CN1105781C - 酶法改变人参皂甙糖基制备人参皂甙的方法 - Google Patents
酶法改变人参皂甙糖基制备人参皂甙的方法 Download PDFInfo
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Abstract
本发明系酶法改变人参皂甙糖基制备稀有人参皂甙的方法，采用皂甙β-葡萄糖苷酶、α-、β-***糖苷酶、α-鼠季糖苷酶、β-糖醛酸苷酶或β-半乳糖苷酶等皂甙酶，对于人参属的所有种类参的人参皂甙进行水解处理，可使皂甙分子甙元上的部分糖基被水解掉成、为新的稀有皂甙。优点是：它可让人以们简单且经济的方式，将自然界中广泛存在的一些人参皂甙“改造”成Rh1、Rh2之类稀有的、经济价值很高的皂甙。
Description
本发明涉及一种改变人参皂甙糖基的生物方法，特别是采用酶法改变人参皂甙糖基制备稀有人参皂甙的方法。
人参是植物五加科(araliaceae)中的人参(Panax)属，有四个不同种和其他变种，如人参(P.ginseng)、西洋参(P.quenquefolinus)、田七参(P.notoginseng)、竹节参(P.uaponicus)等以及其他一些变种。人参是驰名中外的滋补强壮的贵重补药，人参的主要有效成份是皂甙。已发现的人参皂甙有三十四种；人参皂甙根据皂甙元的不同，可分为三种，即二醇类皂甙类(protopanaxdill-type Gisenosides)、三醇皂甙类(Protopanaxtriol-typeGisenosides)。其中二醇类皂甙占整个皂甙的50～60％，其次为三醇类皂甙30～35％，还有齐墩果酸类皂甙。
人参中占皂甙总量50～60％的二醇类皂甙Ra1、Ra2、Ra3、RB1、Rb2、Rb3、Rc、Rd及Rg3和稀有人参皂甙Rh2皂甙元相同，只是糖链有所不同。其中Rb、Rc、Rd等皂甙在人参中含量高，容易得到。但抗癌活性很高的Rh2皂甙，在白参中几乎不存在，只有在红参含有十万分之一。Rh2与R6、Rc、Rd一样皂甙分子的甙元第3碳上有一个β-葡萄糖基，因此只要将Rb、Rc、Rd等皂甙分子上的部分糖基去掉，即可得到价值极高的抗癌皂甙Rh2。同样，对于人参中占皂甙总量40～50％人参三醇类皂甙Re、Rf、Rg1和Rg2皂甙，去掉部分糖基，即可得到价值极高的皂甙Rh1。
本发明目的是：利用生物技术，提供一种酶法改变人参皂甙糖基制备稀有人参皂甙的方法。
本发明是这样实现的：酶法改变人参皂甙糖基制备稀有人参皂甙的方法，其特征是：采用皂甙β-葡萄糖苷酶、α-、β-***糖苷酶、α-鼠李糖苷酶、β-糖醛酸苷酶或β-半乳糖苷酶等皂甙酶，对于人参属的所有种类参的人参皂甙进行水解处理，可使皂甙分子甙元上的部分糖基被水解掉、成为新的稀有皂甙。
以上述方案实现本发明时，所述的各种皂甙酶可由细菌、链霉菌、霉菌、酵母菌、担子菌等微生物或植物、植物种子加工提取。
应用本发明，可以β-葡萄糖皂甙酶水解人参皂甙Rg3得到稀有人参皂甙Rh2。也可以α-鼠李糖皂甙酶水解人参皂甙Rg2得到稀有人参皂甙Rh1。亦可将可产皂甙酶的菌类直接接种到人参粉或片上培养，得到稀有人参皂甙成份含量高的人参粉或片。
本发明的核心是利用皂甙糖苷酶水解在各种参中含量较高的人参皂甙的部分糖基，从而获得稀有人参皂甙。事实上，一般的纤维素酶或半纤维素酶的外切酶很难水解皂甙糖基，因为皂甙与纤维素不同，带大的甙元环，只有皂甙糖苷酶才能实现水解皂甙糖基。
本发明优点在于：它可让人们以简单且经济的方式，将自然界中广泛存在的一些人参皂甙成份“改造”成Rh1、Rh2之类稀有的、经济价值又很高的人参皂甙成份。
下面结合实施例对本发明做进一步叙述：
实施例1：
以高温好氧的Bacillus sp.JF菌，在含3％玉米粉、1％人参浸出物的0.01MgSO4培养基中，温度60℃下通风培养30～40小时，然后离心除菌、70％乙醇沉淀酶蛋白、收集蛋白，冻干，得到能水解β-葡萄糖键的皂甙糖苷酶。
取3克上述皂甙酶和140毫升生理水混合；10克人参皂甙Rg3、20毫升生理盐水和40毫升乙醇混合均匀。两种上述混合液再混合，在温度60℃下搅拌反应12小时。反应后加入800毫升乙醇，过滤除去蛋白沉淀，过量乙醇洗沉淀、滤液减压蒸干，干品蒸馏水洗去盐三次，减去蒸干，得到7克含40～95％Rh2皂甙。
实施例2：
曲霉菌培养后除菌，培养液用乙醇沉淀酶蛋白，用pH5的0.02M醋酸钠缓冲液溶解，用DEAE-Cullulose离子交换树脂盐梯度洗脱法分离α-鼠李糖皂甙酶，冻干。
将10克三醇类人参皂甙Re溶于200毫升用PH5的0.02M醋酸钠缓冲液溶解，加入0.05克α-鼠李糖皂甙酶在温度40℃搅拌反应12小时，加热至100℃30分钟，过滤出去蛋白沉淀，大孔树脂200克AB-8吸附，500毫升70％乙醇洗脱，其乙醇液减压蒸干得粗Rg1皂甙。其粗Rg1皂甙经硅胶柱分离法分离，减压蒸馏得5.5克Rg1。
同样方法，上述酶处理10克三醇人参皂甙Rg2，可以得到贵重的Rh1皂甙4.5～6克。
实施例3：
取芽孢杆菌后培养后除菌，用乙醇沉淀酶蛋白，冻干。将10*****克人参混合皂甙溶解于200毫升用pH5的0.02M醋酸钠缓冲液溶解，加入0.3克上述混合皂甙酶，在温度40℃搅拌反应12小时。经高效液相色谱分析，80％以上皂甙至少失去一个糖基。稀有的抗癌皂甙Rh2、Rh1皂甙含量提高了几百倍。反应液经加热除去酶蛋白，经200克AB-8多孔树脂吸附、500毫90％乙醇洗脱，减压蒸干，可得到8克稀有皂甙含量高的混合皂甙。
实施例4
将产皂甙酶的菌曲霉菌(不产毒或者对人体有害物)接种到水分含量25～150％的人参片上，在28～38℃培养2～7天，干燥即得到高含稀有皂甙的人参。经高效液相色谱分析，80％以上皂甙至少失去一个糖基。稀有的抗癌皂甙Rh2、Rh1皂甙含量提高了几百倍。
Claims (7)
1、酶法改变人参皂甙糖基制备人参皂甙的方法，其特征是：采用皂甙β-葡萄糖苷酶、α-、β-***糖苷酶、α-鼠李糖苷酶、β-糖醛酸苷酶或β-半乳糖苷酶皂甙酶，对于人参属的所有种类参的人参皂甙进行水解处理，可使皂甙分子甙元上的部分糖基被水解掉、得到皂甙Rh1或Rh2、或人参皂甙Rg1。
2、根据权利要求1所述的方法，其特征是：所述的各种皂甙酶可由细菌、链霉菌、霉菌、酵母菌、担子菌微生物或植物、植物种子加工提取。
3、根据权利要求1所述的方法，其特征是：以β-葡萄糖皂甙酶水解人参二醇类皂甙得人参皂甙Rh2。
4、根据权利要求3所述的方法，其特征是：以β-葡萄糖皂甙酶水解人参皂甙Rg3得人参皂甙Rh2。
5、根据权利要求1所述的方法，其特征是：以α-鼠李糖皂甙酶水解人参三醇类皂甙得人参皂甙Rh1。
6、根据权利要求5所述的方法，其特征是：以α-鼠李糖皂甙酶水解人参皂甙Rg2得人参皂甙Rh1。
7、根据权利要求2所述的方法，其特征是：将可产皂甙酶的菌类直接接种到人参粉或片上培养，得到稀有人参皂甙Rh1或Rh2成份含量高的人参粉或片。
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| KR1019990045180A KR100329259B1 (ko) | 1999-03-17 | 1999-10-14 | 효소(酵素)로 인삼 사포닌 당기(糖基)를 변화시켜서 희소한 인삼사포닌을 제조하는 방법 |
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| US9976167B2 (en) | 2012-12-06 | 2018-05-22 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | Group of glycosyltransferases and use thereof |
| EP3985110A1 (en) | 2012-12-06 | 2022-04-20 | CAS Center for Excellence in Molecular Plant Sciences | Group of glycosyltransferases and use thereof |
| EP4043560A2 (en) | 2012-12-06 | 2022-08-17 | CAS Center for Excellence in Molecular Plant Sciences | Group of glycosyltransferases and use thereof |
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| KR20000062140A (ko) | 2000-10-25 |
| KR100329259B1 (ko) | 2002-03-20 |
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