WO2003040383A1 - Preparation of 20-$g(b)-d-pyranoglucosyl-protopanaxadiol by enzymatic hydrolysis of ginsenoside - Google Patents
Preparation of 20-$g(b)-d-pyranoglucosyl-protopanaxadiol by enzymatic hydrolysis of ginsenoside Download PDFInfo
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- the present invention relates to the preparation of 20- ⁇ -D-glucopyranosylprotopanaxadiol from ginsenosides.
- C-K Compound-K
- Ginsenoside c- ⁇ is a metabolite of diol-type ginsenosides in the intestine after oral administration. Studies have shown that c- ⁇ has antitumor and immunomodulatory effects, improves microcirculation, regulates digestive functions, soothe the nerves, anti-aging, anti-aging Various biological activities such as stress, prevention of gastrointestinal ulcers, improvement of quality of life, enhancement of memory and learning ability. Therefore, c- ⁇ production methods have attracted much attention. Kyobashi Kobashi (Chem Pharm Bull 21 (3): 245-249, 1988) dissolved 500 mg of ginsenoside Rbl and 250 mg of naringinase in 32 ml of phosphate buffer solution (pH 4.0, containing 10% ethanol).
- reaction solution was centrifuged into a precipitate and a mother liquor, and the precipitate was dissolved in 3.5 ml of ethanol, and added to the mother liquor with 32 ml of a buffer solution and 250 mg of naringinase. Hydrolyzed for 7 days.
- the reaction solution was extracted with ether, and the ether phase was evaporated to dryness to obtain ginsenoside C-K.
- the mother liquor was extracted with n-butanol, and the n-butanol phase was washed with water, dried, and evaporated to obtain a residue; the residue was separated by preparative TLC to separate C-K; and combined C-K to obtain 253 mg.
- This method has a high yield (86%), but has the following disadvantages: long hydrolysis time; large amount of enzyme, and the source of the enzyme is inconvenient and uneconomical; it requires extraction with explosive ether, so it is not suitable for mass production .
- Technical content of the invention :
- the present invention provides a method for preparing CK by enzymatic hydrolysis of ginsenosides, including hydrolysis and collection steps, and is characterized in that the diol is hydrolyzed by a snail enzyme in a buffer solution.
- Type ginsenoside the weight ratio of diol type ginsenoside to snail enzyme is 1: 1 ⁇ 10: 1, pH value is between 4.0 ⁇ 5.0, hydrolysis temperature is 30 ⁇ 45 ° C, and hydrolysis time is 2 ⁇ 3 days.
- the collecting process is as follows: the reaction precipitate is washed with water and then repeatedly washed with ethanol. After the combined ethanol solutions were evaporated to dryness, the collected residue was C-K.
- the washing liquid can be combined with the mother liquor and concentrated for reuse in enzymatic hydrolysis.
- the snail enzyme used may be a crude snail enzyme.
- the optimum temperature for hydrolyzing diol-type ginsenosides by crude snail enzyme is 40 ° C, and the optimum pH is 4.5.
- the type of buffer solution has little effect on the enzyme activity.
- the pH of the invention is 4.5. Phosphoric acid-citrate buffer with a strength of 0.01.
- a certain amount of organic solvent should be added to the reaction solution.
- the organic solvent assists the intermediate product to facilitate the normal progress of the enzymatic hydrolysis reaction.
- the type and amount of organic solvents are based on those that do not affect enzyme activity. After screening, 5 ⁇ 15% ethanol is the best.
- the ratio of diol saponin to enzyme is 6: 1; generally speaking, after 8 hours of hydrolysis, the hydrolysis time is prolonged, and the yield of CK is extremely increased. Less, so it is not necessary to lengthen the hydrolysis time.
- the invention provides a convenient and low-cost enzyme for preparing ginsenoside C-K by enzymatic hydrolysis of ginseng glycol saponin.
- the process for preparing C-K using this enzyme is simple, convenient, low cost and high recovery rate.
- the C-K content prepared by this method is 95%, and the aglycon yield is 91%.
- FIG. 1 shows the effect of pH on C-K production
- Figure 2 shows the effect of material ratio on C-K output
- FIG. 3 shows the effect of temperature on C-K production
- Figure 4 shows the effect of ionic strength on C-K production
- FIG. 5 shows the change of C-K output over time
- Example 1 '500 mg of diol-type ginsenoside, 84 mg of snail enzyme was dissolved in 32 ml of phosphate-citrate buffer (pH 4.5, ionic strength 0.01, containing 10% ethanol), and hydrolyzed in a water bath at 40 ° C for 1 day The reaction solution was collected by centrifugation. The precipitate was repeatedly washed with water (10ml X 3). The washing solution was combined with the mother liquor and concentrated to 32ml for reuse. The precipitate was repeatedly washed with ethanol (10ml X 5). The ethanol solution was combined, evaporated to dryness, and collected. The residue gave 106 mg of CK.
- phosphate-citrate buffer pH 4.5, ionic strength 0.01, containing 10% ethanol
- Diol ginsenoside and snail enzyme were dissolved in 3.2ml phosphate-citrate buffer (pH4.5, ionic strength 0.01, containing 10% ethanol) in different proportions, and hydrolyzed in a 40 ° C water bath for 1 day; reaction The precipitate was collected by centrifugation. The precipitate was repeatedly washed with water (lml X 3). The washing solution was combined with the mother liquor and concentrated to 3.2 ml for reuse. The precipitate was repeatedly washed with ethanol (lml X 5). The ethanol solution was combined, evaporated to dryness, and the residue was collected. The CK yield is shown in Figure 2.
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Abstract
A process for preparing 20-β-D-pyranoglucosyl-protopanaxadiol by enzymatic hydrolysis of ginsenoside, which comprises steps of hydrolysis, collection and is caracterized in glycosyl in the molecular of diol type of ginsenoside being site-directed excising by snail-enzyme to generate objective compound 20-β-D-pyranoglucosyl-protopanaxadiol snail-enzyme used in the present invention is available conveniently and cheap. The process for preparing C-K is simple, convenient, low-cost, and high yield. it can satisfy the need of preparing anti-tumor drug.
Description
技术领域： Technical field:
本发明涉及由人参皂甙制备 20- β -D-吡喃葡萄糖基原人参二醇 The present invention relates to the preparation of 20-β-D-glucopyranosylprotopanaxadiol from ginsenosides.
(俗称 Compound-K,以下简称 C-K)的方法， 特别是涉及粗蜗牛酶水解 制备方法。 背景技术： (Commonly known as Compound-K, hereinafter referred to as C-K), and particularly relates to a method for preparing a crude snail by enzymatic hydrolysis. Background technique:
人参皂甙 c-κ是二醇型人参皂甙经口服后在肠道中的代谢产物， 研究表明： c-κ有抗肿瘤和免疫调节作用、 改善微循环作用、 调节消 化机能、 安神、 抗衰老、 抗紧张、 预防消化道溃疡、 提高生命质量、 增强记忆力和学习能力等多种生物活性。 因此， c-κ的生产方法备受 关注。小桥恭一等（ Chem Pharm Bull 21(3): 245-249, 1988 )将 500mg 人参皂甙 Rbl和 250mg柚皮甙酶溶于 32ml磷酸缓冲液 (pH4.0,含 10% 的乙醇）中，于 40°C水浴中水解 3天；将反应液离心分为沉淀和母液， 沉淀溶于 3.5ml乙醇中， 并与 32ml缓冲液和 250mg柚皮甙酶一起加 入母液中， 于 40°C水浴中再水解 7天。用乙醚萃取反应液， 乙醚相蒸 干得人参皂甙 C-K。 母液用正丁醇萃取， 正丁醇相经水洗、 干燥、 蒸 干得残余物； 残余物经制备 TLC分离出 C-K; 合并 C-K得 253mg。 该法收率高（86%)， 但有下列缺陷： 水解时间长； 酶的用量大， 并且 酶的来源不方便， 也不经济； 需使用易爆品乙醚萃取， 因此， 不适于 大批量生产。 发明的技术内容： Ginsenoside c-κ is a metabolite of diol-type ginsenosides in the intestine after oral administration. Studies have shown that c-κ has antitumor and immunomodulatory effects, improves microcirculation, regulates digestive functions, soothe the nerves, anti-aging, anti-aging Various biological activities such as stress, prevention of gastrointestinal ulcers, improvement of quality of life, enhancement of memory and learning ability. Therefore, c-κ production methods have attracted much attention. Kyobashi Kobashi (Chem Pharm Bull 21 (3): 245-249, 1988) dissolved 500 mg of ginsenoside Rbl and 250 mg of naringinase in 32 ml of phosphate buffer solution (pH 4.0, containing 10% ethanol). It was hydrolyzed in a 40 ° C water bath for 3 days; the reaction solution was centrifuged into a precipitate and a mother liquor, and the precipitate was dissolved in 3.5 ml of ethanol, and added to the mother liquor with 32 ml of a buffer solution and 250 mg of naringinase. Hydrolyzed for 7 days. The reaction solution was extracted with ether, and the ether phase was evaporated to dryness to obtain ginsenoside C-K. The mother liquor was extracted with n-butanol, and the n-butanol phase was washed with water, dried, and evaporated to obtain a residue; the residue was separated by preparative TLC to separate C-K; and combined C-K to obtain 253 mg. This method has a high yield (86%), but has the following disadvantages: long hydrolysis time; large amount of enzyme, and the source of the enzyme is inconvenient and uneconomical; it requires extraction with explosive ether, so it is not suitable for mass production . Technical content of the invention:
为了简单、 方便、 低成本、 大批量地制备 C-K， 本发明提供了一 种用酶水解人参皂甙制备 C-K的方法， 包括水解、 收集步骤， 其特征 在于： 用蜗牛酶在缓冲液中水解二醇型人参皂甙， 二醇型人参皂甙与 蜗牛酶重量比为 1 : 1~10: 1， pH值在 4.0〜5.0之间， 水解温度 30〜45 °C， 水解时间 2〜3天。 In order to prepare CK in a simple, convenient, low-cost and large-scale manner, the present invention provides a method for preparing CK by enzymatic hydrolysis of ginsenosides, including hydrolysis and collection steps, and is characterized in that the diol is hydrolyzed by a snail enzyme in a buffer solution. Type ginsenoside, the weight ratio of diol type ginsenoside to snail enzyme is 1: 1 ~ 10: 1, pH value is between 4.0 ~ 5.0, hydrolysis temperature is 30 ~ 45 ° C, and hydrolysis time is 2 ~ 3 days.
本发明用酶水解人参皂甙制备 C-K的方法中， 收集过程是： 反应 沉淀物用水洗涤后， 再用乙醇反复洗涤。 合并乙醇液蒸干后， 收集的 残余物即为 C-K。 In the method for preparing C-K by enzymatically hydrolyzing ginsenosides in the present invention, the collecting process is as follows: the reaction precipitate is washed with water and then repeatedly washed with ethanol. After the combined ethanol solutions were evaporated to dryness, the collected residue was C-K.
收集过程中反应沉淀物用水反复洗涤后， 可将洗液与母液合并， 浓缩后再次用于酶水解。
本发明用酶水解人参皂甙制备 c-κ的方法中，所用蜗牛酶可以为 粗蜗牛酶。 After the reaction precipitate is repeatedly washed with water during the collection, the washing liquid can be combined with the mother liquor and concentrated for reuse in enzymatic hydrolysis. In the method for preparing c-κ by enzymatic hydrolysis of ginsenosides in the present invention, the snail enzyme used may be a crude snail enzyme.
粗蜗牛酶水解二醇型人参皂甙的最适温度为 40°C，最适 pH为 4.5， 离子强度越低越好， 缓冲液的种类对酶活性影响不大； 本发明选用 pH4.5, 离子强度为 0.01的磷酸-柠檬酸缓冲液。 The optimum temperature for hydrolyzing diol-type ginsenosides by crude snail enzyme is 40 ° C, and the optimum pH is 4.5. The lower the ionic strength, the better. The type of buffer solution has little effect on the enzyme activity. The pH of the invention is 4.5. Phosphoric acid-citrate buffer with a strength of 0.01.
本发明用酶水解人参皂甙制备 C-K的方法中，反应液中应该加入 一定量的有机溶剂， 有机溶剂对中间产物起助溶作用， 有利于酶水解 反应的正常进行。 有机溶剂的种类和加入量以不影响酶活性为准。 经 筛选， 以 5〜15%的乙醇最佳。 In the method for preparing C-K by enzymatically hydrolyzing ginsenosides in the present invention, a certain amount of organic solvent should be added to the reaction solution. The organic solvent assists the intermediate product to facilitate the normal progress of the enzymatic hydrolysis reaction. The type and amount of organic solvents are based on those that do not affect enzyme activity. After screening, 5 ~ 15% ethanol is the best.
本发明用酶水解人参皂甙制备人参皂甙 K化合物的方法中，二醇 型皂甙与酶的比例以 6: 1最为经济； 一般地说 ， 水解 8小时后， 再 延长水解时间， C-K的产量增加极少， 因此， 勿需加长水解时间。 In the method for preparing ginsenoside K compound by enzymatically hydrolyzing ginsenoside in the present invention, the ratio of diol saponin to enzyme is 6: 1; generally speaking, after 8 hours of hydrolysis, the hydrolysis time is prolonged, and the yield of CK is extremely increased. Less, so it is not necessary to lengthen the hydrolysis time.
本发明为人参二醇皂甙酶解制备人参皂甙 C-K提供了一种来源方 便、 价格低廉的酶， 用该酶制备 C-K工艺简单方便、 成本低、 回收率 高。 使用本方法制备的 C-K含量 95%， 甙元收率 91%。 下面通过 实施例详述本发明。 附图说明： The invention provides a convenient and low-cost enzyme for preparing ginsenoside C-K by enzymatic hydrolysis of ginseng glycol saponin. The process for preparing C-K using this enzyme is simple, convenient, low cost and high recovery rate. The C-K content prepared by this method is 95%, and the aglycon yield is 91%. The present invention is described in detail by the following examples. Brief description of the drawings:
图 1为 pH对 C-K产量的影响； Figure 1 shows the effect of pH on C-K production;
图 2为物料比对 C-K产量的影响； Figure 2 shows the effect of material ratio on C-K output;
图 3为温度对 C-K产量的影响；； Figure 3 shows the effect of temperature on C-K production;
图 4为离子强度对对 C-K产量的影响； Figure 4 shows the effect of ionic strength on C-K production;
图 5为 C-K产量随时间的变化关系； Figure 5 shows the change of C-K output over time;
图 6为缓冲液种类对对 C-K产量的影响。. 具体实施方式： Figure 6 shows the effect of buffer type on C-K production. . detailed description:
实施例 1 ' 500mg二醇型人参皂甙， 84mg蜗牛酶溶于 32ml磷酸 -柠檬酸缓冲 液 （pH4.5,离子强度为 0.01， 含 10%的乙醇） 中， 于 40°C水浴中水 解 1天； 反应液经离心收集沉淀； 用水反复洗涤沉淀 （10ml X 3 )， 洗 液与母液合并，浓缩至 32ml供再次使用；用乙醇反复洗涤沉淀（10ml X 5)， 合并乙醇液、 蒸干、 收集残余物得 C-K 106mg。 Example 1 '500 mg of diol-type ginsenoside, 84 mg of snail enzyme was dissolved in 32 ml of phosphate-citrate buffer (pH 4.5, ionic strength 0.01, containing 10% ethanol), and hydrolyzed in a water bath at 40 ° C for 1 day The reaction solution was collected by centrifugation. The precipitate was repeatedly washed with water (10ml X 3). The washing solution was combined with the mother liquor and concentrated to 32ml for reuse. The precipitate was repeatedly washed with ethanol (10ml X 5). The ethanol solution was combined, evaporated to dryness, and collected. The residue gave 106 mg of CK.
实施例 2 Example 2
5mg二醇型人参皂甙， 2.5mg蜗牛酶溶于 3.2ml pH不同的磷酸- 柠檬酸缓冲液（离子强度为 0.01， 含 10%的乙醇）中， 于 40°C水浴中
水解 1 天； 反应液经离心收集沉淀； 用水反复洗涤沉淀 （lml X 3 ), 洗液与母液合并，浓缩至 3.2ml供再次使用；用乙醇反复洗涤沉淀（ lml X 5)， 合并乙醇液、 蒸干、 收集残余物得 C-K产量见图 1。 5mg diol-type ginsenoside, 2.5mg snail enzyme dissolved in 3.2ml phosphate-citrate buffer solution with different pH (0.01, 10% ethanol) in 40 ° C water bath Hydrolysis for 1 day; The reaction solution was collected by centrifugation. The precipitate was repeatedly washed with water (lml X 3). The washing solution was combined with the mother liquor and concentrated to 3.2 ml for reuse. The precipitate was repeatedly washed with ethanol (lml X 5). The yield of CK after evaporation to dryness and collection of the residue is shown in Figure 1.
实施例 3 Example 3
二醇型人参皂甙与蜗牛酶按不同比例溶于 3.2ml磷酸 -柠檬酸缓冲 液 （pH4.5, 离子强度为 0.01， 含 10%的乙醇） 中， 于 40°C水浴中水 解 1 天； 反应液经离心收集沉淀； 用水反复洗涤沉淀 （lmlX 3)， 洗 液与母液合并，浓缩至 3.2ml供再次使用； 用乙醇反复洗涤沉淀（lml X 5), 合并乙醇液、 蒸干、 收集残余物得 C-K产量见图 2。 Diol ginsenoside and snail enzyme were dissolved in 3.2ml phosphate-citrate buffer (pH4.5, ionic strength 0.01, containing 10% ethanol) in different proportions, and hydrolyzed in a 40 ° C water bath for 1 day; reaction The precipitate was collected by centrifugation. The precipitate was repeatedly washed with water (lml X 3). The washing solution was combined with the mother liquor and concentrated to 3.2 ml for reuse. The precipitate was repeatedly washed with ethanol (lml X 5). The ethanol solution was combined, evaporated to dryness, and the residue was collected. The CK yield is shown in Figure 2.
实施例 4 Example 4
3mg二醇型人参皂甙， 0.5mg蜗牛酶溶于 3.2ml磷酸 -柠檬酸缓冲 液（pH4.5, 离子强度为 0.01， 含 10%的乙醇）中， 于不同温度的水浴 中水解 1天； 反应液经离心收集沉淀； 用水反复洗涤沉淀（lmlX 3)， 洗液与母液合并,浓缩至 3.2ml供再次使用；用乙醇反复洗涤沉淀（ lml X 5)， 合并乙醇液、 蒸干、 收集残余物得 C-K产量见图 3。 3mg diol-type ginsenoside, 0.5mg snail enzyme was dissolved in 3.2ml phosphate-citrate buffer (pH4.5, ionic strength 0.01, containing 10% ethanol), and hydrolyzed in water bath at different temperatures for 1 day; reaction The precipitate was collected by centrifugation; the precipitate was repeatedly washed with water (lml X 3); the washing solution was combined with the mother liquor and concentrated to 3.2 ml for reuse; the precipitate was repeatedly washed with ethanol (lml X 5); the ethanol solution was combined, evaporated to dryness, and the residue was collected The CK output is shown in Figure 3.
实施例 5 Example 5
3mg二醇型人参皂甙， 0.5mg蜗牛酶溶于 3.2ml.磷酸 -柠檬酸缓冲 液（pH4.5, 含 10%的乙醇）中， 离子强度变化， 于 40°C水浴中水解 1 天； 反应液经离心收集沉淀； 用水反复洗涤沉淀 （lmlX 3 )， 洗液与 母液合并，浓缩至 3.2ml供再次使用；用乙醇反复洗涤沉淀（lml X 5)， 合并乙醇液、 蒸干、 收集残余物得 C-K产量见图 4。 3mg diol-type ginsenoside, 0.5mg snail enzyme was dissolved in 3.2ml. Phosphate-citrate buffer (pH4.5, containing 10% ethanol), the ionic strength was changed, and hydrolyzed in a water bath at 40 ° C for 1 day; reaction The precipitate was collected by centrifugation. The precipitate was repeatedly washed with water (lmlX 3). The washing solution was combined with the mother liquor and concentrated to 3.2ml for reuse. The precipitate was repeatedly washed with ethanol (lml X 5). The ethanol solution was combined, evaporated to dryness, and the residue was collected. The CK output is shown in Figure 4.
实施例 6 Example 6
3mg二醇型人参皂甙， 0.5mg蜗牛酶溶于 3.2ml磷酸 -柠檬酸缓冲 液 （pH4.5,离子强度为 0.01， 含 10%的乙醇） 中， 于 40 °C水浴中水 解； 反应液经离心收集沉淀； 用水反复洗涤沉淀 （lmlX 3 )， 洗液与 母液合并，浓缩至 3.2ml供再次使用；用乙醇反复洗涤沉淀（lml X 5)， 合并乙醇液、 蒸干、 收集残余物得 C-K， 产量与水解时间的关系见图 5。 3mg diol-type ginsenoside, 0.5mg snail enzyme was dissolved in 3.2ml phosphate-citrate buffer (pH4.5, ionic strength 0.01, containing 10% ethanol), and hydrolyzed in a water bath at 40 ° C; The precipitate was collected by centrifugation; the precipitate was repeatedly washed with water (lmlX 3), the washing solution was combined with the mother liquor, and concentrated to 3.2ml for reuse; the precipitate was repeatedly washed with ethanol (lml X 5), the ethanol solution was combined, evaporated to dryness, and the residue was collected to obtain CK The relationship between yield and hydrolysis time is shown in Figure 5.
实施例 7 Example 7
3mg二醇型人参皂甙，一 0.5mg蜗牛酶溶于 3.2ml pH4.5离子强度为 3mg diol type ginsenoside, 0.5mg snail enzyme dissolved in 3.2ml pH4.5 ionic strength is
0.01含 10%的乙醇的不同缓冲液中， 于 40°C水浴中水解 8小时； 反应 液经离心收集沉淀； 用水反复洗涤沉淀（lmlX3 )， 洗液与母液合并， 浓缩至 3.2ml供再次使用； 用乙醇反复洗涤沉淀（lmlX 5)， 合并乙醇 液、 蒸干、 收集残余物得 C-K产量见图 6。 其中 1为甘氨酸 -HC1, 2 为邻苯二甲酸氢钾 -HC1， 3为 Na2HP04-柠檬酸， 4为柠檬酸 -柠檬酸钠， 5为醋酸-醋酸钠。
In different buffers containing 10% ethanol, hydrolyzed in a 40 ° C water bath for 8 hours; the reaction solution was collected by centrifugation; the precipitate was repeatedly washed with water (lmlX3), the washing solution was combined with the mother liquor, and concentrated to 3.2ml for reuse ; The precipitate was repeatedly washed with ethanol (1 ml × 5), the ethanol solution was combined, evaporated to dryness, and the residue was collected to obtain the CK yield as shown in FIG. 6. 1 is glycine-HC1, 2 is potassium hydrogen phthalate-HC1, 3 is Na 2 HP0 4 -citric acid, 4 is citric acid-sodium citrate, and 5 is acetic acid-sodium acetate.
Claims
1、一种用酶水解人参二醇型皂甙制备 20- β 吡喃葡萄糖基原人 参二醇的方法， 包括水解、 收集步骤， 其特征在于： 用蜗牛酶在缓冲 液中水解二醇型人参皂甙， 二醇型人参皂与蜗牛酶的重量比为 1 :1〜10:1， ρΗ值在 4.0~5.0之间， 水解温度 30-45°C， 水解时间 2小时 ~3天。 1. A method for preparing 20-β glucopyranosylprotopanaxadiol by enzymatic hydrolysis of ginsenodiol type saponin, comprising the steps of hydrolysis and collection, which is characterized in that the diol type ginsenoside is hydrolyzed by a snail enzyme in a buffer solution. The weight ratio of diol-type ginseng soap to snail enzyme is 1: 1 to 10: 1, the ρΗ value is between 4.0 to 5.0, the hydrolysis temperature is 30-45 ° C, and the hydrolysis time is 2 hours to 3 days.
2、 按照权利要求 1所述用酶水解人参二醇型皂甙制备 20- β -Ο- 吡喃葡萄糖基原人参二醇的方法， 其特征在于所用酶为蜗牛酶。 2. The method for preparing 20-β-O-glucopyranosylprotopanaxadiol by enzymatic hydrolysis of ginsenodiol-type saponins according to claim 1, characterized in that the enzyme used is a snail enzyme.
3、 按照权利要求 1所述用酶水解人参二醇型皂甙制备 20- e -D- 吡喃葡萄糖基原人参二醇的方法， 其特征在于所述的收集过程是： 反 应沉淀物用水洗涤后； 再用乙醇反复洗涤， 合并乙醇液蒸干后， 收集 的残余物即为人参皂甙 C-K。 3. The method for preparing 20-e-D-glucopyranosylprotopanaxadiol by hydrolyzing ginsenodiol-type saponins according to claim 1, characterized in that the collection process is: after washing the reaction precipitate with water After repeated washing with ethanol, the combined ethanol solution was evaporated to dryness, and the collected residue was ginsenoside CK.
4、 按照权利要求 3所述用酶水解人参二醇型皂甙制备 20- β -ϋ- 吡喃葡萄糖基原人参二醇的方法， 其特征在于所述反应沉淀物用水反 复洗涤后， 洗液与母液合并， 浓缩后再次用于酶水解。 4. The method for preparing 20-β-ϋ-glucopyranosylprotopanaxadiol by enzymatic hydrolysis of ginsenodiol-type saponins according to claim 3, characterized in that after the reaction precipitate is repeatedly washed with water, the washing solution and The mother liquors were combined and concentrated again for enzymatic hydrolysis.
5、 按照权利要求 1所述用酶水解人参二醇型皂甙制备 20- e -D- 吡喃葡萄糖基原人参二醇的方法，其特征在于所用缓冲液中含有 5〜15 %的乙醇。 5. The method for preparing 20-e-D-glucopyranosylprotopanaxadiol by enzymatic hydrolysis of ginsenodiol-type saponins according to claim 1, characterized in that the buffer used contains 5 to 15% ethanol.
6、 按照权利要求 1所述用酶水解人参二醇型皂甙制备 20- P -D- 吡喃葡萄糖基原人参二醇的方法， 其特征在于所用溶液为缓冲液， 其 pH为 4.5， 溶液离子强度为 0.01。 6. The method for preparing 20-P-D-glucopyranosyl-proto-ginsenodiol by enzymatic hydrolysis of ginsenodiol-type saponins according to claim 1, characterized in that the solution used is a buffer solution, the pH of which is 4.5, and the solution ion The intensity is 0.01.
7、 按照权利要求 1所述用酶水解人参二醇型皂甙制备 20- β -ϋ- 吡喃葡萄糖基原人参二醇的方法， 其特征在于水解温度为 40°C。 7. The method for preparing 20-β-ϋ-glucopyranosylprotopanaxadiol by enzymatic hydrolysis of ginsenodiol-type saponins according to claim 1, characterized in that the hydrolysis temperature is 40 ° C.
8、 按照权利要求 7所述用酶水解人参二醇型皂甙制备 20- β -ϋ- 吡喃葡萄糖基原人参二醇的方法， 其特征在于： 二醇型人参皂甙与蜗 牛酶的重量比为 6: 1。 8. The method for preparing 20-β-ϋ-glucopyranosyl-proto-ginsengdiol by enzymatic hydrolysis of ginsenodiol-type saponins according to claim 7, wherein the weight ratio of diol-type ginsenoside to snail enzyme is: 6: 1.
9、 按照权利要求 1所述用酶水解人参二醇型皂甙制备 20- P -D- 吡喃葡萄糖基原人参二醇的方法， 其特征在于： 水解时间 2〜8小时。
9. The method for preparing 20-P-D-glucopyranosylprotopanaxadiol by enzymatic hydrolysis of ginsenodiol-type saponins according to claim 1, characterized in that the hydrolysis time is 2 to 8 hours.
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| CN01133410.X | 2001-11-06 | ||
| CNB01133410XA CN1167805C (en) | 2001-11-06 | 2001-11-06 | Ginsenoside enzymatically hydrolyzing process to prepare 2o-beta-D-pyranoglucose protopanoxadiol |
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| PCT/CN2002/000786 WO2003040383A1 (en) | 2001-11-06 | 2002-11-05 | Preparation of 20-$g(b)-d-pyranoglucosyl-protopanaxadiol by enzymatic hydrolysis of ginsenoside |
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| CN1508146A (en) * | 2002-12-13 | 2004-06-30 | 中国科学院大连化学物理研究所 | Anti-tumour ginseng saponin |
| CN1305479C (en) * | 2003-09-28 | 2007-03-21 | 中国科学院大连化学物理研究所 | Low polarity ginsenoside combination possessing anti-tumor activity |
| US8541035B2 (en) | 2009-05-14 | 2013-09-24 | Il Hwa Co., Ltd. | Methods for preparing a fermented ginseng concentrate or powder |
| EP2432891B1 (en) * | 2009-05-19 | 2018-04-04 | Il Hwa Co., Ltd. | Methods for preparing a fermented ginseng concentrate or powder |
| CN101768619A (en) * | 2010-02-10 | 2010-07-07 | 华侨大学 | Method for preparing rare ginsenoside IH-901 with Rd as substrate |
| CN102154417A (en) * | 2010-12-13 | 2011-08-17 | 天津中医药大学 | Method for preparing periplocymarin |
| CN102251009A (en) * | 2011-06-09 | 2011-11-23 | 华侨大学 | Simple production method of rare ginsenoside IH-901 |
| CN103360441B (en) * | 2012-04-01 | 2016-08-10 | 浙江海正药业股份有限公司 | Multi-crystal form of ginsenoside C-K and preparation method thereof |
| CN103360450B (en) * | 2012-04-01 | 2016-05-11 | 浙江海正药业股份有限公司 | Two kinds of crystal formations of ginseng saponin C-K and preparation method thereof |
| CN103073611B (en) * | 2013-01-10 | 2015-10-28 | 天津大学 | A kind of ginseng saponin C-K hemi-hydrate crystalline and preparation method |
| CN104622928A (en) * | 2015-01-04 | 2015-05-20 | 中国药科大学 | Total ginsenoside achieving immune regulation effect by activating IL-1beta/ IL-18 activation secretion |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0648946A (en) * | 1991-06-21 | 1994-02-22 | Takuo Kosuge | Tonic agent containing saponin derivative |
| CN1184851A (en) * | 1996-11-29 | 1998-06-17 | 株式会社一和 | Process for prepn. of metabolites of ginseng saponins |
| CN1190098A (en) * | 1998-01-22 | 1998-08-12 | 白求恩医科大学基础医学院 | Preparation, medicinal composition and application of grouped gensenoside |
-
2001
- 2001-11-06 CN CNB01133410XA patent/CN1167805C/en not_active Expired - Fee Related
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0648946A (en) * | 1991-06-21 | 1994-02-22 | Takuo Kosuge | Tonic agent containing saponin derivative |
| CN1184851A (en) * | 1996-11-29 | 1998-06-17 | 株式会社一和 | Process for prepn. of metabolites of ginseng saponins |
| CN1190098A (en) * | 1998-01-22 | 1998-08-12 | 白求恩医科大学基础医学院 | Preparation, medicinal composition and application of grouped gensenoside |
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| CN1417345A (en) | 2003-05-14 |
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