药用石斛鉴定方法的系统研究

【作者】 白音；

【导师】 阎玉凝；

【作者基本信息】 北京中医药大学 ， 中药学， 2007， 博士

【摘要】 石斛为兰科石斛属(Dendrobium)金钗石斛、铁皮石斛或马鞭石斛及其近似种的新鲜或干燥茎,具益胃生津和滋阴清热的功能。目前,我国药用石斛约有50余种,然而市场上把兰科石仙桃属Pholidota、石豆兰属Bulbophyllum和金石斛属Flickingeria的数十种植物也作为药用石斛混入商品流通和临床应用,对药用石斛的临床疗效和用药安全产生了很多不良影响。笔者认为药用石斛鉴定方法的不够完善是导致上述混乱问题的主要原因,为此本文以42种药用石斛及其混淆品为研究材料,对药用石斛的性状鉴定、显微鉴定、红外光谱(IR)鉴定、高效液相色谱(HPLC)鉴定和DNA分子标记鉴定方法进行系统研究,建立其综合鉴定方法,为药用石斛及其混淆品的鉴别提供有效手段和应用平台。其主要研究结果如下:(1)性状鉴定结果表明,在16个性状特征中较有专属性的性状特征有7个,分别是茎形状、茎分枝、干品质地、叶和叶鞘被黑毛、叶鞘套叠、叶片形状和叶鞘条纹;该鉴定方法能够鉴别金钗石斛D. nobile、流苏石斛D. fimbiatum和苏瓣石斛D. harveyanum等21种药用石斛及其混淆品。(2)显微鉴定结果表明,在22个显微特征中,表皮细胞大小及细胞壁增厚、皮下层细胞大小和细胞壁厚度、草酸钙方晶、草酸钙针晶以及簇状硅晶等17个显微特征的鉴别意义比较突出;该鉴定方法能够鉴别流苏石斛、金钗石斛和黑毛石斛D. williamsonii等16种药用石斛及其混淆品。本文补充和更正了金钗石斛和流苏石斛(马鞭石斛)茎显微构造特征,并对未报道的8种药用石斛及其混淆品(小黄花石斛D. jenkinsii、长苏石斛D. brymerianum、叉唇石斛D. stuposum、肿节石斛D. pendulum、滇桂石斛D. guangxiense、翅梗石斛D. trigonopus、栽培石斛D. sp. cv.和密花石豆兰Bulbophyllum odoratissimum)的茎横切面显微构造进行描述。(3)IR鉴定结果表明,42种药用石斛及其混淆品具有丰富的IR指纹特征,其IR指纹峰共493个,平均每种有9～15个指纹峰,其氯仿提取物的总IR指纹峰为708个,平均每种有10～28之间;IR鉴定方法能够鉴别32种药用石斛及其混淆品。另外,IR技术还可以用于药用石斛药效组分的红外指纹特征的表征。(4)在42种药用石斛及其混淆品中,每种共选用7～15个HPLC色谱峰,用于其HPLC鉴别。结果表明,该鉴定法能够鉴别40种药用石斛及其混淆品,其中包括药典(2005版)收载的3种药用石斛(金钗石斛、流苏石斛和铁皮石斛)。(5)利用筛选出来的5种随机引物对41种药用石斛及其混淆品进行RAPD分析,结果显示,共扩增出222个多态性位点,平均每种药用石斛及其混淆品的多态性位点为12～26个之间。分析结果表明,RAPD分子标记鉴定方法能够鉴别39种药用石斛及其混淆品。(6)利用筛选出来的8种EcoR I和Mse I选择性引物组合对41种药用石斛及其混淆品进行AFLP分析,结果显示,8种选择性引物共扩增出1648个位点,其中多态性位点有1643个,特异性条带有107个。分析结果表明,AFLP分子标记鉴定方法能够鉴别39种药用石斛及其混淆品。对一份未知样品(DSP.)的AFLP鉴别结果表明,该鉴定方法适用于那些性状显微鉴定方法难以鉴别的药用石斛的DNA分子鉴定。说明上述5种鉴定方法都不能完全鉴别42种药用石斛及其混淆品,为此本文在5种鉴定方法进行综合评价的基础上,建立了药用石斛综合鉴定方法,为药用石斛及其混淆品的鉴定提供了较为可行的技术和方法。(7)利用性状、显微、IR、HPLC和DNA分子标记鉴别数据,借助Access数据库软件,初步建立了药用石斛检索数据库。该数据库的主要特点是检索速度快,操作简便,并可以利用ASP应用程序打开和远程网络访问,为药用石斛及其混淆品的鉴别提供了良好的检索工具和应用平台。(8)测定结果显示,生长年限为一年、二年和三年的美花石斛多糖含量分别是18.45%,23.54%和15.71%;5个不同产地美花石斛多糖含量为12.21～22.62%之间,平均含量为17.80%。39种药用石斛的多糖含量为7.69%～24.13%之间,平均含量为15.56%,其中铁皮石斛多糖含量最高(24.13%),剑叶石斛为最低(7.69%)。说明药用石斛多糖含量无论在种间还是种内均有较大差异,该分析结果对药用石斛质量标准的制度和评价提供科学依据。更多还原

【Abstract】 Herba Dendrobii(Shi-hu) is the fresh or dried stem of Dendrobium nobile, D. candidum(D. officinale) or D. fimbriatum var. oculatum(D. fimbriatum) and their allied species according to the Chinese Pharmacopoeia(2005) for nourishing“Yin”and clearing heat. Now there are more than 50 kinds of medical Dendrobii used as“shi-hu”, but some other species are also used as medical Dendrobii in the circulataion of commodity and clinical application, including the plant of Pholidota, Bulbophyllum and Flickingeria of Orchidaceae etc. It has harmful effects in the clinical effect and the safety of the application. The writer presume that the imperfect identification methods of medical Dendrobii is the main cause of these confusions. Therefore this research had a systemic investigation on 42 kinds of Dendrobium(Shihu) and their adulterants used in our country by mesans of character and micro-identification, IR, HPLC, DNA molecular identification, and then used synthesis identification to establish a platform and supply a good device of identifying medical Dendrobii and their adulterant . The main findings as follows:(1) The results of character identification revealed that there are 7 specific identification characters among the 16 ones, which are the shape of stem, the branch of the stem, the texture of the dried, the leaves and vaginas covering with black floss, the vagina’s cascade, the shape of the leaf, and the stripe of the vagina. This method can identify 21 kinds of medical Dendrobii and their adulterants, such as D. nobile, D. fimbiatum and D. harveyanum etc.(2) The results of microscopical identification indicated that the identifying meaning of 17 characters in 22 microscopical characters are outstanding such as the size and the type of sclerosis of epidermal cell, the size and thickness of stratum subcutaneum cell, the calcium oxalate of prism and acicular crystal and the cluster silicon crystal ect. The method can identify 16 kinds of medical Dendrobii and their adulterants such as D. fimbriatum, D. nobile and D. williamsonii. This paper corrected and replenished the microstructure of D. nobile and D. fimbriatum, and described the microstructure of cross section of the other 8 kinds of medical Dendrobii and their adulterant which are not reported. They are D. jenkinsii, D. brymerianum, D. Stuposum, D. pendulum, D. guangxiense, D. trigonopus, D. sp. cv. and Bulbophyllum odoratissimum.(3) The results of IR identification revealed that there are profuse IR fingerprint characters among the 42 kinds of medical Dendrobii and their adulterants. The total IR fingerprint peaks of them are 493 and the average are between 9 and 15 in every species; the IR fingerprint peaks of extractive of chloroform are 708 and the average are between 10 and 28. The analytic result indicated that this method of identification can be used to identify 32 kinds of medical Dendrobii species and their adulterants. And IR can be used in drug action composition’s superficial syndrome and offer evidence to quality evaluation of medical Dendrobii as well as.(4) Among 42 kinds of medical Dendrobii and their adulterants we choose 7~15 HPLC chromatographic peaks as HPLC identification characters for them. The results indicated that this method can identify 40 kinds of medical Dendrobii including 3 kinds of medical Dendrobii which in the Chinese Pharmacopoeia(2005).(5) We have sieved 5 RAPD random primer to analyze 41 kinds of medical Dendrobii and their adulterant. The results showed that the 5 random primers amplified 222 polymorphic loci, the average of polymorphic loci are among 12～26. The results indicated that the RAPD molecule mark identification method can identify 39 kinds of medical Dendrobii and their adulterants.(6) We have sieved 8 kinds of EcoRI and MseI selective primers to analyze 41 kinds of medical Dendrobii and their adulterants. The results showed that the 8 kinds of AFLP selective primers are amplified 1648 loci, among them there are 1643 polymorphic loci and 107 special straps. The analytic result indicated that the method of AFLP molecular identification can identify 39 species medical Dendrobii and their adulterant. The result of the AFLP identification of a unkonwn sample (DSP.) indicated that the method refer to the DNA molecular identification of medical Dendrobii which other method hard to identifyed.It indicated that the 5 methods could not completely identify 42 kinds of medical Dendrobii and their adulterants. So this paper based on the synthesis estimate of 5 identification methods to establish the synthesis identification of medical Dendrobii. It can apply a feasible technique and method to identify medical Dendrobii.(7) We have set up the search database of medical Dendrobii initially by Access database software through the data of character, micro-identification, IR and DNA molecular identification which has fast speed, convenient operation, and it can also be unfolded with ASP application program and long range access through network. It is an application platform for identification to share together.(8) The contents of polysaccharides of D. loddigesii in different growth stages (one year, two years and three years) are 18.45%,23.54% and 15.71% respectivly, and the total content of polysaccharides of D. loddigesii from five areas lies between 12.21 and 22.62%, average 17.80%. The contents of polysaccharide of 39 kinds of medical Dendrobii are between 7.69%～24.13%, and the average is 15.56%. Among them, D. officinale has the highest content(24.13%), while D. acinaciforme has the lowest(7.69%). All of these revealed that there is obviously difference on the contents of polysaccharide among medical Dendrobii and their adulterants. The polysaccharide assaying of different species can offer reference to the formulating of quality criteria of medical Dendrobii.更多还原