【作者】 刘继永；

【导师】 王英平；

【作者基本信息】 中国农业科学院 ， 药用植物资源学， 2010， 博士

【摘要】 中药材栽培、炮制加工与质量评价是中药现代化的重要组成部分,也是本学科研究的方向之一。其中,针对人参的研究,一直是本领域的重点研究课题。人参（Panax ginseng C.A.Mey）是吉林省主要道地药材,人参做为吉林的优势产业,在国际市场上占有举足轻重的份额,人参产业的发展状况,直接影响其在吉林特种农业发展中的引领地位。近年来,随着人参不同用药部位及其加工产品在医药及保健品领域的研究应用,众多人参制品应运而生,又因为人参种植环境与产品需求的长期矛盾,使得人参在不久的将来将出现资源短缺的态势。因此,寻找新的人参药用资源已成为当务之急。蒸参水是红参加工过程中的副产物,是在红参加工过程中,水蒸气对人参蒸熏、浸渍,冷却后,溶于蒸锅内的一种暗棕色、混浊、粘稠状水溶液,含有部分人参皂苷,人参多糖以及多种氨基酸。红参的世界年产量约为7000吨,中国约为4000吨,吉林省约为3400吨,每加工1Kg红参干品,可得蒸参水干物质48.6g。因此,吉林省蒸参水年产量极为可观,生产上一般将其做为废物弃掉,势必造成资源的浪费。因此,系统研究蒸参水的化学成分和生物活性,对吉林省人参的产业化开发,具有现实指导意义。为此,开展本项研究,并取得如下进展:1.蒸参水化学成分研究。本项研究采用有机溶剂提取、萃取、大孔吸附树脂、ODS柱层析、硅胶柱层析、凝胶层析等常规的植物化学方法和手段,结合制备型HPLC等先进的仪器设备,通过多种不同的提取、分离制备工艺路线,从蒸参水中分离制备了11个人参皂苷单体化合物。通过物理常数、化学方法和MS波谱分析鉴定了11个化合物归属。分别为原人参二醇型皂苷Rb1、Rb2、Rc、Rd、Rg3的二个构型异构体;原人参三醇型皂苷Re、Rf;齐醇果酸型人参皂苷Ro;及红参中特有人参皂苷Rg5和Rk1。2.采用中压柱层析色谱建立蒸参水中Rb₁、Rb₂、Rc、Rd、Re、Rf,6个人参单体皂苷的快速分离制备工艺。3.采用HPLC-ESI-MS对鲜人参、红参甲醇提取物和蒸参水萃取物的水相、正丁醇相及正丁醇相D101大孔树脂的水洗脱相、10%乙醇洗脱相、30%乙醇洗脱相、50%乙醇洗脱相和70%乙洗脱相的共计21个提取物组分的人参皂苷类成分进了对比分析。红参中,水相分离鉴定了14个人参皂苷,正丁醇相分离鉴定了9个人参皂苷,水相较正丁醇相多鉴定出Rb1、Rc、Rb2、Rb3、Rd,5个皂苷。D101大孔树脂水洗脱相、10%、30%乙醇洗脱相均分离到5个人参皂苷且种类相同;50%乙醇洗脱相仅得到Rg₁、Rf、Rg₂,3个人参皂苷。鲜人参中,除D101大孔树脂30%和50%洗脱相皂苷种类相同外,其他组分皂苷种类或数量均存在差异;蒸参水各组分中,正丁醇萃取相、D101大孔树脂30%乙醇洗脱相和50%乙醇洗脱相中均分离出人参皂苷F4、Rg₆和Rh₄,以上3种人参皂苷在该实验的鲜人参和红参中未曾检测到。4.采用GC-MS对蒸参水、鲜人参和红参醚溶性成分进行了对比分析。分离蒸参水中24个醚溶性成分,鉴定了10个醚溶性化合物;分离红参中95个醚溶性成分,鉴定33个化合物;分离鲜人参中94个醚溶性成分,鉴定31个化合物,鲜人参和红参中鉴定了15个相同的化合物。5.对鲜人参、红参和蒸参水21个提取物组分免疫活性进行筛选,通过体外培养小鼠腹腔巨噬细胞,以巨噬细胞产生TNF-α的量确定提取物组分活性,以蒸参水D101大孔树脂10%乙醇洗脱组分活性最强,主要含有:ginsenoside-Rg₁、Re、Ro、Rf、Rg₂、Rb₁、Rc、Rb₂、Rb₃、F₄、Rg₆、20（S）-Rg₃、20（R）-Rg₃、Rg₅、Rk₁,17种人参皂苷,主要为原人参三醇型皂苷、原人参二醇型和齐墩果酸型皂苷及红参特有的稀有人参皂苷Rg₅和Rk₁。体外培养小鼠脾淋巴细胞,以脾淋巴细胞增殖率为指标确定组分中活性最强者为鲜人参D101大孔树脂30%乙醇洗脱相,皂苷种类主要包括:ginsenoside-Rg₁、Re、Ro、Rf、Rg₂、Rc、Rb₂、Rb₃、Rd等人参皂苷。6.21个提取物组分体外培养小鼠脾淋巴细胞、巨噬细胞和L929成纤维细胞,测定其细胞毒性。鲜参70%乙醇相,红参70%乙醇相对小鼠腹腔巨噬细胞有细胞毒性,但毒性较低;对于小鼠脾淋巴细胞,鲜人参70%乙醇相和红参70%乙醇相有较高的细胞毒性;鲜人参70%乙醇相对L929小鼠成纤维细胞也具有较低的细胞毒性。7.对鲜人参、红参和蒸参水甲醇提取物及21个提取物组分清除DPPH自由基、超氧阴离子自由基和羟自由基的抗氧化活性研究,甲醇提取物尤以蒸参水的效果最好,其IC50值:清除DPPH:26 mg/mL;清除超氧阴离子自由基:14 mg/mL;清除羟自由基:10 mg/mL。对21个提取物组分进行抗氧化筛选,初步筛选了3个活性较强的组分,即9号组分:“鲜人参正丁醇萃取相”,10号组分:“鲜人参正丁醇萃取相D101大孔树酯水洗脱相”和16号组分:“蒸参水正丁醇萃取相”。分别比较测试9、10、16号组分对DPPH自由基、超氧阴离子自由基和羟自由基清除率,结果3种提取物组分对DPPH自由基清除率以组分16活性最强,其IC50值为8.0 mg/mL;3种提取物组分对超氧阴离子自由基清除率以组分10活性最强,其IC50值为13.0 mg/mL; 3种提取物组分对羟自由基清除率以组分16活性最强,其IC50值为11.0 mg/mL。本文创新点如下:1.首次采用GC-MS对蒸参水、鲜人参和红参醚溶性成分进行了对比分析。NIST08.L标准图谱库自动检索,分离鉴定了蒸参水中10个醚溶性化合物;红参中33个醚溶性化合物;鲜人参中31个醚溶性化合物。首次从鲜人参和红参中鉴定了多炔类抗癌活性成分镰叶芹醇和具有免疫兼美容活性的成分角鲨烯。2首次对鲜人参、红参和蒸参水21个提取物组分免疫活性进行体外筛选,并进行细胞毒性研究,并确定细胞毒性较强组分,为蒸参水开发利用提供参考;3首次对鲜人参、红参、蒸参水清除DPPH自由基、超氧阴离子自由基及羟自由基活性筛选研究,筛选出清除DPPH自由基和羟自由基活性最强者为“蒸参水正丁醇萃取相”;清除超氧阴离子自由基活性最强者为“鲜人参正丁醇萃取相D101大孔树酯水洗脱相”。综合以上研究结果,蒸参水做为红参加工过程中的副产物,具有较好的利用价值,可以做为化妆品、健康食品添加剂及动物生物制品免疫佐剂开发,必将为我国参业发展产生新的经济增长点。更多还原

【Abstract】 The cultivation, processing and quality evaluation of Chinese Medicinal Materials is an important part of traditinal Chinese medicine modernization, and also the one of the reasearch areas in the professional studies. Among these, about ginseng research still plays an important role in the field. Panax ginseng C.A.Mey.is one of a genuine herbs in Jilin province.Jilin ginseng as the dominant industries, holds a key in the international market share. The condition of ginseng indutry development directly impacting on its paticular industries leading position in Jilin’s agricultural development.In recent years, as part of ginseng drugs and their processed products in the field of medcine and health products reasearch and appliccation, here came into being the many ginseng products, here should be occur the situation of the ginseng resources shortage because long term confliicts of the ginseng cultivation and demond. Therefore,the search for new ginseng medicinal resources has become a priority. Steamed ginseng water is the byproduct of the red ginseng processing, in the process of red ginseng, water vapor steamed ginseng, after dipping into the steaming pot at the bottom of solution, is a dark drown, turbid, viscous liquid, containing part of the ginseng saponins, ginseng polysaccharides and amino acids. World annual production of red ginseng is about 7,000 tons, China is about 4,000 tons, about 3,400 tons in Jilin Province, to process 1Kg dryed red ginseng there should be produce dryed steamed ginseng water 8.6g . Therefore, steamed ginseng water has Considerable annual output in Jilin Province, steamed ginseng water generally be discarded as waste, thus, should be inevitably cause a waste of resources.Therefore, study on the chemistry and biological of steamed ginseng water systematicly has a practical guide on the industrialization development of Jiling ginseng. To carry out this study and made the following progress.1 Study on chemical constituents of steamed ginseng water. Eleven ginsenosides were isolated from the steamed ginseng water by the different process of extraction and isolation,and with the extraction of organic solvent, Macro-reticular resins, ODS column chromatorgraphy, silica gel column chromatography, and other isolation method. On the basis of the physical constants, chemical evidence and MS identyfied the attribution of 11 ginsenosids, there are protopanaxadiol saponins, ginsenoside- Rb1,Rb2,Rc, Rd, 20(R)-Rg3 and 20(S)-Rg3, Protopanaxtriol saponins, ginsenoside-Re, Rf, Oleanolic acid type saponins, ginsenoside- Ro, and the unique red ginseng ginenosides Rg5 and Rk1.2 Using medium pressure column chromatography to establish the saponins’saperation and preparation process of steamed ginseng water. Six saponins ginsenosid- Rb1 , Rb2, Rc, Rd, Re and Rf were prepared.3 Isolated the saponins of fresh ginseng, red ginseng methanol extracts and steamed ginseng water extracted by organic solvents phase, include aqua phase, n-butanol phase and the n-butanol phase D101 macroporous resin washed by aqua , 10%ethanol, 30%ethanol, 50%ethanol and 70 ethanol, total 21 components compare analysed by HPLC-ESI-MS. In red ginseng , 14 ginsenosids were identified in aqua phase, 9 ginsenosids were identified in n-butanol phase, compared with aqua phase , n-butanol phase have no ginsenoside- Rb1、Rc、Rb2、Rb3 and Rd. D101 macroporous resin washed by water, 10%ethanol, 30%ethanol, were all identified the same five ginsenosids, 50% ethanol phase only identified gisenoside- Rg1, Rf and Rg2. In fresh ginseng, all extracts has different ginsenosids except D101 macroporous resin 30% and 50% ethanol phase. Ginsenoside- F4, Rg6, Rh4 were identified in n-butanol extract phase, D101 macroporous resin 30% and 50% ethanol phase of steamed ginseng water,, fresh ginseng and red ginseng haden’t identified them.4 Fresh ginseng, red ginseng and steamed ginseng water ether soluble components were analyzed by GC-MS. In steamed ginseng water isolated 24 components, 10 of them were identified, In red ginseng isolated 95components, 33of them were identified, In fresh ginseng isolated 94 components, 31 of them were identified,15 same components were identified in fresh ginseng and red ginseng。5 The extracts immune activity of the Fresh ginseng, red ginseng and steamed ginseng water 21 components were screened. By cultured mouse macrophages, to determine the activity of extract components by the amount of TNF-α, the result is steamed ginseng water D101 macroporous resin 10% ethanol fraction has the strongest activity, the generation rarte of TNF-αwas 78.8%, in this fraction contains ginsenoside- Rg1、Re、Ro、Rf、Rg2、Rb1、Rc、Rb2、Rb3、F4、Rg6、20(S)-Rg3、20(R)-Rg3、Rg5、Rk1,17kinds of ginseng saponin,both conains protopanaxadiol saponins, Protopanaxtriol saponins, Oleanolic acid type saponins also the rare and unique ginsenosides- Rg5、Rk1 of red ginseng. By cultured mouse spleen lymphocytes, to determine the activity of extract components by the generation rate of mouse spleen lymphocytes, the result is fresh ginseng D101 macroporous resin 30% ethanol fraction has the strongest activity, the generation rarte was 66.9%. In this fraction contains ginsenoside-Rg1、Re、Ro、Rf、Rg2、Rc、Rb2、Rb3、Rd。6 Cultured mouse macrophages, mouse spleen lymphocytes, mouse fibroblast L929 by 21 extract frections, to determine their cytotoxicity. The result is, fresh ginseng 70% ethanol phase and red ginseng 70% ethanol phase have lower cytotoxicity to mouse macrophages, but higher cytotoxicity to mouse spleen lymphocytes, fresh ginseng 70% ethanol phase has lower cytotoxicity to mouse fibroblast L929.7 Studied on the clearance of DPPH radical , superoxide anion radical, hydroxyl radical by fresh ginseng, red ginseng and steamed ginseng water methanol extracts and their 21 extract fractions. The result was, the steamed ginseng water methanol extract has the strongest antioxidant activity, the IC50 were, DPPH 26 mg/mL, superoxide anion14 mg/mL, hydroxyl 10 mg/mL, to 21 components of the extracts for antioxidant screening, initial screening of the three samples have stronger activity. They are NO.9 fresh ginseng n-butanol extract, No.10 fresh ginseng D101 macroporous resin aqua phase, No.16 steamed ginseng water n-butanol extract.Compared the activity of NO.9, NO. 10, NO.16 samples to the three kinds of radical. The result was, the strongest clearance of DPPH was NO.16, the IC50 is 8.0 mg/mL, the strongest clearance of superoxide anion was NO.10, the IC50 is 13.0 mg/mL, the strongest clearance of hydroxyl was NO.16, the IC50 is 11.0 mg/mL.The innovation is as follows 1 For the first time, fresh ginseng, red ginseng and steamed ginseng water ether soluble components were analyzed by GC-MS. NIST08.L standard atlas automatic retrieval, idenyified 10 ether soluble components of steamed ginseng water, 33 ether soluble components of red ginseng, and 31 ether soluble components of fresh ginseng. For the first time isolated anticancer component falcarinol from fresh ginseng and red ginseng.Immune and Cosmetology active ingredient squalene isolated in red ginseng , 2,6,10,15,19,23-hexamethyl-, (all-E)- isolated in fresh ginseng.2 For the first time, the extracts immune activity and Cytotoxicity of the Fresh ginseng, red ginseng and steamed ginseng water 21 components were screened.3 For the first time, studied on the clearance of DPPH radical , superoxide anion radical, hydroxyl radical by fresh ginseng, red ginseng and steamed ginseng water methanol extracts and their 21 extract fractions were screened.Based on the above findings, the seamed ginseng water as the byproduct in the process of red ginseng, has a high-value in development and utilization, can be used as cosmetics, food additives and animal health adjuvant development of biological products.Should be generating new economic growth point for china ginseng industry.更多还原