【作者】 赵云鹏；

【导师】 傅承新；

【作者基本信息】 浙江大学 ， 植物学， 2006， 博士

【摘要】 猫人参为华东地区治疗各类消化道肿瘤的习用中草药，是目前本地区抗肿瘤处方中最多用的品种之一。目前中药学及植物学专著多记载其原植物为对萼猕猴桃（又称镊合猕猴桃）（Actinidia valvata Dunn），但民间也有以大籽猕猴桃（A．macrosperma C．F．Liang）为猫人参的原植物，甚至认为后者入药疗效更佳。而且因猫人参用量较大而野生资源有限，时常有同属其它种类如黑蕊猕猴桃（A．melanandra Franch）、小叶猕猴桃（A．lanceolata Dunn）、中华猕猴桃（A．chinensis Planch．）等混用为猫人参，影响了本品的品质和疗效。因此，当务之急首先要确定正品猫人参的原植物，并准确鉴别其与混淆品；其次猫人参的近缘种能否代用，以扩大目前严重紧缺的药源。基于此目的，本文考证了猫人参原植物，系统鉴别了猫人参及其混淆品，研究了猫人参有效部位HPLC图谱与体外抗肿瘤活性，分析比较了猫人参及其混淆品的矿质元素，确认了猫人参猫草反应的化学物质。1、原植物考证通过民间与市场调查、药材性状与原植物形态鉴定、文献查阅，根据原植物形态、药用历史和学名发表时间，明确了正品猫人参的主要来源应为大籽猕猴桃，而对萼猕猴桃可能只是其次要来源。2、种质鉴别从原植物形态、生药性状、荧光反应、紫外光谱、荧光光谱、傅立叶变换红外光谱（FT-IR）、PCR-RFLP等角度系统地鉴别了大籽猕猴桃及其近缘种，尤其是形态相似而一直被认为是猫人参原植物的对萼猕猴桃。大籽猕猴桃与对萼猕猴桃等近缘种的形态差异主要在繁殖器官花果上，因此营养生长期较难鉴别。以生药性状为基础，结合9种猕猴桃生药的蒸馏水、乙醇、氯仿、石油醚提取液在254nm、365nm紫外光激发下的荧光颜色，4种提取液的紫光光谱，水提液和醇提液的荧光光谱，完全、准确鉴别了9种药用猕猴桃，编制了鉴定检索表。9种猕猴桃红外谱图总体较相似，但中华猕猴桃和毛花猕猴桃有强烈末端吸收，明显区别于其它7种猕猴桃，进一步比较大籽猕猴桃等7种猕猴桃与中华猕猴桃红外谱图的相减谱图，完全区分了9种猕猴桃，编制了鉴定检索表。限制性内切酶DdeI、DraI对大籽猕猴桃叶绿体trnK片段的酶切式样具有特异性，建立了大籽猕猴桃的PCR-RFLP分子指纹图谱，可有效鉴别猫人参混淆品的原植物。3、体外抗肿瘤活性比较体外抗肿瘤试验结果表明，大籽猕猴桃对供试的3种肿瘤细胞株均有较强的抑制作用，但敏感性存在差异，抑制效果为肝癌SMMC-7221(IC₅₀=54.22μg／ml)＞胃癌SGC-7901（133.16μg／ml）＞肺癌A549（206.63μg／ml）。大籽猕猴桃和对萼猕猴桃对人肝癌细胞株SMMC-7221抑制活性差异显著，后者（217.85μg／ml）仅为前者的1／4。异色猕猴桃、长叶猕猴桃、毛花猕猴、小叶猕猴桃桃对SMMC-7221的抑制效果均显著优于大籽猕猴桃，尤其是小叶猕猴桃的IC₅₀达到6.53μg／ml，活性相当于大籽猕猴桃的8倍，是本研究的重大发现，值得进一步研究。在人胃癌细胞株SGC-7901、人肺癌细胞株A549试验中，9种供试材料仅大籽猕猴桃和对萼猕猴桃有一定的抑制作用，而且二者活性相当，差异不显著。基于活性分析结果，我们建议在治疗胃癌和肺癌时，大籽猕猴桃和对萼猕猴桃活性接近，均可作猫人参入药，其它种类不宜混用；而治疗肝癌时，大籽猕猴桃的活性明显高于对萼猕猴桃，对萼猕猴桃不宜作猫人参入药，或者至少须用量加倍，验证了富阳草药医认为“白货”对萼猕猴桃药效不及“红货”大籽猕猴桃，需加倍使用的说法。结合HPLC图谱和体外抗肿瘤活性结果，推测猫人参中起抗肿瘤作用的有效组分可能并不是其含量最高的几个成分，而可能是本实验条件下吸收强度较弱、分离度不佳的化合物。本试验的HPLC图谱仍需进一步改进洗脱条件，改善图谱，以更加真实地反映乙酸乙酯部位的化学成分组成。4、矿质元素分析矿质元素（包括微量元素）与有效成分是中药气味的两大物质基础，采用电感耦合等离子体原子发射光谱法（ICP-AES）测定、比较了猫人参及其混淆品和浙江地区常见的其它药用猕猴桃的矿质元素含量。目前公认的与肿瘤发生、发展有关的微量元素有Cu、Zn、Se、Fe。大籽猕猴桃Cu、Zn含量显著高于其它8种猕猴桃，而Se含量较低，Fe含量中等，说明猫人参的抗肿瘤作用还可能与高含量的Cu、Zn有关。基于矿质元素的聚类图（加大了Cu、Fe、Se、Cu的权重）表明，大籽猕猴桃与其它8种猕猴桃均有较大差异，因此从抗肿瘤微量元素含量的角度，其它种类猕猴桃代用大籽猕猴桃作猫人参似乎并不适宜。5、猫草反应研究猫人参引起人们的关注并用于临床治疗癌症，始于其对猫的特异性吸引和嚼食作用，本文采用GC-MS法分析了野生与组培大籽猕猴桃叶片挥发油成分，确定了大籽猕猴桃引起家猫特异性取食现象（猫草反应）的化合物为二氢荆芥内酯、二氢猕猴桃内酯及阿根廷蚁素三种化合物。这三种化合物在野生植株和人工繁殖植株中均存在，且含量相似，推测这是大籽猕猴桃的遗传性状。更多还原

【Abstract】 Mao-Ren-Shen （cat ginseng） is one of the most frequently used traditional Chinese medicine （TCM） for anti-tumor therapy, especially against cancers of alimentary canal, in East China. It was recorded to be made from Actinidia valvata according to most TCM dictionaries and Flora. However, A. macrosperma, a morphologically similar species to A. valvata, has been believed to be the genuine resource of Mao-Ren-Shen and to be more effective than A. valvata in first and regularly applied area, based on our folk investigation. Enormous demands of this TCM led to over-exploitation of wild resource and confusion of original plant. Adulterants, such as A. melanandra, A. lanceolata, A. chinensis, were frequently observed to be misused as Mao-Ren-Shen resulted from wrong identification and profitable purpose. The quality and clinic effect were seriously jeopardized. The crucial issues are: i) What is the genuine resource of Mao-Ren-Shen; ii) how to identify it; iii) whether other Actinidia species can be used as it. To deal with these questions, we carried out a series of researches including original plant confirmation; morphological, chemical & physical, and molecular identification; inhibition activity on in vitro tumor cell line; analysis of mineral elements; analysis of essential oil. The results were obtained below.1. Confirmation of genuine resource of Mao-Ren-ShenA. macrosperma was proved to be the main resource of Mao-Ren-Shen, while the originally recorded resource A. valvata maybe the minor resouce at most, according to the results of folk and market survey, identification of plant and raw material, and history of medicinal application and scientific name.2. Germplasm identificationA. macrosperma was discriminated from adulterants comprehensively by methods of morphology of original plant and raw material, fluorescent reaction, UV spectrum, fluorescence spectrum, FT-IR, and PCR-RFLP. Raw material of A. macrosperma was completely and accurately identified by combination of morphology, fluorescent colors of 4 solvent extracts excited by 254nm and 365nm UV light, UV spectra of 4 solvent extracts, and fluorescent spectra of 2 solvent extracts. A discrimination key was presented finally. FT-IR spectra and subtraction spectra can distinguish raw materials of nine Actinidia species rapidly. Specific restriction patterns of trnK sequence from A. macrosperma digested by DdeI, DraI were obtained by PCR-RFLP. Molecular fingerprint of A. macrosperma was established.3. Comparison of inhibition activity on in vitro tumor cell linesThe results of tumor cell inhibition indicate A. macrosperma is effective to the studied three cell lines, but differ in intensity: SMMC-7221 （IC50=54.22μg/ml） > SGC-7901 （133.16μg/ml） > A549 （206.63μg/ml）. In assessment of activity on liver cancer SMMC-7721, A. callosa var. discolor, A. hemsleyana, A. eriantha and A. lanceolata are more active than A. macrosperma, especially A. lanceolata with IC₅₀ of 6.53μg/ml, about 8 times that of A. macrosperma. With regard to stomach cancer SGC-7901 and lung cancer A549, A. macrosperma （IC50=133.16μg/ml, 206.63μg/ml） is as effective as A. valvata （136.46μg/ml, 224.83μg/ml）, while the other species were ineffective. Therefore, our suggestion for application of Mao-Ren-Shen is that A. macrosperma and A. valvata can both be used as Mao-Ren-Shen for therapy of stomach and lung cancers, but A. valvata and other adulterants should not be used as Mao-Ren-Shen for liver cancer treatment. The high activity of A. lanceolata on liver cancer should be concerned.Based on the combination of HPLC profile and inhibition activity, it was speculated that the active components responsible for the cytotoxocity may be not the ones that the highest peaks in HPLC profile represent, while the ones unobviously detected. HPLC profiles need to be improved by modify elution methods.4. Analysis of mineral elementsMineral elements are deemed to benefit the effects of TCM, as well as bioactive fragment. Mineral elements of raw materials from nine Actinidia species were analyzed by ICP-AES. At least four mineral elements, Cu, Zn, Se, Fe, are believed to inhibit formation and growth of cancer. A. macrosperma is with the highest contents of Cu and Zn, relatively low content of Se, and middle content of Fe. It indicated that the effect of anti-tumor of A. macrosperma may be partly related to high contents of Cu and Zn. Cluster analysis was conducted based on mineral elements data with enlarged weights of Cu, Zn, Se, Fe. Cluster dendrograms showed A. macrosperma was quite different from the other eight species. Therefore, from the angle of mineral elements related to anti-tumor, it seemed other Actinidia species are not suitable to be used as Mao-Ren-Shen.5. Research on catnip responseMao-Ren-Shen first caught people’s interests because of its attraction to cat. Cats preferred eating its fresh leaves and twigs to excite themselves and cure wounds. The reaction was named catnip response. Essential oils from fresh leaves of both wild and regenerated plants of A. macrosperma were analyzed by GC-MS. Three compounds, dihydronepetalactone, iridomyrmecin, and dihydroactinidiolide, were detected in essential oils from leaves of both wild plant and artificially cultured plant with similar contents. These compounds have been recognized to be responsible for catnip response.更多还原