【作者】 李军华；

【导师】 赵寿经；

【作者基本信息】 吉林大学 ， 农产品加工及贮藏工程， 2005， 硕士

【摘要】 人参发根是经发根农杆菌A4 菌株浸染人参外植体遗传转化而成,属非天然植物。人参发根在液体培养基上生长速率快,培养四周人参总皂苷含量就达到栽培三年生人参根总皂苷含量,而发根中人参皂苷Rb1含量接近栽培六年生人参根中Rb1含量,且其在总皂苷中的比例很高,达到50%左右。本文是人参发根的下游工程,主要是为了从人参发根中分离得到人参皂苷Rb1单体,利用柱层析技术对人参(西洋参)及人参发根中人参皂苷Rb1进行了分离研究。本文对人参发根进行了继代和扩大培养,为提取分离人参皂苷Rb1提供材料。同时进行了人参(西洋参)总皂苷提取,应用柱层析法分离人参皂苷Rb1,通过洗脱对比实验,确定洗脱剂最终比例为氯仿∶甲醇=7.5∶2.5。然后进行人参发根总皂苷提取,应用西洋参所确定的最终洗脱剂比例分离人参发根总皂苷中人参皂苷Rb1。经高效液相色谱--质谱检测,其结构与西洋参中人参皂苷Rb1 的结构相同。最后对两种材料所得Rb1 进行对比,结果人参发根更有利于人参皂苷Rb1的产业化生产,可全面降低成本,提高利用价值。本文主要侧重研究柱层析法分离提纯应用生物技术得到的人参发根中人参皂苷Rb1,此前未曾报导。本文的研究成果可为大规模制成单体皂苷制剂,实现真正意义上的中药现代化做些基础工作。更多还原

【Abstract】 Ginseng（Panax ginseng C. A. Mey） is a famous and precious Chinesetraditional medicinal material. Ginsenoside is the main effective composition ofginseng, and the pharmacology activation that ginseng displays is mainly thefruiting of the ginsenoside function. Ginsenoside is a secondary supersession resultof ginseng. Ginsenoside Rb1 is a kind of ginsenosides among them, having muchpharmacology function, for example, ginsenoside Rb1 not only has function oftreating the disease in the nervous system of centre, but also hurts to have theresult of treatment to the memory that senile dementia and medicinecause. Meanwhile, ginsenoside Rb1 is still forebody thing and raw materials ofanticancer drug Rg₃ , Rh₂ , Compound K ,etc by biochemistry transform.The ginseng hairy root was induced from the root explants of ginseng byAgrobacterium rhizogenes A4 with directly inoculating, non-natural plant. Theginseng hairy root could grow rapidly on liquid culture. After trainning fourseeks , the total ginsenoside content in the hairy root came up to that in thegingseng root planted for three years. Furthermore the content of ginsenoside Rb1closed to that in the ginseng root planted for six years, and its proportion was veryhigh in the total ginsenoside, up to 50% or so, and success in the small-scaleginsenoside factory produce was obtained. The transformed hairy root could growrapidly on MS medium without phytohormones and was characterized by stronglybranching, clustering and non-geotropism,etc. This thesis was the downstreamengineering of the ginseng hairy root, mainly was for the sake of separating thesingle body of ginsenoside Rb1 from the ginseng hairy root, going on researchof separating ginsenoside Rb1 from the ginseng and the ginseng hairy root bymaking use of column chromatography technology.This thesis studied as the main material with the ginseng and the ginseng hairyroot. First, generation and extension culture of the hairy root was carried on inorder to offer enough material for extracting and separating ginsenoside Rb1. At thesame time, organic solvent and macroreticular resin were used to draw the totalginsenoside in the ginseng for separating ginsenoside Rb1 by columnchromatography. Through eluting experiment of comparation, final proportion ofeluting pharmaceutical chloroform to methanol is 7. 5:2. 5. Then organic solventand macroreticular resin were used to draw the total ginsenoside in the hairy root,utilizing final proportion of eluting pharmaceutical to separate ginsenoside Rb1from the hairy root. The main contents and conclusions are as follows:1) This thesis studied culture of the hairy root. The hairy root grew vigorous.Through incessant generation and extension culture, the ginseng hairy rootmaterials that follow-up’s testing needed. were obtained.2) Through marinating with alcohol , degreasing, dividing sugar anddecoloring to ginseng fibrous root powder of 300g, the purer ginseng total saponinsof 13.5121g was obtained finally, and the purity was 75.3% by vanillic aldehydecolorimetry. Seeing that the weight of ginseng fibrous root powders was 300g, sothe extraction rate of the total ginsenoside was 3.39%.3) The same, through marinating with alcohol , degreasing, dividing sugar anddecoloring to the ginseng hairy root powder of 300g, the purer ginseng totalsaponin of 4.4123g was obtained finally, and the purity was 72.1% by vanillicaldehyde colorimetry. Seeing that the weight of the ginseng hairy root powder was300g, so the extraction rate of the total ginsenoside was 1.06%.4) Before dividing sugar to the total ginsenoside of ginseng, AB-8 resin to thecapacity of absorbing in the degreasing liquid of the ginseng total saponin wasdetermined, and the capacity of absorbing was 59.7mg saponin/g waterish resin;Because the composition and content of saponin monomer in the degreasing liquidof the ginseng total saponin and the gingseng hairy root total saponin were different,so before dividing sugar to the total ginsenoside of ginseng hariy root, AB-8 resinto the capacity of absorbing in the degreasing liquid of the ginseng hairy root totalsaponin was determined again, and the capacity of absorbing was 50.1mgsaponin/g waterish resin.5) Ginsenoside Rb1 was separated from the total ginseng saponin by columnchromatography. At first, the different proportion chloroform and methyl alcohol toelute was adopted, and It was confirmed that the rough proportion of elutingpharmaceutical is between 7.8:2.2 and 7.3:2.7. Then 7.3:2.7 and 7.5:2.5 wereselected to elute further and two experiment results were compared. And it wasfound that because chloroform and methyl alcohol are the liquid apt to volatilize ,while the vacuum was spent greatly , eluting pharmaceutical volatilized fast, andthere was little volume collected , then consumption was much in volume , andeluting times were many too. Through the control on vacuum , it was found thatwhile it was in 0.03～0.04, the result of eluting was better. And it was found that20ml was too few in eluting amount each time , there were so much ones that lostthat eluting times were too many.6) As the proportion was 7.3:2.7, ginsenoside Rb1 of 0.2412g was obtainedfrom the ginseng total saponin of 2.0008g （purity was 75.3%）. The purity was82.25% by HPLC; As the proportion was 7.5:2.5, ginsenoside Rb1 of 0.2689g wasobtained from the ginseng total saponin of 2.0007g （purity was 75.3%）. The puritywas 82.25% by HPLC7) As the proportion was 7.3:2.7, the separation speed was fast , but there waslittle quantity of obtaining ginsenoside Rb1 and the purity is low; As the proportionwas 7.5:2.5, the separation speed was slow, but there was much quantity ofobtaining ginsenoside Rb1 and the purity is high. So compared with two kinds ofexperimental results and considered from two respects of the economic benefitsand result of separating , 7.5:2.5 is comparatively suitable.8) Ginsenoside Rb1 was separated from the total ginseng hairy root saponin bycolumn chromatography. The final proportion 7.5:2.5 confirmed by the ginsengwas adopted. Ginsenoside Rb1 of 0.5024g was obtained from the ginseng hairy roottotal saponin of 2.0005g （purity is 72.1%）. Measured by high performeance liquidchromatography and mass spectrum, the structure of the extracted ginsenoside Rb1from the ginseng hairy root was the same as that of the extracted ginsenoside Rb1from the ginseng. The purity was 89.95%by HPLC. The purpose of purification wasachieved tentatively, so better separation of ginsenoside Rb1 was obtained from theginseng hairy root by column chromatography.9) The more important thing is that in this thesis , ginsenoside Rb1 ofseparating by column chromatography was compared with the above two kinds ofmaterial. According to above-mentioned experimental datas, ginsenoside Rb1 of1.7491g could be obtained from the ginseng of 300g （purity was 75.3%）, thus theyield rate is 0.49%, and ginsenoside Rb1 of 1.1745g could be obtained from theginseng hairy root of 300g （purity was 72.1%）, thus the yield rate was 0.35%.Because ginseng hairy root could grow rapidly and the content of the totalginsenoside and ginsenoside Rb1 was high, so the ginseng hairy root is morefavorable to industrialization production of ginsenoside Rb1, The cost is reduced in anall-round way,and value is relatively high.This thesis studied mainly on separating ginsenoside Rb1 from the ginseng hairyroot that was transformed by biotechnology , have being not reported before this , which更多还原