CN102260304B - Method for purifying rebaudioside A - Google Patents

Abstract

The invention provides a method for refining and purifying rebaudioside A, comprising the following steps: dispersing the raw material containing rebaudioside A in a solvent system composed of absolute ethanol, acetone and water, and collecting insoluble matter by filtration to obtain Crude rebaudioside A: disperse the crude rebaudioside A in a solvent system composed of absolute ethanol and water, filter and recrystallize the filtrate, collect crystals, and dry to obtain purified rebaudioside A. The method of the invention has simple steps, has the advantages of low production cost, high conversion rate and environmental protection, and is suitable for industrial production.

Description

A method for refining and purifying rebaudioside A

technical field

The application relates to the field of pharmacognosy, in particular to a method for extracting and purifying crude drugs, especially a method for refining and purifying rebaudioside A from stevioside.

Background technique

Stevia sugar is a high-sweetness and low-calorie non-nutritive natural sweetener extracted from stevia leaves. At present, eight kinds of glycosides have been discovered from stevioside, namely stevioside (slevioside), rebaudioside (Rebaudioside A, B, C, D, E), dulcoside A (Dul～ide and Si Teviboside (steviobi～siti). Among them, rebaudioside A (hereinafter referred to as RA) has the highest sweetness, 300 times greater than sucrose, and the best taste without any bad aftertaste. It is the most ideal natural sweetener.

The extraction and preparation of stevioside products with high rebaudioside A content is a research hotspot in the stevioside production industry at home and abroad.

In the existing patented technology, the disclosed methods for obtaining high-purity RA are as follows:

The method of the publication number CN101270138A starts from stevia, and obtains RA crystals with a purity greater than 90%-98% through steps such as extraction, resin separation, decolorization, crystallization, recrystallization, and washing.

The first method of the publication number CN101200480A starts from stevioside, through silica gel chromatography, RA-containing fractions are combined, concentrated, and crystallized to obtain RA with a purity ≥ 99%.

The second method is to obtain RA with a purity of ≥99% from stevia rebaudiana through extraction, ultrafiltration, resin separation and crystallization.

The method of Publication No. CN101100477A starts from stevioside containing 30% RA, crystallizes and recrystallizes with methanol and isopropanol (volume ratio 99:1), and obtains RA with a purity >90% through membrane separation.

There are also some deficiencies in the above method. These methods or many steps are not conducive to industrialized production; or the purity of RA obtained is not high, only >90%, and the solvents used at the same time are not conventional industrially produced solvents, such as methanol is toxic, and isopropanol is very expensive. Use solvents.

Contents of the invention

In order to improve the production process of RA, make its steps simple, and have the advantages of low production cost, high conversion rate and environmental protection, etc., and propose the present invention. The method of the invention is suitable for industrial production.

According to the technical scheme provided by the present invention, the method for refining and purifying rebaudioside A from stevioside comprises the following steps:

- Disperse the raw material containing rebaudioside A in a solvent system composed of absolute ethanol, acetone and water, and collect the insoluble matter by filtration to obtain the crude product of rebaudioside A;

- Disperse the crude rebaudioside A in a solvent system composed of absolute ethanol and water, filter and recrystallize the filtrate, collect the crystals, and dry to obtain purified rebaudioside A.

The volume ratio of various solvents in the solvent system composed of described dehydrated alcohol, acetone and water is: 3～8 parts of dehydrated alcohol: 5～10 parts of acetone: 0.4～0.8 part of water; preferably, in the described solvent system The volume ratio of various solvents is: 4-6 parts of absolute ethanol: 6-8 parts of acetone: 0.4-0.6 parts of water. Under the preferred ratio, the product can have higher conversion rate and purity. Unless otherwise specified, the conversion rate in the present invention=the final product quality/theoretical content of the product in the raw material×100%, and the purity=pure product quality/final product quality×100%.

According to a preferred solution of the present invention, there is a fixed proportional relationship between the amount of the raw material containing rebaudioside A and the above-mentioned solvent system. In the ratio relationship, the raw material is calculated by mass, and the solvent is calculated by volume. The mass unit is mg and volume The unit ml is equivalent, for example, when 1 part of raw material refers to 1 mg of raw material, 1 part of solvent refers to 1 ml of solvent. The specific proportioning of this preferred scheme is: 1 part of raw material: 3 to 8 parts of absolute ethanol: 5 to 10 parts of acetone: 0.4 to 0.8 part of water; preferably, the volume ratio of various solvents in the solvent system is: 1 Parts of raw materials: 4-6 parts of absolute ethanol: 6-8 parts of acetone: 0.4-0.6 parts of water, that is, the amount of the aforementioned solvent system matches 1 part of the raw material containing rebaudioside A. Under the preferred ratio, the product can be made to have higher conversion rate and purity.

The volume ratio of the two solvents in the solvent system composed of absolute ethanol and water is: 12 to 16 parts of absolute ethanol: 1.5 to 1.6 parts of water; preferably, the volume ratio of various solvents in the solvent system is: 14 parts absolute ethanol: 1.6 parts water. Under the preferred ratio, the product can be made to have higher conversion rate and purity.

According to a preferred solution of the present invention, there is a fixed ratio relationship between the amount of the crude rebaudioside A and the above-mentioned solvent system. In the ratio relationship, the crude rebaudioside A is calculated by mass, and the solvent is calculated by volume. The mass unit mg is equivalent to volume unit ml, for example, when 1 part of crude rebaudioside A refers to 1 mg of crude rebaudioside A, 1 part of solvent refers to 1 ml of solvent. The specific ratio of this preferred solution is: 1 part of crude product: 12 to 16 parts of absolute ethanol: 1.5 to 1.6 parts of water; preferably, the volume ratio of various solvents in the solvent system is: 1 part of crude product: 14 parts of anhydrous ethanol: Water ethanol: 1.6 parts of water, that is, the amount of the aforementioned solvent system matches 1 part of crude rebaudioside A. Under the preferred ratio, the product can be made to have higher conversion rate and purity.

The raw material containing rebaudioside A in the present invention adopts stevioside with RA content of 20%-80%, preferably 40%-60%.

The dispersion in the present invention refers to the process of dispersing the particles of one substance into another substance, that is, dispersing the dispersed phase into the dispersion medium to form a dispersion system. Those skilled in the art can choose various means according to actual needs, for example, one or more means such as stirring and shaking, ultrasonic dispersion, heating and reflux can be used for dispersion. Preferably, the technical solution of the present invention adopts the method of heating and refluxing.

The advantages of the present invention are:

1. The process is simple and suitable for industrial production. This process only needs reflux and recrystallization. Other patented processes need to go through silica gel column chromatography or macroporous adsorption resin column and then recrystallize. The process is more cumbersome.

2. The production time is short, which can save the production cost.

3. High conversion rate. The process can use steviol glycosides with RA content of 40%-60% as raw materials to produce more than 97% RA, and the conversion rate is greater than 50%.

4. Environmental protection. This process only uses 2 kinds of organic solvents, which are cheap and most of them can be recycled and reused. No toxic and expensive organic solvents are used.

Description of drawings

Fig. 1 is the HPLC collection of illustrative plates of the RA that experiment 3. makes in the comparative example;

Fig. 2 is the HPLC collection of illustrative plates that contains 50% RA raw material in embodiment 2;

Fig. 3 is the HPLC collection of illustrative plates of RA crude product in embodiment 2;

Fig. 4 is the HPLC collection of illustrative plates of the final product of embodiment 2.

Detailed ways

comparative example

Carry out the following experiments according to the conditions known in the prior art

1. Investigate the influence of different multiples of 90% ethanol on refining 50% RA

①Precisely weigh 5g of 50% RA, add 50mL of 90% ethanol, reflux until completely dissolved, filter, and leave the filtrate to crystallize.

②Precisely weigh 5g of 50% RA, add 40mL of 90% ethanol, reflux until completely dissolved, filter, and place the filtrate for crystallization.

③Precisely weigh 5g of 50% RA, add 30mL of 90% ethanol, reflux until completely dissolved, filter, and place the filtrate for crystallization.

④Precisely weigh 5g of 50% RA, add 20mL of 90% ethanol, reflux until completely dissolved, filter, and place the filtrate until crystallization occurs.

When the above solution was left for 6 days, a small amount of crystals precipitated in ②③④. After 15 days, no crystals precipitated in ①. Filter ②③④ to collect the crystals, dry them, and weigh them. The results are shown in Table 2.

The HPLC spectrum of RA prepared in experiment ③ is shown in Fig. 1 . It can be seen from Fig. 1 that according to the method of the prior art, the purity of RA is relatively low, less than 97%.

Through the above experiments, it is verified that 97% of RA cannot be obtained through the patented method, and the conversion rate is not high at the same time.

2. Investigate the influence of different concentrations of ethanol on refining 50% RA

①Precisely weigh 5g of 50% RA, add 30mL of absolute ethanol, reflux until completely dissolved, filter, and place the filtrate for crystallization.

②Precisely weigh 5g of 50% RA, add 30mL of 96% ethanol, reflux until completely dissolved, filter, and place the filtrate until crystallization occurs.

③Precisely weigh 5g of 50% RA, add 30mL of 92% ethanol, reflux until completely dissolved, filter, and place the filtrate until crystallization occurs.

When the above solution was placed for 6 days, only ③ a small amount of crystals precipitated out.

Through the above experiments, it is verified that through the patented method, there is no crystallization, and 97% of RA cannot be obtained.

Conclusion: The method of direct crystallization with high-concentration ethanol takes a long time, the conversion rate is low, and the purity of RA cannot reach 97%, which is not suitable for industrial production.

97% RA Example

Embodiment 1: get 100g containing the raw material of 40%RA, add dehydrated alcohol, acetone and water by the ratio of 1:5:7:0.5 (raw material is calculated by mass, solvent is calculated by volume, hereinafter expressed as m/v), Heat and reflux at 80°C for 1 hour, filter, take insoluble matter, and dry to obtain crude RA; weigh the crude RA, add absolute ethanol and water at a ratio of 1:14:1.6 (m/v), reflux for 1 hour, and filter , the filtrate was placed for 72 hours to recrystallize, filtered, collected crystals, dried, and obtained. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Table 1 RA refining experiment results

Example 2: Get 100 g of raw material containing 50% RA, add absolute ethanol, acetone and water in a ratio of 1:5:7:0.5 (m/v), heat and reflux at 80° C. for 1 hour, filter, and take the insoluble matter, Dry to obtain crude RA; weigh crude RA, add absolute ethanol and water at a ratio of 1:14:1.6 (m/v), reflux for 1 hour, filter, leave the filtrate for 72 hours to recrystallize, filter, and collect crystals. Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1. Figures 2 to 4 show the HPLC spectrum of the raw material containing 50% RA, the HPLC spectrum of the crude RA product, and the HPLC spectrum of the final product, respectively. From the HPLC chromatograms of Fig. 2～Fig. 4, it can be seen intuitively that before the method of this embodiment is not processed, in the chromatogram containing 50% RA raw material, there are 6 obvious impurity peaks except the main peak; After the treatment in the first step of the example, the visible impurity peaks in the HPLC spectrum of the obtained RA crude product become two; There are again obvious impurity peaks.

Example 3: Get 100g of raw material containing 60% RA, add absolute ethanol, acetone and water in a ratio of 1:5:7:0.5 (m/v), heat and reflux at 80°C for 1 hour, filter, and get the insoluble matter, Dry to obtain crude RA; weigh crude RA, add absolute ethanol and water at a ratio of 1:14:1.6 (m/v), reflux for 1 hour, filter, leave the filtrate for 72 hours to recrystallize, filter, and collect crystals. Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Example 4: Get 100 g of raw material containing 50% RA, add absolute ethanol, acetone and water in a ratio of 1:4:6:0.4 (m/v), heat and reflux at 80° C. for 1 hour, filter, and get the insoluble matter, Dry to obtain crude RA; weigh crude RA, add absolute ethanol and water at a ratio of 1:14:1.6 (m/v), reflux for 1 hour, filter, leave the filtrate for 72 hours to recrystallize, filter, and collect crystals. Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Example 5: Get 100 g of raw material containing 50% RA, add absolute ethanol, acetone and water in a ratio of 1:5:7:0.5 (m/v), heat and reflux at 80° C. for 1 hour, filter, and take the insoluble matter, Dry to obtain crude RA; weigh the crude RA, add absolute ethanol and water at a ratio of 1:14:1.6 (m/v), reflux for 2 hours, filter, leave the filtrate for 72 hours to recrystallize, filter, collect crystals, Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Example 6: Get 100 g of raw material containing 50% RA, add dehydrated alcohol, acetone and water in a ratio of 1:6:8:0.6 (m/v), heat and reflux at 80° C. for 1 hour, filter, and get the insoluble matter, Dry to obtain the crude product of RA; weigh the crude product of RA, add absolute ethanol and water according to the ratio of 1:14:1.6 (m/v), reflux for 3 hours, filter, leave the filtrate for 72 hours to recrystallize, filter, collect crystals, Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Example 7: Get 100 g of raw material containing 50% RA, add absolute ethanol, acetone and water in a ratio of 1:3:5:0.8 (m/v), heat and reflux at 80° C. for 1 hour, filter, and take the insoluble matter, Dry to obtain crude RA; weigh crude RA, add absolute ethanol and water at a ratio of 1:14:1.6 (m/v), reflux for 1 hour, filter, leave the filtrate for 72 hours to recrystallize, filter, and collect crystals. Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Example 8: Get 100 g of raw material containing 50% RA, add absolute ethanol, acetone and water in a ratio of 1:8:10:0.4 (m/v), heat and reflux at 80° C. for 1 hour, filter, and take the insoluble matter, Dry to obtain crude RA; weigh crude RA, add absolute ethanol and water at a ratio of 1:14:1.6 (m/v), reflux for 1 hour, filter, leave the filtrate for 72 hours to recrystallize, filter, and collect crystals. Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Example 9: Get 100 g of raw materials containing 50% RA, add absolute ethanol, acetone and water in a ratio of 1:4:6:0.4 (m/v), heat and reflux at 80° C. for 1 hour, filter, and take the insoluble matter, Dry to obtain crude RA; weigh the crude RA, add absolute ethanol and water at a ratio of 1:12:1.5 (m/v), reflux for 1 hour, filter, leave the filtrate for 72 hours to recrystallize, filter, collect crystals, Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Example 10: Take 100 g of raw materials containing 50% RA, add absolute ethanol, acetone and water in a ratio of 1:4:6:0.4 (m/v), heat and reflux at 80°C for 1 hour, filter, and take the insoluble matter, Dry to obtain crude RA; weigh the crude RA, add absolute ethanol and water at a ratio of 1:16:1.6 (m/v), reflux for 1 hour, filter, leave the filtrate for 72 hours to recrystallize, filter, collect crystals, Dried, that is. Sampling was carried out for purity detection by HPLC method, and the results are shown in Table 1.

Claims (8)

1. A method for refining and purifying rebaudioside A, characterized in that, comprising the following steps: - Disperse the raw material containing rebaudioside A in a solvent system composed of absolute ethanol, acetone and water, and collect the insoluble matter by filtration to obtain the crude product of rebaudioside A; - dispersing the crude rebaudioside A in a solvent system composed of absolute ethanol and water, recrystallizing the filtrate after filtration, collecting the crystals, and drying to obtain purified rebaudioside A; the absolute ethanol, The volume ratio of various solvents in the solvent system made of acetone and water is: 3-8 parts of absolute ethanol 5-10 parts of acetone 0.4～0.8 parts of water; The volume ratio of various solvents in the solvent system that described dehydrated alcohol and water constitute is: 12-16 parts absolute ethanol 1.5-1.6 parts of water. 2. according to the method for claim 1, it is characterized in that, described dehydrated alcohol, the volume ratio of various solvents in the solvent system that acetone and water form are: 4-6 parts of absolute ethanol 6-8 parts of acetone 0.4 to 0.6 parts of water. 3. The method according to claim 1 or 2, wherein the amount of the solvent system matches 1 part of the raw material containing rebaudioside A, wherein the solvent system is calculated by volume, containing rebaudioside A The raw materials of A are calculated by mass, and the mass unit mg is equivalent to the volume unit ml. 4. according to the method for claim 1, it is characterized in that, the volume ratio of various solvents in the solvent system that described dehydrated alcohol and water form is: 14 parts absolute ethanol 1.6 parts water. 5. The method according to claim 4, wherein the amount of the solvent system matches 1 part of the crude product of rebaudioside A, wherein the solvent system is calculated by volume, and the crude product of rebaudioside A is calculated by mass , the mass unit mg is equivalent to the volume unit ml. 6. The method according to claim 1, characterized in that, the raw material containing rebaudioside A adopts stevioside with RA content of 20%-80%. 7. The method according to claim 1, characterized in that, the raw material containing rebaudioside A uses 40%-60% stevioside. 8. The method according to claim 1, characterized in that the dispersion is carried out by heating to reflux.