCR扩增后对16SrRNA基因扩增片段克隆,并对克隆片段进行DNA序列测定,根据序列结果分析细菌组成。同时在不同培养条件下进行细菌培养及鉴定。结果16SrRNA基因测序结果表明儿童近端小肠粘膜相关菌群包括6个细菌门类,厚壁菌门、变形菌门和放线菌门占总细菌的88.8%;拟杆菌门、梭杆菌门和TM7占11.2%。链球菌属和奈瑟菌属为儿童近端小肠粘膜相关菌群的主要菌属;韦荣菌属、孪生菌属、放线菌属、罗氏球菌属、嗜血菌属、普雷沃菌属和颗粒链菌属占较高比例。细菌培养结果与16SrRNA基因测序结果类似。结论儿童近端小肠粘膜具有独特的菌群定殖分布。     

三七的18S rRNA,matK基因序列和HPLC化学指纹图谱分析研究

目的分析中药三七Panaxnotoginseng的18SrRNA和matK基因的分子特征和三七的化学指纹特征,为三七的正品药材基原鉴定提供分子和化学依据。方法采用PCR直接测序技术测定三七及其7种伪品的18SrRNA和matK基因部分核苷酸序列以及不同产地三七的DNA分子特征。利用HPLC的化学分析技术,明确产地对三七化学成分的影响,以及三七不同部位的化学指纹特征。结果(1)三七及其7种常见伪品的核糖体18SrRNA基因序列存在很大的差异。(2)不同产地的三七的核糖体18SrRNA和叶绿体matK基因序列特征完全一致,分别与GenBank上已报道的R1型(D85171)和M1型(AB027526)序列吻合。(3)不同产地的三七HPLC指纹图谱相似。(4)三七不同部位均具有其相对稳定的HPLC指纹特征,其中花、叶具有特有的指纹区,根、须根、剪口、筋条等不同商品规格的HPLC指纹图谱比较相似。结论基因序列标记能从分子水平定性分辨三七及其伪品的遗传背景差异,为中药品种标准化提供了先进可行、稳定可靠的分子标准;HPLC指纹图谱分析可以直观地为三七的化学成分定性,三七不同商品规格的特征性指纹有望成为以其为原材料的各种产品的质控标准,而三七不同部位(尤其是花和叶)的HPLC指纹图谱将有望成为制定三七花、三七叶新药用资源质控标准的依据。     

广藿香的基因序列与挥发油化学型的相关性分析

目的探讨“南药”广藿香Pogostemon cablin (Blanco) Benth.不同产地间的叶绿体和核基因组的基因型与挥发油化学型的关系，为广藿香道地性品质评价、规范化种植提供分子依据。方法用PCR直接测序技术对广藿香6个产地样本的叶绿体matK基因和核18S rRNA基因核苷酸序列进行测序分析研究。结果广藿香6个样本的matK基因序列长均为1 245 bp，编码415个氨基酸成熟酶。18S rRNA基因序列长为1 803～1 805 bp。根据排序比较，广藿香6个样本间的matK基因序列存在47个变异位点，18S rRNA基因存在17个变异位点，非加权组平均法构建的系统分支树表明广藿香基因序列分化与其产地、所含挥发油化学变异类型呈良好的相关性。结论结合挥发油分析数据，基因测序分析技术可作为广藿香道地性品质评价方法这一以及规范化种植过程关键技术“物种鉴定”的强有力工具。     

中药材龟甲的分子鉴定研究

用PCR产物直接测序法对中药材龟甲(板)进行鉴别。从乌龟 Chinemys revesii 和其他20种产地为中国或东南亚国家的龟类的组织材料中提取DNA,扩增约110bp的线粒体12SrRNA,基因片段并进行序列分析,构建了21种龟类的12SrRNA基因片段序列数据库。序列比较的结果表明乌龟与其它20种龟类的这段序列均有差别,序列差异在3.7～15.7%之间。从江苏省药品检验所提供的19块龟甲检品上各取样0.1～0.5g提取 DNA,扩增与上述相同的基因片段,与构建的数据库进行比较,结果表明19块龟甲中只有3块的原动物为乌龟,其余的龟甲均为混淆品。本文的结果为药材龟甲的鉴定找到了有效、可靠的分子遗传标记方法。     

现代生物技术在中药鉴定方面的应用及前景

近几年,在分子生物学领域中,DNA分子遗传标记(DNAMoleuclar Gene-tic Marker)技术迅猛发展,新方法、新技术不断涌现。继同工酶(isozyme)、 RFLP (Restriction FragmentLength Polymorphi - sm)和DNA指纹(DNA fingerprinting)技术以后,90年代又发展了RAPD(Random Amplifed Polymor-phic DNA)、CAPS (Cleaved Amplified Polymorphic Sequence)、AFLP(AmPlified Fragment Length Polymorphic)技术等,它们已越来越多地用于药材品种鉴别的研究。在已有的研究报道中使用的鉴别方法可归为三类：(1)基于PCR反应的方法,包括RAPD、AP-PCR、微卫星DNA等;(2)限制性片段长度多态性和PCR产物的RFLP分析(PCR-RFLP);(3)DNA测序,已报道了5SrRNA间隔区、ITSI、ITS2、Cytb和12S rRNA等基因片段的序列分析。以下对在中药材品种鉴别中应用较多的RAPD和高特异性PCR鉴别技术作一简单介绍。     

蒙药悬钩子木的rbcL序列分析

目的 通过对蒙药悬钩子木DNA进行PCR扩增,测序分析不同产地悬钩子木的rbcL序列,并与悬钩子木混伪品的rbcL序列进行比较。方法 采用植物基因组DNA提取试剂盒,从悬钩子木20个样本中提取总DNA,对rbcL序列进行PCR扩增并测序;从GenBank上下载悬钩子木常见混伪品的rbcL序列,鉴定不同产地悬钩子木及其混伪品的rbcL序列。结果 悬钩子木的种内最大遗传距离小于混伪品的种间最小遗传距离,NJ系统发育树显示：悬钩子木20个样品聚为一支,能与同属的混伪品红泡刺藤、紫色悬钩子、拟复盆子、直立悬钩子、藏南悬钩子、多腺悬钩子、秀丽莓、无腺白叶莓、粉枝莓进行区分。结论 rbcL序列可用于鉴定悬钩子木及其混伪品的条形码序列。     

基于454FLX高通量技术的厚朴叶绿体全基因组测序及应用研究

 叶绿体基因组序列在中药DNA条形码鉴定及基因工程生物制药方面具有广泛的应用前景。本文应用454高通量测序技术对厚朴进行了叶绿体全基因组测序, 建立了标准测序流程。生物信息学分析共获得有效序列重叠群 (contig) 14个, 测序覆盖度达到99.99%。厚朴叶绿体基因组大小为160 183 bp, 大 (LSC)、小 (SSC) 单拷贝区大小分别为88 210 bp和18 843 bp, 反向互补重复区 (IR) 大小为26 565 bp, 共注释叶绿体基因126个, 其中每个IR区17个。厚朴与木兰亚纲其他物种的叶绿体编码基因在种类、排列顺序及结构大小上基本一致。采用叶绿体81个蛋白编码基因对厚朴及同属5个种9个样本进行了分子鉴定, 鉴定效率100%, 并且可以成功区分不同产地的凹叶厚朴。以上结果表明, 本研究建立的标准测序流程适用于叶绿体基因组测序, 叶绿体基因组序列可有效区分厚朴及近缘物种。     

HPLC-ELSD测定穿心莲超分子提取物中三种穿心莲内酯的含量

目的 建立穿心莲超分子提取物中穿心莲内酯、脱水穿心莲内酯和14-去氧穿心莲内酯的含量测定方法.方法 HPLC-ELSD;色谱柱:Agilent ZORBAX Extent-C18 (4.6 mm×250 mm,5 μm);检测器:蒸发光散射检测器(ELSD);流动相:乙腈-水,线性梯度洗脱,流速0.8 mL·min-1,柱温:40 ℃.结果 穿心莲内酯、脱水穿心莲内酯和14-去氧穿心莲内酯线性范围分别为0.920～9.200 μg(r=0.999 6),0.397～3.952 μg(r=0.999 3)和0.395～3.936 μg(r=0.999 3);三者的回收率分别为98.9%、97.8%和98.4%,RSD分别为1.1%、2.2%和2.1%(n＝6).结论 本法简便,快捷,结果真实可靠,可用于穿心莲超分子提取物穿心莲内酯等有效成分的含量测定.     

Stability of andrographolide in powdered Andrographis herb under accelerated conditions

The stability of andrographolide in powdered Andrographis Herb--the aerial part of Andrographis paniculata (Burm. f.) Nees (Acanthaceae)--was determined using a heat-accelerated experiment to reveal a second-order kinetics of degradation. The fast decomposition was observed regardless of the method of analysis. The rate constant of the decomposition of andrographolide at 25 degrees C ( K(25)( degrees C)), predicted from the Arrhenius plot, was 6.58 x 10 (-6) d (-1).     

穿心莲中穿心莲内酯、脱水穿心莲内酯在茎和叶中的分布

采用薄层扫描法分别测定了12批穿心莲商品药材中茎和叶所含穿心莲内酯和脱水穿心莲内酯的含量,并进行了比较,结果其总量在茎中平均占0.29%,在叶中平均占2.26%.因此建议穿心莲中叶所占比例应不少于35%.     

Antidiabetic and antihiperlipidemic effect of Andrographis paniculata (Burm. f.) Nees and andrographolide in high-fructose-fat-fed rats

Objectives:

Andrographis paniculata (Burm. f.) Nees originates from India and grows widely in many areas in Southeast Asian countries. Andrographis paniculata (Burm. f.) Nees has shown an antidiabetic effect in type 1 DM rats. The present study investigates the purified extract of the plant and its active compound andrographolide for antidiabetic and antihyperlipidemic effects in high-fructose-fat-fed rats, a model of type 2 DM rats.

Materials and Methods:

Hyperglycemia in rats was induced by high-fructose-fat diet containing 36% fructose, 15% lard, and 5% egg yolks in 0.36 g/200 gb.wt. 55 days. The rats were treated with the extract or test compound on the 50^th day. Antidiabetic activity was measured by estimating mainly the pre– and postprandial blood glucose levels and other parameters such as cholesterol, LDL, triglyceride, and body weight.

Results:

The purified extract and andrographolide significantly (P<0.05) decreased the levels of blood glucose, triglyceride, and LDL compared to controls. However, no changes were observed in serum cholesterol and rat body weight. Metformin also showed similar effects on these parameters.

Conclusions:

Andrographis paniculata (Burm. f.) Nees or its active compound andrographolide showed hypoglycemic and hypolipidemic effects in high-fat-fructose-fed rat.KEY WORDS: Andrographis paniculata (Burm. f.) Nees, andrographolide, diabetes, fructose, hyperlipidemia     

穿心莲提取物对凝固酶阴性葡萄球菌的体外抗菌活性研究

目的:观察穿心莲提取物对凝固酶阴性葡萄球菌的体外抗菌活性。方法:采用新的中药抑菌实验方法对穿心莲提取物进行100株凝固酶阴性葡萄球菌的最低抑菌浓度MIC测定。结果:穿心莲提取物对57株耐甲氧西林的凝固酶阴性葡萄球菌和43株甲氧西林敏感凝固酶阴性葡萄球菌的MIC50、MIC90分别为0.072,0.072mg/mL和0.288,0.144mg/mL。结论:穿心莲提取物对100株凝固酶阴性葡萄球菌具有较强的抑菌力,说明穿心莲是极具开发前景的抗感染中药。     

Suppression of rat neutrophil reactive oxygen species production and adhesion by the diterpenoid lactone andrographolide

The present study was to examine whether andrographolide, a diterpenoid lactone isolated from the anti-inflammatory herbal medicine Andrographis paniculata (Burm. f.) Nees. (Acanthaceae), has the ability to prevent phorbol-12-myristate-13-acetate (PMA)-induced reactive oxygen species (ROS) production, as well as N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced adhesion by rat neutrophils. Results demonstrated that PMA (100 ng/ml) induced rapid accumulation of H2O2 and O2. in neutrophils within 30 minutes. Andrographolide (0.1 to 10 microM) pretreatment (10 min, 37 degrees C) significantly attenuated the accumulation of these two oxygen radical metabolites. Administration of andrographolide also significantly prevented fMLP-induced neutrophil adhesion. These data suggest that preventing ROS production and neutrophils adhesion may confer andrographolide the ability to be an anti-inflammatory drug.     

Hepatoprotective effects ofAndrographis paniculata against carbon tetrachloride-induced liver damage

Alcoholic extract of the leaves of Andrographis paniculata Ness (= AAP) was obtained by cold maceration. A dose of 300 mg/kg (1/6 of LD50) of the extract was selected to study hepatoprotective action against carbon tetrachloride-induced liver damage. The extract was found to be effective in preventing liver damage which was evident by morphological, biochemical and functional parameters.     

Characters of nrDNA ITS region sequences of fruits of Alpinia galanga and their adulterants.

The fruits of Alpinia galanga (L.) Sw. are used as a traditional Chinese medicine; but the dry fruits of A. conchigera, A. suishaensis, A. maclurei and A. polyantha are also used as the medicine in local areas. Because dry fruits of these related plants are similar to those of Alpinia galanga (L.) Sw. in odor, morphological characters and chemical components, and even anatomical characters, it is difficult to identify the medicine. Nuclear ribosomal DNA internal transcribed spacer (ITS) regions of the five taxa were directly sequenced using an automated sequencer. Sequence analysis showed that the ITS 1 ranges from 177 to 178 base pairs (bp), and the ITS 2 from 225 to 234 bp. The size of the 5.8S coding region is 164 bp for all species. Also, the pairwise sequence divergence is higher and some molecular markers were determined. According to these molecular markers, Alpinia galanga (L.) Sw. and the related species can easily be distinguished from each other. Therefore, evidence from nrDNA ITS sequence variation can identify the medicine at the DNA level.     

The effects of Andrographis paniculata (Burm.f.) Nees extract and diterpenoids on the CYP450 isoforms’ activities,a review of possible herb–drug interaction risks

Andrographis paniculata (Burm.f.) Nees is a popular medicinal plant and its components are used in various traditional product preparations. However, its herb-drug interactions risks remain unclear. This review specifically discusses the various published studies carried out to evaluate the effects of Andrographis paniculata (Burm.f.) Nees plant extracts and diterpenoids on the CYP450 metabolic enzyme and if the plant components pose a possible herb-drug interaction risk. Unfortunately, the current data are insufficient to indicate if the extracts or diterpenoids can be labeled as in vitro CYP1A2, CYP2C9 or CYP3A4 inhibitors. A complete CYP inhibition assay utilizing human liver microsomes and the derivation of relevant parameters to predict herb-drug interaction risks may be necessary for these isoforms. However, based on the current studies, none of the extracts and diterpenoids exhibited CYP450 induction activity in human hepatocytes or human-derived cell lines. It is crucial that a well-defined experimental design is needed to make a meaningful herb-drug interaction prediction.