CN1324043C - Prepn and use of high-purity momordica glycoside V - Google Patents

Abstract

The present invention relates to a preparation method of high-purity mogroside V, which is characterized in that: firstly, the mogroside V is crushed, heated and refluxed with ethanol for extraction, and then the extract is separated by a D101 macroporous resin column, and deionized water and ethanol are separated Eluted to colorless, then collected the eluate, concentrated under reduced pressure to an extract, then separated with a normal phase silica gel column, added chloroform, methanol, and water as eluents, collected the eluate, concentrated under reduced pressure, and then Separation with a reverse-phase silica gel C-18 column, and use HPLC detection for each eluate collected, and combine mogroside V components with the same retention time and a purity between 90% and 98% and above 98% , and concentrated under reduced pressure to obtain mogroside V with a purity between 90% and 98% and mogroside V components with a purity greater than 98%. The production method can not only improve the purity of mogroside V, but also reduce the production cost.

Description

Preparation method and application of high-purity mogroside V

technical field

The invention relates to a method for preparing mogroside V, in particular to a method for preparing mogroside V with a purity greater than 98%.

Background technique

Luo Han Guo (Momordica grosvenori swingle), which belongs to the fruit of perennial herbaceous vines of Cucurbitaceae, is a unique plant in southern my country. It is mainly produced in northern Guangxi, Guangxi, and a small amount of wild in some subtropical areas south of the Yangtze River, but in Guilin, Guangxi Luo Han Guo in the region is the best. In the past, Luo Han Guo has been used as traditional Chinese medicine and dried fruit for a long time. Luo Han Guo has the effects of quenching thirst, reducing heat and heat, relieving cough and reducing phlegm, cooling blood and nourishing lungs, and lowering blood pressure. It can be used to treat tuberculosis, asthma, gastritis, pertussis, acute and chronic Tracheitis and acute and chronic tonsillitis and other diseases. In recent years, people have not only studied the strong sweet substance mogroside V in Luo Han Guo as a sweetener, but also paid attention to its pharmacology.

Public literature reports many methods of extracting mogrosides, such as solvent extraction, resin adsorption, microwave extraction, and microporous membrane filtration, such as public literature reports: 1. [Title] Research on the Extraction Process of Mogrosides [Author] Li Yanqun Wang Ce 【Title】Research and Development of Natural Products. 1995, 7(4). 【Abstract】The saponin with strong sweet taste and health care effect was extracted from Luo Han Guo. Experiments have proved that Ca(OH)2 is a good clarifier, D280, D290, D61 and D151 three domestic separation resins have good decolorization ability, and AB08 adsorption resin can effectively separate this saponin. The yield of saponin using water as a solvent is higher than that of ethanol aqueous solution, and the amount of solvent is 6 times of the weight of the raw material. Ca(OH) ₂ is more effective than alum and AlCl ₃ as clarifier, and it will not cause a great loss of saponin. Clarification should be carried out at room temperature. The decolorization effect of strong base resin D290 and D280 is better than that of acid resin, and the decolorization effect of D280 is better than that of D290. The separation and decolorization should be operated at 25°C. The operation of A8-8 adsorption resin to adsorb mogroside should be operated at a flow rate of SV2 at a room temperature of about 20°C. Mogroside can be effectively desorbed from AB-8 adsorption resin with 50% ethanol aqueous solution. 2. [Title] Study on the Extraction Technology of Mogroside by Orthogonal Method [Author] Li Jun, Lu Cheng, Guangxi Normal University [Title] Chemical World. 1999, 40(2).-92-94 [Abstract] The ethanol extraction process of mogroside in Luo Han Guo was systematically studied by using an orthogonal test design, and the process parameters were optimized. The results showed that: using 30% ethanol 30 times the weight of the raw material, extracting at 75-80 ° C for 3 hours in a slightly boiling state Optimal conditions. 3.【Title】Study on the refinement of mogroside (V)【Author】Liu Zhongdong, Zhengzhou Grain Institute 【Title】Ion Exchange and Adsorption. 1999, 15(4). 【Abstract】Using domestic resin to study mogroside (V) The refining method, the effect of concentration and pH on the refining process and the physical, chemical and spectral properties of the product were studied. 4. Mogroside extraction process, Chinese Patent: Application (Patent) No.: 02128065.7 Applicant (Patent Right): Guilin Hongye Green Plant Products Co., Ltd. Address: No. 6, Shijiayuan Road, Qixing District, Guilin City, Guangxi Zhuang Autonomous Region Abstract: The invention is to crush the Luo Han Guo, add ethanol solution, and soak for 5-15 hours at a temperature of 30-60°C. The filtrate is filtered, the temperature drops to 20-35°C, and the filtrate is sequentially sent to three membrane separation modules through a pressure pump at a working pressure of 0.01-0.2 MPa; the first stage intercepts and separates the concentrated Luo Han Guo fiber, protein, pectin and impurities , used to make organic fertilizers; the second stage is concentrated mogroside solution, which is vacuum dried to obtain mogroside products; the third stage is concentrated mogroside polysaccharides and trace elements, which is mogroside sugar paste; the remaining filtrate is recovered and recycled . 5. A method for extracting mogroside from Luo Han Guo Chinese patent: application (patent) number: 03117430.2, applicant (patent right): Guilin Laiyin Biological Products Co., Ltd. Address: No. 18, Xiangjiang Road, Xing'an County, Guilin City, Guangxi Zhuang Autonomous Region Abstract: The present invention relates to a method for extracting mogroside from Luo Han Guo, which uses fresh Luo Han Guo as the raw material, uses water as the extraction solvent, and adopts microfiltration, ultrafiltration and nanofiltration as the main extraction processes, wherein the microfiltration membrane adopts 0.05 Microporous membrane with pore size of μm～0.3μm; ultrafiltration membrane adopts hollow fiber ultrafiltration membrane with cut-off relative molecular weight of 20000-80000; nanofiltration adopts nanofiltration membrane with cut-off relative molecular weight of 100-400 or RO reverse osmosis equipment for concentration . Since this method is operated under low temperature conditions, the color, solubility, clarity and taste of the product are better. In addition, the membrane separation technology is a high-precision molecular screening, which avoids the loss of active ingredients, reduces energy consumption and cost, improved product yield and quality, and this method integrates microfiltration, ultrafiltration, and nanofiltration technologies for membrane separation, using fresh fruits as raw materials and water as the extraction solvent to form an advanced separation and purification process to complete mogroside The separation and concentration of simplifies the production process and shortens the production cycle. 6. Name: Method for Extracting and Separating Multiple Components from Luo Han Guo Application (Patent) No.: 02113548.7 Applicant (Patent Right) Person: He Weiping Address: Guilin Strength Company, Bali Street Economic and Technological Development Zone, Guilin City, Guangxi Zhuang Autonomous Region Abstract: A method from The method of extracting and separating various components from Luo Han Guo is to treat the broken Luo Han Guo with water and then alkaline water to obtain the extract, and then treat it with fatty alcohol to obtain the precipitate (I) and the solution (II). The precipitate (I) is anionized Resin column combined with fatty alcohol treatment and purification to obtain Luo Han Guo polysaccharide. The solution (II) is passed through a polyamide resin column, and the effluent from the column is purified through a macroporous resin column combined with fatty alcohol to obtain mogroside. The components adsorbed by the polyamide resin column are washed out with alkaline fatty alcohol, concentrated and dried to obtain Luo Han Guo flavonoids. All three extracted components are suitable for health products and food additives. 7. 【Title】Microwave-assisted extraction of mogrosides 【Author】 Li Haibin Li Lin et al. 【Institution】School of Food and Bioengineering, South China University of Technology, 【Title】Food Science. 2003, 24(2).-92-95【 Keywords: Microwave-assisted extraction of mogrosides from Luo Han Guo Process conditions Medicinal edible 【Abstract】Using dried Luo Han Guo as raw material, under microwave radiation conditions, water as solvent to extract mogrosides. The microwave power, microwave radiation time, solid-liquid ratio, Extraction time, the influence of factors such as extraction stages on the extraction rate, the determined optimal extraction process conditions are: microwave power is 638W, microwave radiation time is 25min, solid-liquid ratio is 1:30, water bath extraction time is 2h, The extraction level is two levels. Under these conditions, the extraction rate of mogroside was 90.35%. 8. [Title] Study on the Extraction of Mogroside by Macroporous Adsorbent Resin [Author] Li Haibin, Li Lin et al. [Institution] School of Food and Bioengineering, South China University of Technology, [Title] Food Industry Science and Technology. 2003, 24(2).-19 -21 [Abstract] The method of extracting mogroside by macroporous adsorption resin was studied, including the screening of resin, the concentration of mogroside solution, the influence of pH and salt ion concentration on the adsorption process and the selection of desorbent, and the suitable adsorption and Desorption conditions. 9. 【Title】The application of microwave technology in the extraction of fresh mogrosides 【Author】Zhu Xiaoyun, He Chaowen 【Organization】Guangxi Guilin Shili Natural Food Co., Ltd., College of Biotechnology and Sugar Engineering, Guangxi University 【Publication Title】Guangxi Light Industry.2002 (2). [Abstract] The influence of the ratio of raw material to liquid, microwave output power, and extraction time on the extraction efficiency of Mogroside in the process of extracting mogroside by microwave technology was investigated by orthogonal experiment method, and the optimal scheme of microwave extraction was optimized. This program was used as the experimental group, and the conventional boiling method was used as the control group to carry out parallel experiments. The results showed that the efficiency of microwave extraction of mogroside was significantly better than that of conventional boiling method. It is a new extraction method that saves time, energy and is easy to operate. 10. 【Title】Preparation of Mogroside V Standard by High Performance Liquid Chromatography 【Authors】Yu Lijuan, Chen Quanbin, Yi Xianghui, Yang Ruiyun, Zhang Yizheng 【Organization】Department of Resources and Environment, Guangxi Normal University, Sichuan University Molecular Biology and Biology Technical Key Laboratory [Public Name] Chromatography. 2003 (7). [Abstract] In order to further study the pharmacological effects of functional components in Luo Han Guo and further develop the active ingredients in Luo Han Guo, the water extract of Luo Han Guo fresh fruit was adsorbed with AB-8 After resin separation and D-280 ion exchange resin decolorization, semi-preparative high-performance liquid chromatography was used to obtain the standard product of triterpenoid saponin compound mogroside V with a purity of 98.5%. The chromatographic column is Alltech Econosphere NH ₂ column; the mobile phase is acetonitrile-water (68:32 by volume) solution, the flow rate is 5mL/min; the detection wavelength is 203nm; the column temperature is 40°C. This method has the advantages of simple operation, good reproducibility and high product purity.

The above-mentioned methods discuss the extraction and separation of mogrosides (glucosides) from different angles. Some of them have high separation purity and simple methods, but some have complicated operation techniques and cannot be industrialized. Moreover, due to the high polarity of mogroside, it is not easy to separate from the pigment. For the preparation of high-purity mogroside, there is currently a semi-preparative high-performance liquid chromatography method for separation. Although the purity of mogroside V obtained by this method is very high, the instruments used are relatively expensive and the production components are relatively high, which is not suitable for Because of the production of mogroside V with a purity greater than 90% in large quantities.

Contents of the invention

In order to overcome the existing disadvantages of low purity, low yield or high production cost of mogroside V, the present invention provides a method for preparing high-purity mogroside V, which can not only improve the purity of mogroside V, but also Can reduce production cost.

The technical solution adopted by the present invention to solve its technical problems is:

(1) First crush the Luo Han Guo, heat and reflux extract with 20%-70% ethanol for 8-18h, filter the extract, add 30%-60% ethanol to heat and reflux for extraction for 4-8h, combine the extracts and Concentrate under reduced pressure to a paste to obtain A;

(2) Separate A with a D101 type macroporous resin column, first elute with deionized water and 20% to 30% ethanol until colorless, then elute with 30% to 50% ethanol eluent and collect the eluted Deliquified, concentrated under reduced pressure to an extract to obtain B;

(3) In the normal phase silica gel column separation, use chloroform-methanol-water (60:40:1～60:40:10) as the eluent to make the collected eluent easier to recover; For separation on a silica gel C-18 column, acetone-water ratio of 25:75 to 35:65 is used as the eluent. The solvent used is relatively low in toxicity, and general industrial grade acetone can be used, so the production cost is relatively low.

The HPLC detection chromatographic column mentioned above is an amino column; mobile phase: EtCN:H2O=75:25～60:40; detection wavelength: 203nm; flow rate: 0.6ml/min～1ml/min.

The beneficial effects of the present invention are: the mogroside standard product is obtained by separating and obtaining the mogroside V by the above method, which can not only improve the purity of mogroside V, but also reduce the production cost, the method is simple and easy, and the product has good purity. This provides convenience for the study of the activity of mogroside V monomer and the pharmacological process in vivo, and also provides a useful reference for the preparation of other Mogroside triterpene glycoside standard products. At the same time, the determination of the content of mogroside V in the fruit is of great significance for determining the quality of Luo Han Guo, guiding the purchase of Luo Han Guo and the production of mogroside products.

The high-purity mogroside V of the present invention can be used not only as a standard product, but also as a high-grade sweetener. The sweet substance with extremely high sweetness is a low-calorie ideal natural sweetener.

Detailed ways

Embodiment one: (1). Get 5 kg of Luo Han Guo, pulverize, use 20% ethanol 5000ml to heat and reflux extract for 10h, filter the extract, add 60% ethanol to the dregs and add 60% ethanol for 4000ml to heat and reflux to extract for 6h, combine the extract and concentrate under reduced pressure to Paste, get A.

(2). Separate A with a D101 type macroporous resin column (about 15 cm in inner diameter, 5 kg of D101 type macroporous resin), first deionized water and 20% ethanol were eluted to colorless, and then 35% ethanol eluent Eluted and collected the eluate, concentrated under reduced pressure to extract, and B was obtained.

(3). Separate B with a normal-phase silica gel column (about 10 cm in inner diameter, 4 kg of silica gel, dry packing, dry loading), and use chloroform-methanol-water (60:40:1) as the eluent to collect The eluate of the spot with Rf=0.23 (the stationary phase is a normal phase plate, and the developing solvent is n-butanol-acetic acid-water=4:1:1) is concentrated under reduced pressure to obtain C.

(4). Separate C with a reverse-phase silica gel C-18 column (about 5cm in inner diameter, reverse-phase silica gel C-180.5kg, wet packing, wet loading), and acetone-water (25:75) is the elution reagent, collect the eluate of the spot with Rf=0.23 (normal phase plate, n-butanol-acetic acid-water=4:1:1), concentrate under reduced pressure, and obtain mogroside V between 85% and 98%.

Embodiment two: (1). Get grosvenoria grosvenori 4kg, pulverize, use 30% ethanol 5000ml to heat and reflux extract for 16h, filter the extract, add 35% ethanol to the dregs and add 35% ethanol to heat and reflux to extract for 6h, combine the extract and concentrate under reduced pressure to Paste, get A.

(2). Separate A with a D101 type macroporous resin column (about 15 cm in inner diameter, 5 kg of D101 type macroporous resin), first deionized water and 25% ethanol were eluted to colorless, and then 40% ethanol eluent Eluted and collected the eluate, concentrated under reduced pressure to extract, and B was obtained.

(3). Separate B with a normal-phase silica gel column (about 10 cm in inner diameter, 4 kg of silica gel, dry packing, dry loading), and use chloroform-methanol-water (60:40:5) as the eluent to collect The eluate of the spot with Rf=0.23 (the stationary phase is a normal phase plate, and the developing solvent is n-butanol-acetic acid-water=4:1:1) is concentrated under reduced pressure to obtain C.

(4). Separate C with a reversed-phase silica gel C-18 column (about 5 cm in inner diameter, 0.5 kg of reversed-phase silica gel C-18, wet packing, wet loading), and acetone-water (30:70) as the washing Remove the reagent, collect the eluate of the spot with Rf=0.23 (normal phase plate, n-butanol-acetic acid-water=4:1:1), concentrate under reduced pressure, and obtain mogroside V between 85% and 98%. .

Embodiment three: (1). Get Luo Han Guo 5kg, pulverize, use 65% ethanol 5000ml to heat and reflux extract for 10h, filter the extract, add 55% ethanol to the dregs and add 5000ml of ethanol to heat and reflux for 5h, combine the extract and concentrate under reduced pressure to Paste, get A.

(2). Separate A with a D101 type macroporous resin column (about 15 cm in inner diameter, 5 kg of D101 type macroporous resin), first deionized water and 30% ethanol were eluted to colorless, and then 50% ethanol eluent Eluted and collected the eluate, concentrated under reduced pressure to extract, and B was obtained.

(3). Separate B with a normal-phase silica gel column (about 10 cm in inner diameter, 4 kg of silica gel, dry packing, dry loading), and use chloroform-methanol-water (60:40:3) as the eluent to collect The eluate of the spot with Rf=0.23 (the stationary phase is a normal phase plate, and the developing solvent is n-butanol-acetic acid-water=4:1:1) is concentrated under reduced pressure to obtain C.

(4). Separate C with a reverse-phase silica gel C-18 column (about 5 cm in inner diameter, 0.5 kg of reverse-phase silica gel C-18, wet packing, wet loading), and acetone-water (30:70) Remove the agent, collect the eluate of the spot of Rf=0.23 (normal phase plate, n-butanol-acetic acid-water=4:1:1), and utilize HPLC detection (chromatographic column : amino column; mobile phase: EtCN: H ₂ O = 75: 25; detection wavelength: 203nm; flow rate: 1ml/min), the mogroside V components with the same retention time and a purity greater than 98% were combined, concentrated under reduced pressure, Mogroside V with a purity of more than 98% is obtained.

Embodiment four: (1). Get grosvenoria grosvenori 4kg, pulverize, use 45% ethanol 5000ml to heat and reflux extract 7h, filter the extract, add 65% ethanol 5000ml to heat and reflux extract 7h for the dregs, combine the extract and concentrate under reduced pressure to Paste, get A.

(2). Separate A with a D101 type macroporous resin column (about 15 cm in inner diameter, 5 kg of D101 type macroporous resin), first deionized water and 20% ethanol were eluted to colorless, and then 40% ethanol eluent Eluted and collected the eluate, concentrated under reduced pressure to extract, and B was obtained.

(3). Separate B with a normal-phase silica gel column (about 10 cm in inner diameter, 5 kg of silica gel, dry packing, dry loading), and use chloroform-methanol-water (60:40:2) as the eluent to collect The eluate of the spot with Rf=0.23 (the stationary phase is a normal phase plate, and the developing solvent is n-butanol-acetic acid-water=4:1:1) is concentrated under reduced pressure to obtain C.

(4). Separate C with a reversed-phase silica gel C-18 column (about 5 cm in inner diameter, 0.5 kg of reversed-phase silica gel C-18, wet packing, wet loading), and acetone-water (27:73) as the washing Remove the agent, collect the eluate of the spot of Rf=0.23 (normal phase plate, n-butanol-acetic acid-water=4:1:1), and utilize HPLC detection (chromatographic column : amino column; mobile phase: EtCN: H ₂ O = 60: 40; detection wavelength: 203nm; flow rate: 0.6ml/min), the mogroside V components with the same retention time and a purity greater than 98% were combined and concentrated under reduced pressure , to obtain mogroside V with a purity of more than 98%.

Claims (2)

1. the preparation method of a high-purity momordica glycoside V is characterized in that:

(1) earlier Grosvenor Momordica is pulverized, with the alcohol heating reflux extraction 8-18h of 20%-70%, leaching extracting solution, the dregs of a decoction add 30%～60% alcohol heating reflux extraction 4-8h again, and united extraction liquid also is evaporated to paste, obtains A;

(2) A is separated with D101 type macroporous resin column, is eluted to earlier colourlessly with deionized water and 20%～30% ethanol respectively,, be evaporated to the medicinal extract shape again with 30%～50% ethanol elution agent wash-out and collect its elutriant, B;

(3) B is separated with normal phase silicagel column, with chloroform: methyl alcohol: water is eluent according to 60: 40: 1～60: 40: 10 ratio, collects Rf=0.23, stationary phase is positive phase-plate, developping agent is the elutriant of the spot of propyl carbinol-acetic acid-water=4: 1: 1, and concentrating under reduced pressure obtains C;

(4) C is separated with reverse phase silica gel C-18 post, acetone-water is an eluent according to 25: 75～35: 65 ratio, collect Rf=0.23, stationary phase is positive phase-plate, developping agent is the elutriant of the spot of propyl carbinol-acetic acid-water=4: 1: 1, and utilize HPLC to detect to collected each part elutriant, merge the identical and purity of retention time in the momordica glycoside V component between 90%～98% or more than 98%, concentrating under reduced pressure, can obtain respectively purity between 90%～98% momordica glycoside V or purity greater than 98% momordica glycoside V component.

2. the preparation method of high-purity momordica glycoside V according to claim 1 is characterized in that: it is nh 2 column that HPLC detects chromatographic column; Moving phase: EtCN: H ₂O=75: 25～60: 40; Detect wavelength: 203nm; Flow velocity: 0.6ml/min～1ml/min; Obtain purity in the momordica glycoside V between 90%～98% or purity at the momordica glycoside V more than 98%.