WO2012068832A1

WO2012068832A1 - Method for preparing mogroside iv

Info

Publication number: WO2012068832A1
Application number: PCT/CN2011/073861
Authority: WO
Inventors: 谢海峰; 胡云岭; 张良蕾
Original assignee: 成都普瑞法科技开发有限公司
Priority date: 2010-11-26
Filing date: 2011-05-10
Publication date: 2012-05-31
Also published as: CN102477455B; CN102477455A

Abstract

Provided is a method for preparing mogroside IV, which comprises hydrolyzing mogroside V, Momordica grosvenori fruits, or extracts of Momordica grosvenori fruits containing mogroside V with β-glucosidase. The content of mogroside IV in powder of Momordica grosvenori fruits is increased from less than 0.05% to 1.5% or more by the method. As to extracts of Momordica grosvenori fruits (the content of mogroside V is about 40% and the content of mogroside IV is about 1.5%), the content of mogroside IV is increased to 20% or more by the method.

Description

制备罗汉果苷 IV的方法技术领域 Method for preparing mogroside IV

本发明涉及制备罗汉果苷 IV的方法，属于中药领域。 The invention relates to a method for preparing mogroside IV, belonging to the field of traditional Chinese medicine.

背景技术 Background technique

罗汉果主产于广西，江西，广东，也被称作僧果，在我国有数百年的种植历史，其在中医中的应用可追溯到 1813年。罗汉果中的葫芦烷三萜甙类化合物，包括赛门苷 I，罗汉果苷 II，罗汉果苷 III，罗汉果苷 IIIE, 罗汉果苷 IV，罗汉果苷 V等。其中罗汉果苷 IV的含量极低，提取难度大成本高，国内外未见有罗汉果苷 IV的制备工艺的相关报道。 Mangosteen is mainly produced in Guangxi, Jiangxi, and Guangdong. It is also known as the capsule. It has hundreds of years of planting history in China, and its application in Chinese medicine can be traced back to 1813. The cucurbitane triterpenoids in Siraitia grosvenii, including serotonin I, mogroside II, mogroside III, mogroside IIIE, mogroside IV, mogroside V and the like. Among them, the content of mogroside IV is extremely low, and the extraction is difficult and costly. There is no report on the preparation process of mogroside IV at home and abroad.

目前，国外医药工作者发现罗汉果苷 IV具有良好的治疗丙肝的作用，可单独或与其他药物共用起到良好的治疗效果。罗汉果苷 IV其毒副作用小，生物利用度高，是新兴的抗病毒天然植物成分，具有广阔的市场价值。 At present, foreign medical workers have found that mogroside IV has a good effect on the treatment of hepatitis C, and can be used alone or in combination with other drugs to achieve a good therapeutic effect. Mogroside IV has small toxic and side effects and high bioavailability. It is an emerging antiviral natural botanical ingredient and has broad market value.

目前制约罗汉果苷 IV应用的瓶颈问题是罗汉果苷 IV的制备，罗汉果苷 IV在罗汉果原料粉末中含量<0.05 % (罗汉果苷 V的含量为 2%左右），所以苷 IV制备困难，成本高。严重的制约了罗汉果苷 IV的开发应用。因此，如何开发出一种高效的制备罗汉果苷 IV的方法成为本领域目前迫切需要解决的技术难题。 At present, the bottleneck problem restricting the application of mogroside IV is the preparation of mogroside IV. The content of mogroside IV in the raw material of Luo Han Guo is <0.05% (the content of mogroside V is about 2%), so the preparation of glycoside IV is difficult and the cost is high. Serious development of the development and application of mogroside IV. Therefore, how to develop an efficient method for preparing mogroside IV has become an urgent technical problem in the field.

发明内容 Summary of the invention

本发明所要解决的技术问题是提供一种成本较低的制备罗汉果苷 IV的方法。 The technical problem to be solved by the present invention is to provide a lower cost method for preparing mogroside IV.

本发明制备罗汉果苷 IV的方法为：采用 β-葡萄糖苷酶水解罗汉果苷 V、罗汉果（可以是罗汉果鲜果或罗汉果干粉）或含有罗汉果苷 V的罗汉果提取物。 The method for preparing mogroside IV according to the present invention is as follows: using β-glucosidase to hydrolyze mogroside V, mangosteen (may be dried mangosteen or dried mangosteen) or mangosteen extract containing mogroside V.

其中，罗汉果水提取物或乙醇溶液提取物均适用于本发明。 Among them, the Lo Han Guo water extract or the ethanol solution extract are suitable for use in the present invention.

进一步的，上述的制备罗汉果苷 IV的方法，优选每 kg罗汉果苷 V采用 5k〜50k万 U β- 葡萄糖苷酶水解；其中， β-葡萄糖苷酶在最适 ρΗ值和温度的条件下， lmin水解 1^%的3 - 葡聚糖溶液产生 1 μ g葡萄糖的酶量为 1个 β-葡萄糖苷酶活力单位 U。 β-葡萄糖苷酶的水解时间优选为 2〜24h。 Further, in the above method for preparing mogroside IV, it is preferred to use 5 k~50 kM U β-glucosidase hydrolysis per kg of mogroside V; wherein β-glucosidase is under optimal conditions of pH and temperature, lmin The amount of enzyme that produces 1 μg of glucose by hydrolyzing 1% by weight of the 3-glucan solution is one β-glucosidase activity unit U. The hydrolysis time of the β-glucosidase is preferably 2 to 24 hours.

其中，上述制备罗汉果苷 IV的方法，其采用 β-葡萄糖苷酶水解罗汉果苷 V或含有罗汉果苷 V的罗汉果提取物的具体步骤如下： Among the above, the method for preparing mogroside IV, which uses β-glucosidase to hydrolyze mogroside V or the mogroside extract containing mogroside V, is as follows:

a、溶解：罗汉果苷、罗汉果或含有罗汉果苷 V的罗汉果提取物加溶剂溶解，使溶液中的罗汉果苷 V的浓度为 0.025〜0.300g/mL; a, dissolved: mogroside, mangosteen or Luo Han Guo extract containing Luo Han Guo V is dissolved in a solvent, so that the concentration of mogroside V in the solution is 0.025~0.300g / mL;

b、水解： a步骤所得溶液中加入 β-葡萄糖苷酶水解，并调节溶液的 ρΗ值和温度使其与 β-葡萄糖苷酶的酶解 ρΗ值和温度相适应； b. Hydrolysis: The solution obtained in step a is hydrolyzed by β-glucosidase, and the pH value and temperature of the solution are adjusted to be compatible with the enzymatic hydrolysis value and temperature of β-glucosidase;

c、分离、纯化，得到罗汉果苷 IV。其中，所述 a步骤中的溶剂可以为常规溶剂，溶剂优选为水或与 β-葡萄糖苷酶的酶解 pH 值相适应的缓冲液。 c. Separation and purification to obtain mogroside IV. Wherein, the solvent in the step a may be a conventional solvent, and the solvent is preferably water or a buffer adapted to the pH of the enzymatic hydrolysis of β-glucosidase.

其中，上述 a步骤酶解时，若罗汉果苷 V浓度过高，会在溶液中形成分子间氢键，降低酶水解效率；若罗汉果苷 V浓度过低，则罗汉果苷 IV的产率较低，还会增加浓缩成本，溶液中的罗汉果苷 V的浓度为 0.025〜0.300g/mL较为合适。 Wherein, in the a step of enzymatic hydrolysis, if the concentration of mogroside V is too high, an intermolecular hydrogen bond is formed in the solution to reduce the enzymatic hydrolysis efficiency; if the concentration of mogroside V is too low, the yield of mogroside IV is low. The concentration cost is also increased, and the concentration of mogroside V in the solution is preferably from 0.025 to 0.300 g/mL.

进一步的，由于 β-葡萄糖苷酶的溶解度有限，加入过多，则会造成浪费； β-葡萄糖苷酶的浓度过低，则水解效率较低，上述 b步骤水解时溶液中的 β-葡萄糖苷酶的浓度优选为 1.5 万〜 5.0万 U/mL (指水解时酶的初始浓度）。 Further, since the solubility of β-glucosidase is limited, excessive addition may cause waste; if the concentration of β-glucosidase is too low, the hydrolysis efficiency is low, and β-glucoside in the solution in the above b step hydrolysis The concentration of the enzyme is preferably from 15,000 to 50,000 U/mL (refer to the initial concentration of the enzyme at the time of hydrolysis).

其中，更优选的水解条件为：水解时溶液中的罗汉果苷 V浓度 0. lg/mL (指水解时罗汉果苷 V的初始浓度）， β-葡萄糖苷酶浓度为 2. 0万 U/mL (指水解时酶的初始浓度），水解时间为 16h。 More preferably, the hydrolysis conditions are: the concentration of mogroside V in the solution at the time of hydrolysis is 0. lg / mL (refers to the initial concentration of mogroside V during hydrolysis), the concentration of β-glucosidase is 2 million U / mL ( Refers to the initial concentration of the enzyme at the time of hydrolysis), and the hydrolysis time is 16 h.

其中，由于 β-葡萄糖苷酶的种类较多，不同的 β-葡萄糖苷酶的最佳酶活的 pH值和温度不同，因此，为了提高水解效率，可以调节水解溶液的 pH值和温度使其与 β-葡萄糖苷酶的酶解 pH值和温度相适应，即调节至 β-葡萄糖苷酶的最佳酶活的 pH值和温度。 Among them, due to the variety of β-glucosidases, the pH and temperature of the optimal enzyme activities of different β-glucosidases are different. Therefore, in order to improve the hydrolysis efficiency, the pH and temperature of the hydrolysis solution can be adjusted. It is compatible with the enzymatic pH and temperature of β-glucosidase, that is, the pH and temperature of the optimal enzyme activity adjusted to β-glucosidase.

其中，上述 C步骤中可以采用常规的分离、纯化方法，如：凝胶柱层析方法、 HPLC纯化法等，分离、纯化得到高纯度的罗汉果苷 IV产品。 Among them, in the above C step, a conventional separation and purification method, such as a gel column chromatography method, an HPLC purification method, or the like, can be used for separation and purification to obtain a high-purity mogroside IV product.

其中，上述的 β-葡萄糖苷酶可以为常规的 β-葡萄糖苷酶，可以采用市售的 β-葡萄糖苷酶或自行制备的 β-葡萄糖苷酶（β-葡萄糖苷酶可按常规方法制备），考虑到成本的因素，上述的 β-葡萄糖苷酶优选为纤维素酶、 1，4- β -ϋ-葡聚糖葡糖苷水解酶、半纤维素酶中至少一种。 Wherein, the above β-glucosidase may be a conventional β-glucosidase, and a commercially available β-glucosidase or a self-prepared β-glucosidase (β-glucosidase can be prepared by a conventional method) may be used. The β-glucosidase is preferably at least one of a cellulase, a 1,4-β-ϋ-dextran glucoside hydrolase, and a hemicellulase in consideration of cost.

本发明方法工艺简单，成本较低，可以将罗汉果粉末中罗汉果苷 IV的含量从< 0. 05 %，提高到 2. 00 %以上；对于罗汉果提取物（罗汉果苷 V含量约为 40 %，罗汉果苷 IV含量约为 1. 5 The method of the invention has the advantages of simple process and low cost, and can increase the content of mogroside IV in the Luo Han Guo powder from < 0.05% to more than 1.00%; for the extract of Luo Han Guo (the content of mogroside V is about 40%, Luo Han Guo 5。 The glycoside IV content is about 1. 5

% ), 本发明方法可以将罗汉果苷 IV的含量提高到 20 %以上。经过分离、纯化，可以使罗汉果苷 IV的纯度达到 98 %以上。本发明方法为罗汉果苷 IV的制备提供了一种新的途径，具有广阔的应用前景。 %), the method of the invention can increase the content of mogroside IV to more than 20%. After separation and purification, the purity of mogroside IV can be over 98%. The method of the invention provides a new way for the preparation of mogroside IV, and has broad application prospects.

附图说明 DRAWINGS

图 1 是酶水解前后罗汉果提取物的 HPLC图，其中，图 Α为酶水解前的罗汉果提取物的 HPLC图，图 B为酶水解后罗汉果提取物的 HPLC图，图中划圈处为罗汉果苷 IV。 Fig. 1 is an HPLC diagram of the extract of Siraitia grosvenorii before and after enzymatic hydrolysis, wherein Fig. 2 is the HPLC diagram of the extract of Siraitia grosvenorii before enzymatic hydrolysis, and Fig. B is the HPLC diagram of the extract of Siraitia grosvenii after enzymatic hydrolysis, in which the circle is the mogroside IV.

图 2 是经纯化后的罗汉果苷 IV的 HPLC图（图 A)及罗汉果苷 IV标准样品的 HPLC图（图 Figure 2 is a HPLC diagram of purified mogroside IV (Figure A) and a HPLC chromatogram of the mogroside IV standard sample (Figure 2

B ) 。 B).

图 3 是纯化后的罗汉果苷 IV的核磁图谱 ^H NMR Figure 3 is the nuclear magnetic resonance spectrum of purified mogroside IV ^H NMR

图 4 是纯化后的罗汉果苷 IV的核磁图谱（¹³C NMR)。 Figure 4 is a nuclear magnetic spectrum ( ¹³ C NMR) of purified mogroside IV.

图 5 是纯化后的罗汉果苷 IV的质谱图。 Figure 5 is a mass spectrum of purified mogroside IV.

具体实施方式罗汉果苷 V的结构如（ I ) 所示，罗汉果苷 IV的结构如（Π)所示。 detailed description The structure of mogroside V is as shown in (I), and the structure of mogroside IV is shown as (Π).

(II) (II)

从结构式（ I ) 和（II)可以看出：罗汉果苷 IV比罗汉果苷 V少一个葡萄糖，理论上，可以用糖基水解酶、酸或碱水解使罗汉果苷 V的糖基减少，从而得到罗汉果苷 IV。但是，经过大量试验发现，采用酸或碱水解罗汉果苷 V，其目标性不强，容易破坏罗汉果苷结构，得不到罗汉果苷 IV或得到的罗汉果苷 IV产率较低，难以满足工业大规模生产罗汉果苷 IV的需要。本发明的发明人通过大量试验，发现调整适当的 β-葡萄糖苷酶用量，以及控制合适的酶解时间，却可以将罗汉果中葫芦烷三萜甙类化合物的苷 IV的含量从<0. 05 %，提高到 2. 00 %以上，对于罗汉果提取物（罗汉果苷 V含量约为 40 %，罗汉果苷 IV含量约为 1. 50% )，本发明方法可以将罗汉果苷 IV的含量提高到 20 %以上。均大幅高出了预计的罗汉果苷 IV的产率的<0. 05 % (以罗汉果粉为原料）和< 1. 50 % (以罗汉提取物为原料），更远远高于酸或碱水解的罗汉果苷 IV的产率 <0. 11 % (以罗汉提取物为原料）和<2. 20 % (以罗汉提取物为原料），达到了工业大规模生产罗汉果苷 IV的要求。 It can be seen from structural formulas (I) and (II) that mogroside IV is one glucose less than mogroside V. In theory, glycosyl hydrolyzase, acid or base hydrolysis can be used to reduce the glycosyl group of mogroside V, thereby obtaining mangosteen. Glycoside IV. However, after extensive experiments, it has been found that the hydrolysis of mogroside V by acid or alkali is not highly targeted and easily destroys the structure of mogroside. The lower yield of mogroside IV or the obtained mogroside IV is difficult to meet the needs of industrial large-scale production of mogroside IV. The inventors of the present invention have found that the content of the glycoside IV of the cucurbitane triterpenoids in the Luo Han Guo is from <0. 05, through a large number of experiments, and found that the appropriate amount of β-glucosidase is adjusted and the appropriate hydrolysis time is controlled. %, increased to more than 2. 00%, for the Luo Han Guo extract (the content of mogroside V is about 40%, the content of mogroside IV is about 1.50%), the method of the invention can increase the content of mogroside IV to 20% the above. Both are significantly higher than the predicted yield of mogroside IV <0.05% (based on Luo Han Guo powder) and < 1. 50% (based on Luo Han extract), far more than acid or base hydrolysis The yield of mogroside IV is 0.1% (based on Rohan extract) and <2. 20% (based on Lohan extract), which meets the requirements for industrial large-scale production of mogroside IV.

本发明制备罗汉果苷 IV的方法为：采用 β-葡萄糖苷酶水解罗汉果苷 V、罗汉果或含有罗汉果苷 V的罗汉果提取物。其中，罗汉果水提取物或乙醇溶液提取物均适用于本发明。 The method for preparing mogroside IV of the present invention comprises: hydrolyzing mogroside V, Luo Han Guo or Luo Han Guo extract containing Luo Han Guo V by β-glucosidase. Among them, the Lo Han Guo water extract or the ethanol solution extract are suitable for use in the present invention.

进一步的，为了提高罗汉果苷 IV的产率，上述的制备罗汉果苷 IV的方法，优选每 kg罗汉果苷 V采用 5k〜50k万 υ β-葡萄糖苷酶水解。；其中， β-葡萄糖苷酶在最适 ρΗ值和温度的条件下， lmin水解 1^ %的 β -葡聚糖溶液产生 1 μ g葡萄糖的酶量为 1个 β-葡萄糖苷酶活力单位 U。 β-葡萄糖苷酶的水解时间优选为 2〜24h。 Further, in order to increase the yield of mogroside IV, the above method for preparing mogroside IV preferably uses 5k to 50 kM υ β-glucosidase hydrolysis per kg of mogroside V. Among them, β-glucosidase, under the optimum pH value and temperature, lmin hydrolyzes 1% of β-glucan solution to produce 1 μg of glucose. The amount of enzyme is 1 β-glucosidase activity unit U. . The hydrolysis time of the β-glucosidase is preferably 2 to 24 hours.

其中，上述制备罗汉果苷 IV的方法，其采用 β-葡萄糖苷酶水解罗汉果苷 V或含有罗汉果苷 V的罗汉果提取物或罗汉果原料本身的具体步骤如下： Among them, the above-mentioned method for preparing mogroside IV, which uses β-glucosidase to hydrolyze mogroside V or the mogroside extract containing mogroside V or the mogroside raw material itself, is as follows:

c、分离、纯化，得到罗汉果苷 IV。 c. Separation and purification to obtain mogroside IV.

其中，所述 a步骤中的溶剂可以为常规溶剂，溶剂优选为水或与 β-葡萄糖苷酶的酶解 pH 值相适应的缓冲液。 Wherein, the solvent in the step a may be a conventional solvent, and the solvent is preferably water or a buffer compatible with the pH of the enzymatic hydrolysis of β-glucosidase.

进一步的，由于 β-葡萄糖苷酶的溶解度有限，加入过多，则会造成浪费； β-葡萄糖苷酶的浓度过低，则水解效率较低，上述 b步骤水解时溶液中的 β-葡萄糖苷酶的浓度优选为 1.5 万〜 5.0万 U/mL。 Further, since the solubility of β-glucosidase is limited, excessive addition may cause waste; if the concentration of β-glucosidase is too low, the hydrolysis efficiency is low, and β-glucoside in the solution in the above b step hydrolysis The concentration of the enzyme is preferably from 15,000 to 50,000 U/mL.

其中，由于 β-葡萄糖苷酶的种类较多，不同的 β-葡萄糖苷酶的最佳酶活的 pH值和温度可能不同，因此，为了提高水解效率，可以调节水解溶液的 pH值和温度使其与 β-葡萄糖苷酶的酶解 pH值和温度相适应，即调节至 β-葡萄糖苷酶的最佳酶活的 ρΗ值和温度。 Among them, due to the variety of β-glucosidases, the pH and temperature of the optimal enzyme activities of different β-glucosidases may be different. Therefore, in order to improve the hydrolysis efficiency, the pH and temperature of the hydrolysis solution can be adjusted. Beta-glucoside The enzymatic pH of the enzyme is adapted to the temperature, ie the pH value and temperature of the optimal enzyme activity of the β-glucosidase.

其中，上述 c步骤中可以采用常规的分离、纯化方法，如：凝胶柱层析方法、 HPLC纯化法等，分离、纯化得到高纯度的得到罗汉果苷 IV产品。 Wherein, in the above c step, a conventional separation and purification method, such as a gel column chromatography method, an HPLC purification method, or the like, can be used for separation and purification to obtain a high purity mouroside IV product.

其中，上述的 β-葡萄糖苷酶可以为常规的 β-葡萄糖苷酶，可以采用市售的 β-葡萄糖苷酶或自行制备的 β-葡萄糖苷酶（β-葡萄糖苷酶可按常规方法制备），考虑到成本的因素，上述的 β-葡萄糖苷酶优选为纤维素酶、 1，4- β _ϋ-葡聚糖葡糖苷水解酶、半纤维素酶中至少一种。 Wherein, the above β-glucosidase may be a conventional β-glucosidase, and a commercially available β-glucosidase or a self-prepared β-glucosidase (β-glucosidase can be prepared by a conventional method) may be used. The β-glucosidase is preferably at least one of a cellulase, a 1,4-β-ϋ-glucan glucoside hydrolase, and a hemicellulase in consideration of cost.

下面结合实施例和试验例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所述的实施例范围之中。 The embodiments of the present invention are further described in conjunction with the examples and the examples of the invention, and the invention is not limited to the scope of the embodiments.

试验例 1 β-葡萄糖苷酶用量的筛选试验 Test Example 1 Screening test for the amount of β-glucosidase

原料为罗汉果苷 V含量约 40. 00 % (本发明中的罗汉果苷 V或罗汉果苷 IV的含量均指质量含量），罗汉果苷 IV含量 < 1. 00 %。采用购至和氏璧生物技术有限公司 β—葡聚糖酶水解，水解时的酶浓度（指初始浓度）分别为 0.5万 U/mL， 2.5万 U/mL， 5.0万 U/mL，水解 12h，测定罗汉果苷 IV的得率。结果如表 1所示。从表 1可以看出，酶浓度为 2.5万〜 5.0万 U/mL 时，罗汉果苷 IV得率较高。 The raw material is mogroside V content of about 40. 00% (the content of mogroside V or mogroside IV in the present invention refers to the mass content), and the content of mogroside IV is 1.00%. The enzyme concentration (initial concentration) at the time of hydrolysis was 0.5 million U/mL, 25,000 U/mL, 50,000 U/mL, and hydrolyzed for 12 h, respectively, by hydrolysis of β-glucanase from Hessian Biotechnology Co., Ltd. , the yield of mogroside IV was determined. The results are shown in Table 1. It can be seen from Table 1 that when the enzyme concentration is 25,000 to 50,000 U/mL, the yield of mogroside IV is higher.

表 1 Table 1

试验例 2 水解时间的筛选试验

Test Example 2 Screening test for hydrolysis time

原料为罗汉果苷 V含量约 40.00%，罗汉果苷 IV含量 < 1.00 %。采用购至和氏璧生物技术有限公司的 β—葡聚糖酶水解，水解时的酶浓度（指初始浓度）为 2.5万 U/mL，罗汉果苷 V 浓度为 0.05 g/mL，分别水解 2、 8、 16、 24、 48、 72h，测定罗汉果苷 IV的得率。结果见表 2。从表 2可以看出，水解时间为 8〜24h时，罗汉果苷 IV的得率较高。 The raw material is mouroside V content of about 40.00%, and mogroside IV content is < 1.00%. Using the β-glucanase hydrolysis from Hessian Biotechnology Co., Ltd., the enzyme concentration (initial concentration) at hydrolysis was 25,000 U/mL, and the concentration of mogroside V was 0.05 g/mL, respectively. 8, 16, 24, 48, 72h, the yield of mogroside IV was determined. The results are shown in Table 2. It can be seen from Table 2 that the yield of mogroside IV is higher when the hydrolysis time is 8 to 24 hours.

表 2 Table 2

水解时罗汉果苷 V浓度的筛选试验原料为罗汉果苷 V含量约 40.00%罗汉果提取物，其中罗汉果苷 IV含量 < 1.00%。采用购至和氏璧生物技术有限公司的 β—葡聚糖酶水解，水解时的酶浓度（指初始浓度）为 2.5万 U/mL, 罗汉果苷 V浓度分别为 0.05g/mL、 0.25g/mL、 0.50g/mL，水解 16h，测定罗汉果苷 IV 的得率。结果见表 3。从表 3可以看出，水解时罗汉果苷 V浓度为 0.05〜0.50 g/mL时，罗汉果苷 IV的得率均较高，其中，罗汉果苷 V浓度为 0.25g/mL时，罗汉果苷 IV的得率最高。 Screening test of mogroside V concentration during hydrolysis The raw material is Mogroside V content of about 40.00% Luo Han Guo extract, wherein the mogroside IV content is < 1.00%. The enzyme concentration (initial concentration) at the time of hydrolysis was 25,000 U/mL, and the concentration of mogroside V was 0.05 g/mL, 0.25 g/, respectively, by hydrolysis of β-glucanase purchased from Hersbital Biotechnology Co., Ltd. The yield of mogroside IV was determined by mL, 0.50 g/mL, and hydrolysis for 16 h. The results are shown in Table 3. It can be seen from Table 3 that when the concentration of mogroside V at the time of hydrolysis is 0.05~0.50 g/mL, the yield of mogroside IV is higher, and when the concentration of mogroside V is 0.25 g/mL, the content of mogroside IV is obtained. The rate is the highest.

表 3 table 3

试验例 4 正交试验

Test Example 4 Orthogonal test

原料为罗汉果苷 V含量约 40%罗汉果提取物，其中罗汉果苷 IV含量 < 1.00%。根据试验例 1〜3的结果决定试验因素与水平，并设计正交表，测定水解的最佳条件。结果见表 4〜6。 The raw material is mogroside V content of about 40% of Luo Han Guo extract, wherein the mogroside IV content is < 1.00%. The test factors and levels were determined according to the results of Test Examples 1 to 3, and an orthogonal table was designed to determine the optimum conditions for hydrolysis. The results are shown in Tables 4 to 6.

表 4 试验因素与水平 Table 4 Test factors and levels

表 5 正交表及试验结果

Table 5 Orthogonal table and test results

罗汉果苷罗汉果苷 Mogroside

因素酶浓度水解时间 Factor enzyme concentration hydrolysis time

V浓度 IV得率（％) V concentration IV yield (%)

实验 1 1 1 1 10.80 实验 2 1 2 2 12.50 实验 3 1 3 3 12.58 实验 4 2 1 2 19.50 实验 5 2 2 3 18.10 实验 6 2 3 1 18.35 实验 7 3 1 3 17.43 实验 8 3 2 1 15.35 实验 9 3 3 2 16.95 均值 1 11.960 15.910 14.833 Experiment 1 1 1 1 10.80 Experiment 2 1 2 2 12.50 Experiment 3 1 3 3 12.58 Experiment 4 2 1 2 19.50 Experiment 5 2 2 3 18.10 Experiment 6 2 3 1 18.35 Experiment 7 3 1 3 17.43 Experiment 8 3 2 1 15.35 Experiment 9 3 3 2 16.95 Mean 1 11.960 15.910 14.833

均值 2 18.650 15.317 16.317 Mean 2 18.650 15.317 16.317

均值 3 16.577 15.960 16.037 Mean 3 16.577 15.960 16.037

极差 6.690 0.643 1.484 表 6 试验结果分析 Very poor 6.690 0.643 1.484 Table 6 Analysis of test results

从表 4 5可以看出，通过分析，理论得到的最佳组合为：罗汉果苷 V浓度 0. 10g/mL 酶浓度 3. 5万 U/mL，水解时间 16h It can be seen from Table 4 5 that the best combination obtained by the theory is: mogroside V concentration 0. 10g/mL enzyme concentration 3. 50,000 U/mL, hydrolysis time 16h

从表 4 6可以看出，水解时，罗汉果苷 V浓度表现为极显著性差异，因浓度过高在罗汉果苷溶液中分子之间形成氢键，阻碍的酶的水解，所以表现出并非浓度越高越好，而是在一定的范围内有最佳取值。水解时间的影响在罗汉果苷 V浓度之后，在酶浓度之前。时间也并非越长越好，因为苷 IV也可以被继续被水解转换。其中酶浓度影响不显著的原因推测为酶浓度在溶液中的溶解度决定了其已经达到饱和状态。又由于酶的影响相对不显著，综合考虑成本原因，酶浓度选择 1. 5至 2. 5万 U/mL较佳。 It can be seen from Table 46 that the concentration of mogroside V is extremely significant when hydrolyzed. Due to the high concentration, hydrogen bonds are formed between the molecules in the molybdenum solution, which hinders the hydrolysis of the enzyme, so it shows that the concentration is not The higher the better, the better the value is within a certain range. The effect of hydrolysis time is after the concentration of mogroside V, before the enzyme concentration. The time is not as long as possible, because glycoside IV can also be continuously hydrolyzed. The reason why the influence of the enzyme concentration is not significant is presumed to be that the solubility of the enzyme concentration in the solution determines that it has reached a saturated state. Since the influence of the enzyme is relatively insignificant, considering the cost reason, the enzyme concentration is preferably 1.5 to 25,000 U/mL.

综上所述，最佳水解条件优选为罗汉果苷 V浓度 0. lOg/mL, 酶浓度 2. 0万 U/mL，水解时间 16h。重现试验结果为罗汉果苷 IV含量为 21. 78% In summary, the optimal hydrolysis condition is preferably a concentration of mogroside V 0.1 lOg/mL, an enzyme concentration of 200,000 U/mL, and a hydrolysis time of 16 h. The recurrence test results showed that the content of mogroside IV was 21.78%.

实施例 1 采用本发明方法制备罗汉果苷 IV Example 1 Preparation of Mogroside IV by the method of the present invention

将鲜罗汉果 1000kg破碎后投入提取罐中，用 4000kg纯化水于微沸状态下提取 3次，提取时间为 1.5h lh lh，每次过滤后的滤渣进入下一次提取，合并滤液浓缩至 4000L。测得罗汉果苷 V含量为罗汉果鲜重的 0.27%，罗汉果苷 IV含量小于 0. 01 %。温度降至 50°C加入 10g P—葡聚糖酶（和氏璧生物技术有限公司生产，下同），调 pH至 5，水解时间 20h。测得罗汉果苷 V含量为 0.10 %，罗汉果苷 IV含量 0.12%。抽滤，滤液浓缩至一定体积，加入 10 % Ca (OH) ₂沉淀杂质，静制，过滤，上清液用***萃取酯溶性杂质，弃去***部分，水相继续采用 D280吸附树脂（青岛海洋化工厂）和 100-200目硅胶（青岛海洋化工厂）精制除去糖类，蛋白质类和色素分子，收集罗汉果苷部分，用 lmol/L盐酸调 pH至 5。采用 201型强碱性离子交换树脂脱色。浓缩至合适体积，经制备型 HPLC反复纯化，浓缩，干燥得到样品。 HPLC 检测得到罗汉果苷 IVO. 97kg，其含量为 98 %以上。经纯化后的罗汉果苷 IV的 HPLC图及罗汉果苷 IV标准样品的 HPLC图如图 2所示。纯化后的罗汉果苷 IV的核磁图谱如图 3所示，纯化后的罗汉果苷 IV的质谱图如图 4所示。 1000 kg of fresh Luo Han Guo was crushed and put into an extraction tank, and extracted with 4000 kg of purified water in a micro-boiling state for 3 times. The extraction time was 1.5 h lh lh, and the filtered residue after each filtration was subjected to the next extraction, and the combined filtrate was concentrated to 4000 L. 01%。 The content of mogroside V was 0.27% of the fresh weight of Luo Han Guo, the content of mogroside IV was less than 0.01%. The temperature was lowered to 50 ° C and 10 g of P-glucanase (produced by Hess Biotech Co., Ltd., the same below) was added, and the pH was adjusted to 5, and the hydrolysis time was 20 h. The content of mogroside V was 0.10%, and the content of mogroside IV was 0.12%. After suction filtration, the filtrate was concentrated to a certain volume, and 10% Ca(OH) _{2 was added to} precipitate impurities, which were allowed to stand, filtered, and the supernatant was extracted with ether to extract ester-soluble impurities, and the ether portion was discarded. The aqueous phase continued to use D280 adsorption resin (Qingdao Ocean) The chemical plant) and 100-200 mesh silica gel (Qingdao Marine Chemical Plant) are refined to remove sugars, protein and pigment molecules, and the mogroside fraction is collected, and the pH is adjusted to 5 with 1 mol/L hydrochloric acid. Decolorization was carried out using a 201 type strong basic ion exchange resin. Concentrate to a suitable volume, repeatedly purified by preparative HPLC, concentrated and dried to give a sample. The content of mogroside IVO. 97 kg was determined by HPLC, and its content was 98% or more. The HPLC chromatogram of the purified mogroside IV and the HPLC chromatogram of the mogroside IV standard sample are shown in FIG. 2 . The nuclear magnetic spectrum of the purified mogroside IV is shown in Fig. 3, and the mass spectrum of the purified mogroside IV is shown in Fig. 4.

实施例 2 采用本发明方法制备罗汉果苷 IV Example 2 Preparation of Mogroside IV by the method of the present invention

将罗汉果干粉 10kg投入提取罐中，用 50L 50%乙醇回流提取 3次，每次提取时间为 2h 每次过滤后的滤渣进入下一次提取，浓缩干燥浸膏 lkg HPLC 检测测得罗汉果苷 V含量为 8.4 % ,罗汉果苷 IV含量小于 0. 1 %。浸膏复溶于 5L水，滤去不溶物，温度升至 50°C加入 10gl， 4- β -D-葡聚糖葡糖苷水解酶，调 ρΗ至 4.8，水解时间 20h，测得罗汉果苷 V含量为 3.4 %，罗汉果苷 IV含量为 4.2 %。采用实施例 1方案纯化，得到罗汉果苷 IVO. 17kg, 其含量为 98 %以上。 10kg of dried mangosteen powder was put into the extraction tank, and extracted with 50L of 50% ethanol for 3 times. The extraction time was 2h. The filtered residue after each filtration was injected into the next extraction. The content of mogroside V was determined by HPLC. 5%。 The content of the mogroside IV is less than 0.1%. The extract was redissolved in 5 L of water, and the insoluble matter was filtered off. The temperature was raised to 50 ° C, 10 g, 4-β-D-glucan glucoside hydrolase was added, and the pH was adjusted to 4.8. The hydrolysis time was 20 h, and the mogroside V was measured. The content was 3.4%, and the content of mogroside IV was 4.2%. Purified by the method of Example 1, to obtain mogroside IVO. 17 kg, and its content was 98% or more.

实施例 3 采用本发明方法制备罗汉果苷 IV Example 3 Preparation of Mogroside IV by the method of the present invention

将 40%罗汉果苷 V原料 1kg投入提取罐中，用 5L水溶解，温度升至 50°C加入 10g β—葡聚糖酶，调 ρΗ至 5，水解时间 16h，测得罗汉果苷 V含量 15.5 %，罗汉果苷 IV含量 17. 3 %。浓缩，经制备型 HPLC反复纯化，浓缩，干燥得到罗汉果苷 IVO. 157kg，其含量为 98 %以上。水解前后罗汉果提取物中罗汉果苷 IV含量的 HPLC图如图 1所示，从图 1可以看出，水解后的罗汉果苷 IV含量明显更高。 1 kg of 40% mogroside V raw material was put into an extraction tank, dissolved in 5 L of water, the temperature was raised to 50 ° C, 10 g of β-glucanase was added, and the pH was adjusted to 5, and the hydrolysis time was 16 h, and the content of mogroside V was 15.5 %. , The content of mogroside IV is 17.3%. The mixture was concentrated, purified by preparative HPLC, concentrated, and dried to obtain 157 kg of mogroside IVO. The content was 98% or more. The HPLC chromatogram of the content of mogroside IV in the extract of Luo Han Guo before and after hydrolysis is shown in Fig. 1. It can be seen from Fig. 1 that the content of mogroside IV after hydrolysis is significantly higher.

实施例 4 采用本发明方法制备罗汉果苷 IV Example 4 Preparation of Mogroside IV by the method of the present invention

同实施例 1，只将其中 β—葡聚糖酶（和氏璧生物技术有限公司）改为纤维素酶（和氏璧生物技术有限公司），最后得到罗汉果苷 IV l. 02kg，其含量为 98 %以上。 In the same manner as in the first embodiment, only β-glucanase (Heshi Biotechnology Co., Ltd.) was changed to cellulase (Heshi Biotechnology Co., Ltd.), and finally, mogroside IV l. 02 kg was obtained, and the content thereof was More than 98%.

实施例 5 采用本发明方法制备罗汉果苷 IV Example 5 Preparation of Mogroside IV by the method of the present invention

同实施例 2，只将其中 β—葡萄糖苷酶（和氏璧生物技术有限公司 β —葡萄糖苷酶）改为半纤维素酶，最后得到罗汉果苷 IV0.16kg，其含量为 98 %以上。 In the same manner as in Example 2, only β-glucosidase (β-glucosidase of Hessian Biotechnology Co., Ltd.) was changed to hemicellulase, and finally 0.16 kg of mogroside IV was obtained, and the content thereof was 98% or more.

Claims

权利要求书 Claim

1、制备罗汉果苷 IV的方法，其特征在于：采用 β-葡萄糖苷酶水解罗汉果苷 V、罗汉果或含有罗汉果苷 V的罗汉果提取物。 A method for producing mogroside IV, which comprises: hydrolyzing mogroside V, mangosteen or a mangosteen extract containing mogroside V by using β-glucosidase.

2、根据权利要求 1所述的制备罗汉果苷 IV的方法，其特征在于：所述的罗汉果提取物为罗汉果水提取物或乙醇溶液提取物。 The method for preparing mogroside IV according to claim 1, wherein the mogroside extract is an extract of Lo Han Guo water or an ethanol solution.

3、根据权利要求 1或 2所述的制备罗汉果苷 IV的方法，其特征在于：每 kg罗汉果苷 V 采用 5k〜50k万 U β-葡萄糖苷酶水解；其中， β-葡萄糖苷酶在最适 ρΗ值和温度的条件下， lmin水解 lwt%的 β -葡聚糖溶液产生 1 μ g葡萄糖的酶量为 1个 β-葡萄糖苷酶活力单位 U。 The method for preparing mogroside IV according to claim 1 or 2, characterized in that: per kg of mogroside V is hydrolyzed with 5 k to 50 kM U β-glucosidase; wherein β-glucosidase is optimal Under the conditions of pH and temperature, the amount of enzyme which hydrolyzes 1 wt% of β-glucan solution to produce 1 μg of glucose is 1 β-glucosidase activity unit U.

4、根据权利要求 1〜3任一项所述的制备罗汉果苷 IV的方法，其特征在于： β-葡萄糖苷酶的水解时间为 2〜24h。 The method for producing mogroside IV according to any one of claims 1 to 3, wherein the hydrolysis time of the β-glucosidase is 2 to 24 hours.

5、根据权利要求 1〜4任一项所述的制备罗汉果苷 IV的方法，其特征在于：采用 β-葡萄糖苷酶水解罗汉果苷 V或含有罗汉果苷 V的罗汉果提取物的具体步骤如下： The method for producing mogroside IV according to any one of claims 1 to 4, wherein the specific steps of hydrolyzing mogroside V or mogroside extract containing mogroside V by using β-glucosidase are as follows:

6、根据权利要求 5所述的制备罗汉果苷 IV的方法，其特征在于：所述 a步骤中的溶剂为水或与 β-葡萄糖苷酶的酶解 ρΗ值相适应的缓冲液。 The method for preparing mogroside IV according to claim 5, wherein the solvent in the step a is water or a buffer compatible with the enzymatic hydrolysis value of β-glucosidase.

7、根据权利要求 5或 6所述的制备罗汉果苷 IV的方法，其特征在于：所述 b步骤水解时溶液中的 β-葡萄糖苷酶的浓度为 1.5万〜 5.0万 U/mL。 The method for producing mogroside IV according to claim 5 or 6, wherein the concentration of the β-glucosidase in the solution during the step b hydrolysis is 15,000 to 50,000 U/mL.

8、根据权利要求 5所述的制备罗汉果苷 IV的方法，其特征在于：水解时溶液中的罗汉果苷 V浓度 0. lg/mL, β-葡萄糖苷酶浓度为 2. 0万 U/mL，水解时间为 16h。 The method for the preparation of mogroside IV according to claim 5, wherein the concentration of mogroside V in the solution is 0. lg/mL, and the concentration of β-glucosidase is 2,000,000 U/mL. The hydrolysis time was 16 h.

9、根据权利要求 1〜8任一项所述的制备罗汉果苷 IV的方法，其特征在于：所述的 β-葡萄糖苷酶为纤维素酶、 1，4- β -ϋ-葡聚糖葡糖苷水解酶、半纤维素酶中至少一种。 The method for preparing mogroside IV according to any one of claims 1 to 8, wherein the β-glucosidase is cellulase, 1,4-β-ϋ-dextran At least one of a glycoside hydrolase and a hemicellulase.