CN101029071B - Method for preparing high-purity mogroside V from Luo Han Guo - Google Patents

Abstract

The invention discloses a method for preparing high-purity mogroside V from Luo Han Guo, which belongs to the extraction method of high-purity substances. The steps of the method are as follows: (1) crush the Luo Han Guo into coarse powder, add ethanol to reflux for extraction, and filter to obtain the alcoholic extract; (2) suspend the alcoholic extract in water, extract it with ethyl acetate once to three times, Recover the ethyl acetate mother liquor; (3) continue to extract the mother liquor with n-butanol for 1-3 times, and reclaim the n-butanol to obtain the n-butanol extract; (4) pass the n-butanol extract through the polyamide column, and concentrate the washing liquid Obtain crude saponin; (5) carry out silica gel column chromatography for crude saponin, chloroform-methanol-water elution, collect mogroside v crude product; (6) take mogroside v crude product and carry out chromatography, wash with water, ethanol elution, collect purity Greater than 95% mogroside V. The invention simplifies the process and has the advantages of high purity, high yield, low production cost and the like.

Description

Method for preparing high-purity mogroside V from Luo Han Guo

technical field

The invention relates to an extraction method in the field of biotechnology, in particular to a method for preparing high-purity mogroside V from Luo Han Guo.

technical background

Luo Han Guo is the dried and mature fruit of the perennial herbaceous vine Momordicagrosvenori Swingle of the cucurbitaceae plant Luo Han Guo. It is a commonly used traditional Chinese medicine. It has the effects of clearing away heat and relieving heat, moistening the lungs and quenching thirst. , whooping cough, acute and chronic bronchitis and acute and chronic tonsillitis and other diseases. At the same time, Luo Han Guo is also a commonly used dried fruit for beverages, and its extract is a commonly used sweetener, which is exported in large quantities and exported to Southeast Asia, Canada, the United States and other places.

The main components in Luo Han Guo and Luo Han Guo extracts are cucurbitane-type triterpene saponins, including mogroside IV (mogroside IV), mogroside V (mogroside V), siamenoside I (siamenoside I), 11-oxo-mogroside V (11-oxo-mogroside V), mogroside II _E (mogroside II _E ), mogroside III _E (mogroside III _F ) and other saponins, among which mogroside V is the main saponin component. Mogroside V is also the main active ingredient of Luo Han Guo, which has various pharmacological effects such as anti-tumor and hypoglycemic. Mogroside V is also the main sweet component of Luo Han Guo, and its sweetness is high, more than 400 times that of sucrose. Due to the development of the pharmaceutical industry and the food industry, the demand for high-purity Mogroside V is very large at home and abroad, but there is no production and sales of high-purity Mogroside V. Although there are many patent applications related to the extraction of mogroside, the method is simple, but the obtained product is total glycosides of mogroside, and the purity of mogroside V in the extract is low, not exceeding 80%, which limits its application range. Although, the purity of mogroside V in the obtained product is not high, and it is not suitable for preparing mogroside V with a purity of more than 95%.

After searching the existing technical literature, it was found that the Chinese invention patent with the publication number CN1706861A, "Preparation method and application of high-purity mogroside V" disclosed on December 14, 2005, used D101 macropores in the preparation process of the alcoholic extract After resin separation, normal phase silica gel column chromatography was used, and then C18 column chromatography was further used, and the eluate was checked by HPLC. This method uses macroporous resin to remove impurities, and the operation is cumbersome; reversed-phase chromatography uses acetone-water elution, which has certain toxicity; the purity and yield have certain limitations.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing high-purity mogroside V from Luo Han Guo, which simplifies the process and has the advantages of high purity, high yield, and low production cost.

The present invention is realized through the following technical solutions, specifically comprising the following steps:

(1) First crush the Luo Han Guo into a coarse powder, add 5-10 times of 71%-95% ethanol to reflux for extraction for 2h-6h, filter, recover the ethanol to obtain the alcoholic extract. The above operation can be repeated 1-2 times.

(2) Suspend the alcoholic extract in water (the amount of water used is 5-10 times the weight of the alcoholic extract), extract 1-3 times with ethyl acetate saturated with water, each time the volume of ethyl acetate is 0.5-1.0 of the volume of the aqueous solution times, the ethyl acetate mother liquor was recovered, and the ethyl acetate extract was discarded.

(3) Following water-saturated n-butanol extraction 1-3 times the mother liquor of the previous step, each n-butanol volume is 0.5-1.0 times of the aqueous solution volume, and n-butanol is recovered to obtain n-butanol extract.

(4) Pass the n-butanol extract through a polyamide column (the amount of polyamide is 20-60 times the weight of the n-butanol extract), and wash with water until no mogroside V is washed out (detection method: silica gel G thin-layer detection, Developing agent CHCl ₃ -MeOH-H ₂ O 65:30:5, Rf of mogroside V is 0.4). The washing solution was concentrated to obtain crude saponin.

(5) Crude saponin is subjected to silica gel column chromatography (the amount of silica gel used is 30-100 times the weight of crude saponin), chloroform-methanol-water elution (65:30:1～65:30:5), and the mogroside V is collected More than 40% components (detection method: high performance liquid chromatography HPLC method, with octadecyl bonded silica gel as filler; with acetonitrile-water (24:76) as mobile phase; detection wavelength is 210nm, flow velocity 1.0 ml/min), combined, and the solvent was recovered to obtain the crude product of mogroside v.

(6) Take the crude product of mogroside v and carry out C18 reverse phase silica gel column chromatography (the amount of reverse phase silica gel is 50-200 times the weight of the crude mogroside v), wash with water, discard the washing solution, and elute with 20% to 50% ethanol, Collect fractions with a purity above 95% (the detection method is the same as in step (5)). Combining components with the same retention time, recovering the solvent, and obtaining mogroside V with a purity greater than 95%. IR, MS, ¹ H-NMR, ¹³ C-NMR data are consistent with mogroside V, and its structural formula is as follows:

Beneficial effects of the present invention: the Luo Han Guo soap V prepared by separating and preparing through the above method has a high purity of more than 95%, and the production cost is low. Ethyl acetate was used to remove most of the fat-soluble components (such as fatty acids, oils, flavonoids, and phytosterols) in the extract, and the purification process of Luo Han Guo soap V was simplified. Using n-butanol to extract mogrosides from Luo Han Guo avoids the interference of a large amount of sugar in the extract, and also avoids the disadvantages of cumbersome operation with macroporous resin column chromatography; for the remaining water-soluble flavonoids and water-soluble pigments in the extract Impurities such as tannin and tannin are removed by polyamide column chromatography, which further simplifies the process. Reversed-phase column chromatography uses dilute alcohol solution as eluting agent, which simplifies the process and has high purity, high yield and low production cost. Through the comprehensive use of polyamide, silica gel, reversed phase silica gel and other media with different separation principles for purification,

Detailed ways

The embodiments of the present invention are described in detail below: this embodiment is implemented under the premise of the technical solution of the present invention, and detailed implementation methods and processes are provided, but the protection scope of the present invention is not limited to the following embodiments.

Example 1

This embodiment 1 is implemented under the following conditions of implementation and technical requirements:

1. Add 1Kg of Luo Han Guo dried fruit, add 5L of 71% industrial ethanol to reflux for extraction for 2 hours, filter, recover ethanol from the filtrate (can be used again for medicinal material extraction), and obtain 94g of alcoholic extract.

2. Take the alcoholic extract, add 470mL of water to suspend, then extract with 235mL of water-saturated ethyl acetate, separate the ethyl acetate layer, and recover ethyl acetate under reduced pressure (which can be used again for extraction) to obtain ethyl acetate Ester extract 13g.

3. The aqueous solution extracted with ethyl acetate was further extracted with n-butanol saturated with water, and the dosage was 235 mL. The n-butanol was separated, and the n-butanol was recovered under reduced pressure (which could be used for extraction again) to obtain 15 g of n-butanol extract.

4. After dissolving the n-butanol extract in water, pass it through a polyamide column equipped with 300g 100-200 mesh (column diameter 4.5cm, dry column loading, wet loading), and wash until no mogroside V flows out (detection method : Silica gel G thin-layer detection, developing agent CHCl ₃ -MeOH-H ₂ O 65:30:5, Rf of mogroside V is 0.4), collect effluent and washing liquid. The solvent was collected under reduced pressure to obtain 12.5 g of crude saponin.

5. The crude saponin was subjected to silica gel column chromatography (silica gel dosage 375g, 300-400 mesh; column diameter 4.5cm, dry packing, dry loading), eluted with chloroform-methanol-water (65:30:1) , flow rate 5ml/min, every part collects 50ml, collects the component that contains mogroside V more than 40% (detection method: high performance liquid chromatography HPLC method, with octadecyl bonded silica gel as filler; with acetonitrile-water 24:76 is the mobile phase; the detection wavelength is 210nm, the flow rate is 1.0ml/min), combined, and the solvent is recovered under reduced pressure to obtain 2.2g of crude mogroside v.

6. Take the crude product of mogroside v for reverse-phase column chromatography, use 110 g of reverse-phase silica gel (silica gel particle size: 400-600 mesh; column diameter: 4.5 cm, wet column loading, wet loading), wash with water, discard the water wash liquid; 20% alcohol washing, flow rate 2ml/min, every part collects 10ml, carries out high-performance liquid chromatography (HPLC) detection (detection method is the same as 5) to each fraction, merges the components with the same retention time and purity greater than 95%, Concentrate under reduced pressure to obtain 464 mg of mogroside V with a purity greater than 95%, and its purity is 96.7% after testing.

Example 2

This embodiment 2 is implemented under the following conditions of implementation and technical requirements:

1. Add 1Kg of Luo Han Guo dried fruit, add 10L of 95% industrial ethanol to reflux and extract for 6 hours, filter, and recover ethanol from the filtrate (which can be used again for medicinal material extraction) to obtain an extract. Repeat the above operation 2 times to obtain 168g of alcoholic extract.

2. Take the alcoholic extract, add 1680mL of water to suspend it, then extract with 1680mL of water-saturated ethyl acetate, separate the ethyl acetate layer, and reclaim the ethyl acetate under reduced pressure (which can be used again for extraction) to obtain the extract. Repeat the above operation 2 times, combine the extracts to obtain 21g of ethyl acetate extract.

3. The aqueous solution extracted with ethyl acetate was further extracted with n-butanol saturated with water, and the dosage was 1680 mL. The n-butanol is separated, and the n-butanol is recovered under reduced pressure (which can be used for extraction again) to obtain an extract. Repeat the above operation 2 times, and combine the extracts to obtain 29g of n-butanol extract.

4. After dissolving the n-butanol extract in water, pass through a 60-100 mesh polyamide column equipped with 1740g (column diameter 8.0cm, wet packing, wet loading), and wash until no mogroside V flows out (detection Method: Silica gel G thin-layer detection, developing agent CHCl ₃ -MeOH-H ₂ O 65:30:5, Rf of mogroside V is 0.4), collect effluent and washing liquid. The solvent was collected under reduced pressure to obtain 21 g of crude saponin.

5. The crude saponin is subjected to silica gel column chromatography, the amount of silica gel is 2100g (column diameter 8.0cm, 100-200 mesh; wet packing, wet loading), chloroform-methanol-water (65:30:5) wash Remove, flow rate 10ml/min, collect 1 part per 100ml, collect mogroside V containing more than 40% components (detection method: high performance liquid chromatography (HPLC) method, using octadecyl bonded silica gel as filler; Acetonitrile-water 24:76 was used as the mobile phase; the detection wavelength was 210nm, and the flow rate was 1.0ml/min), combined, and the solvent was recovered under reduced pressure to obtain 3.4g of crude mogroside v.

6. Take the crude product of mogroside v for reverse-phase column chromatography, the amount of reverse-phase silica gel used is 680g (silica gel particle size: 300-400 mesh; column diameter 6.5cm, wet packing, wet loading), wash with water, discard Washing liquid; 50% alcohol washing, flow rate 5ml/min, every 30ml collects 1 part, carries out high-performance liquid chromatography (HPLC) detection (detection method is the same as 5) to each fraction, merges the groups with the same retention time and purity greater than 95% points, concentrated under reduced pressure to obtain 623 mg of high-purity mogroside V, and its purity was 95.3% after testing.

Example 3

This embodiment 3 is implemented under the following conditions of implementation and technical requirements:

1. Add 1Kg of Luo Han Guo dried fruit, add 7.5L of 80% industrial ethanol to reflux for extraction for 4 hours, filter, and recover ethanol from the filtrate (which can be used again for medicinal material extraction) to obtain an extract. Repeat the above operation once to obtain 133g of alcoholic extract.

2. Take the alcoholic extract, add 998mL of water to suspend it, then extract it with 749mL of water-saturated ethyl acetate, separate the ethyl acetate layer, and reclaim the ethyl acetate under reduced pressure (which can be used again for extraction) to obtain the extract. Repeat the above operation once to obtain 18 g of ethyl acetate extract.

3. The aqueous solution extracted with ethyl acetate was further extracted with n-butanol saturated with water, and the dosage was 749 mL. Isolate n-butanol, recover n-butanol (can be used for extraction again), repeat the above operation once, and combine the extracts to obtain 21 g of n-butanol extract.

4. After dissolving the n-butanol extract in water, wash it with water until no mogroside V flows out (detection method TLC detection Silica gel G TLC detection, developer CHCl ₃ -MeOH-H ₂ O (65:30:5, Rf of mogroside V is 0.4), collect the effluent and washing liquid. The solvent is collected under reduced pressure to obtain crude Saponin 17g.

5. The crude saponin is subjected to silica gel column chromatography, the silica gel is 200-300 mesh, the dosage is 1105g (the column diameter is 6.5cm, the column is packed by wet method, and the column is loaded by dry method), and washed with chloroform-methanol-water (65:30:3) Remove, flow rate 5ml/min, collect 1 part per 50ml, collect mogroside V containing more than 40% components (detection method: high performance liquid chromatography (HPLC) method, use octadecyl bonded silica gel as filler; Acetonitrile-water 24:76 was used as the mobile phase; the detection wavelength was 210nm, and the flow rate was 1.0ml/min), combined, and the solvent was recovered under reduced pressure to obtain 2.9g of crude mogroside v.

6. Take the crude product of Mogroside V and perform reverse-phase column chromatography. The amount of reverse-phase silica gel used is 362.5g (silica gel particle size: 200-300 mesh; column diameter 6.0cm, wet column loading, wet loading), wash with water, discard 35% alcohol wash, flow rate 3ml/min, collect 1 part per 20ml, carry out high performance liquid chromatography (HPLC) detection (detection method is the same as 5) to each fraction, merge retention time identical and purity greater than 95% Components were concentrated under reduced pressure to obtain 561 mg of high-purity mogroside V, and its purity was 96.1% after testing.

Example 4

This embodiment 4 is implemented under the following conditions of implementation and technical requirements:

1. Add 1Kg of Luo Han Guo dried fruit, add 5L of 95% industrial ethanol to reflux and extract for 3 hours, filter, and recover ethanol from the filtrate (which can be used again for medicinal material extraction) to obtain an extract. Repeat the above operation once to obtain 86g of alcoholic extract.

2. Take the alcohol extract, add 860mL of water to suspend it, then extract it with 860mL of water-saturated ethyl acetate, separate the ethyl acetate layer, and reclaim the ethyl acetate under reduced pressure (which can be used again for extraction) to obtain the extract. Repeat the above operation once to obtain 15 g of ethyl acetate extract.

3. The aqueous solution extracted with ethyl acetate was further extracted with n-butanol saturated with water, and the dosage was 430 mL. Isolate n-butanol, recover n-butanol (can be used for extraction again), repeat the above operation once, and combine the extracts to obtain 20 g of n-butanol extract.

4. After dissolving the n-butanol extract in water, pass through a polyamide column equipped with 960g 60-100 mesh (column diameter 6.5cm, dry column loading, wet loading), and wash until no mogroside V flows out (detection method TLC detection Silica gel G TLC detection, developer CHCl ₃ -MeOH-H ₂ O (65:30:5, Rf of mogroside V is 0.4), collect the effluent and washing liquid. The solvent is collected under reduced pressure to obtain crude Saponin 14.5g.

5. The crude saponin is subjected to silica gel column chromatography, the silica gel is 200-300 mesh, the dosage is 1150g (column diameter 6.5cm, wet packing, dry loading), chloroform-methanol-water (65:30:3) wash Remove, flow rate 5ml/min, collect 1 part per 50ml, collect mogroside V containing more than 40% components (detection method: high performance liquid chromatography (HPLC) method, use octadecyl bonded silica gel as filler; Acetonitrile-water 24:76 was used as the mobile phase; the detection wavelength was 210nm, and the flow rate was 1.0ml/min), combined, and the solvent was recovered under reduced pressure to obtain 2.5g of crude mogroside v.

6. Take the crude product of mogroside v for reverse-phase column chromatography, the amount of reverse-phase silica gel is 300g (silica gel particle size: 200-300 mesh; column diameter 6.0cm, wet packing, wet loading), wash with water, discard Washing liquid; 40% alcohol washing, flow rate 3ml/min, every 20ml collects 1 part, carries out high-performance liquid chromatography (HPLC) detection (detection method is the same as 5) to each fraction, merges the groups with the same retention time and purity greater than 95% points, concentrated under reduced pressure to obtain 525 mg of high-purity mogroside V, and its purity was 97.1% after testing.

Claims (9)

1. A method for preparing high-purity mogroside V from Momordica grosvenori, is characterized in that, specifically comprises the steps: (1) First crush the Momordica grosvenori into coarse powder, add 5-10 times of 70%-95% ethanol to reflux for extraction for 2h-6h, filter, recover ethanol to obtain alcoholic extract; (2) Suspend the alcoholic extract in water, extract 1-3 times with ethyl acetate saturated with water, each time the volume of ethyl acetate is 0.5-1.0 times the volume of the aqueous solution, and reclaim the ethyl acetate mother liquor; (3) Following water-saturated n-butanol extraction 1-3 times the mother liquor of the previous step, each n-butanol volume is 0.5-1.0 times of the aqueous solution volume, and n-butanol is recovered to obtain n-butanol extract; (4) passing the n-butanol extract through a polyamide column, washing with water until no mogroside V is washed out, and concentrating the washing solution to obtain crude saponin; (5) The crude saponins were subjected to silica gel column chromatography, eluted with chloroform-methanol-water, and the components containing more than 40% mogroside V were collected, combined, and the solvent was recovered to obtain the crude product of mogroside V; (6) Take the crude product of mogroside v and perform C18 reversed-phase silica gel column chromatography, wash with water, and elute with ethanol, collect fractions with a purity of more than 95%, combine components with the same retention time, and recover the solvent to obtain a purity greater than 95%. Mogroside V. 2. the method for preparing high-purity mogroside V from Momordica grosvenori as claimed in claim 1, is characterized in that, the alcohol extract described in step (2) is suspended in water, and water consumption is 5-5% of the alcohol extract weight. 10 times. 3. the method for preparing high-purity mogroside V from Momordica grosvenori as claimed in claim 1, is characterized in that, n-butanol extractum described in step (4) passes through polyamide column, and the consumption of polyamide is n-butanol 20-60 times the weight of the extract. 4. The method for preparing high-purity mogroside V from Luo Han Guo as claimed in claim 1, characterized in that, in step (4), the detection condition for detecting the washing with water until no mogroside V is washed out is: use silica gel G For thin-layer detection, CHCl ₃ -MeOH-H ₂ O was selected as the developer, the ratio was 65:30:5, and the Rf of mogroside V was 0.4. 5. the method for preparing high-purity mogroside V from Momordica grosvenori as claimed in claim 1, is characterized in that, in the silica gel column described in step (5), the silica gel consumption is 30-100 times of thick saponin weight; Chloroform- The ratio of methanol-water elution is 65:30:1-65:30:5. 6. The method for preparing high-purity mogroside V from Momordica grosvenori as claimed in claim 1, characterized in that, in step (5), the components containing more than 40% mogroside V are collected, and the collection and detection method is selected from high-efficiency liquid phase chromatography. 7. the method for preparing high-purity mogroside V from Luo Han Guo as claimed in claim 6, is characterized in that, the detection condition of described high performance liquid chromatography is: take octadecyl bonded silica gel as filler, with Acetonitrile-water at a ratio of 24:76 was used as the mobile phase, the detection wavelength was 210 nm, and the flow rate was 1.0 ml/min. 8. The method for preparing high-purity mogroside V from Luo Han Guo according to claim 1, characterized in that the ethanol concentration in step (6) is 20%-50%. 9. The method for preparing high-purity mogroside V from Luo Han Guo according to any one of claims 1 to 8, characterized in that step (1) can be repeated 1-2 times.