Efficient extraction and purification process of centella asiatica total glycosides

Abstract

The invention belongs to the technical field of plant extraction, and particularly relates to an efficient extraction and purification process of total glycosides of centella asiatica, which comprises the following preparation steps: crushing dried centella asiatica, adding a specific degreasing agent into the crushed centella asiatica, stirring, centrifuging, drying and collecting centella asiatica powder; adding a mixed functional enzyme and pure water, adjusting the pH value to 5-6.5, carrying out enzymolysis at 45-55 DEG C for 2-4 hours, and carrying out enzyme deactivation, concentration and drying, so as to obtain an enzymolysis centella asiatica solid; adding an extraction solution, heating, stirring and separating to obtain supernate and a solid, adding the extraction solution into the solid, repeatedly extracting for 1-2 times, combining the supernate, adding modified beta-cyclodextrin, and stirring to obtain a centella asiatica extracting solution; concentrating the centella asiatica extracting solution into paste, performing alcohol precipitation, removing ethanol under reduced pressure, and drying to obtain the centella asiatica total glycosides. The asiatic pennywort herb total glycosides prepared by the method are relatively high in purity and have very good biological safety.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a high-efficiency extraction and purification process for total glycosides of Centella asiatica. Background Art

[0002] Modern scientific research has revealed that Centella asiatica contains numerous bioactive substances and organic chemical components of high research value. Current research data indicates that the chemical composition of the whole plant can be divided into four main categories: triterpenoid saponins, polyacetylenes, volatile oils, and other chemical components. Triterpenoid saponins are the most highly utilized active ingredients in Centella asiatica, present in a wide variety and abundant content. Triterpenoid saponins include pentacyclic triterpenoid saponins such as asiaticoside, madecassoside, isoscutaneous glycoside, and boremiside; as well as free triterpenoid acids such as madecassic acid, asiatic acid, boremiside, and madecassic acid. Total glycosides of Centella asiatica not only possess a wide range of biological activities in clinical and biomedical fields, but also have extensive applications in daily life. Because total glycosides of Centella asiatica have excellent nourishing and repairing effects on the skin, can accelerate collagen production, and possess anti-inflammatory and antibacterial properties, their use in skincare products can enhance their overall effectiveness.

[0003] Due to the powerful functions of total Centella asiatica glycosides, more and more research is focusing on the extraction and application of its active ingredients. Patent CN 115813964 A discloses an extraction process and application of a Centella asiatica extract with antioxidant properties. The extraction process comprises the following steps: S1. Grinding the whole plant and stirring for extraction; S2. Pressing the solution and applying it to a resin column for adsorption, eluting the active ingredients from the resin column with ethanol; S3. Decolorizing the solution by applying it to a decolorizing resin column, and concentrating the decolorized solution to a thick paste using a scraper concentrator; S4. Drying in a microwave drying oven in a Class D clean area; S5. Grinding, screening, and mixing to obtain total Centella asiatica glycosides; S6. Dissolving and isolating the asiatica glycosides in ethanol to obtain crude madecassoside; S7. Refining the solution by dissolving it in 95% ethanol, drying it, and grinding it through a sieve to obtain madecassoside. However, this patent still belongs to the traditional extraction process of Centella asiatica glycoside, which has the disadvantages of complicated operation procedures, low extraction efficiency and low purity.

[0004] Based on the shortcomings of the existing technology, it is of great practical significance to develop a method for extracting and purifying total glycosides of Centella asiatica with simple process and high extraction efficiency. Summary of the Invention

[0005] The invention discloses an efficient extraction and purification process for total Centella asiatica glycosides. The prepared total Centella asiatica glycosides have high purity and good biosafety.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides an efficient extraction and purification process for total glycosides of Centella asiatica, comprising the following preparation steps:

[0008] (1) grinding the dried Centella asiatica, adding a specific degreasing agent thereto, stirring, centrifuging, drying, and collecting the Centella asiatica powder;

[0009] (2) adding the mixed functional enzyme and pure water to the Centella asiatica powder of step (1), adjusting the pH value to 5-6.5, performing enzymatic hydrolysis at 45-55° C. for 2-4 hours, inactivating the enzyme, concentrating and drying to obtain an enzymatically hydrolyzed Centella asiatica solid;

[0010] (3) adding an extraction solution to the enzymatically hydrolyzed Centella asiatica solid of step (2), heating and stirring, and separating to obtain a supernatant and a solid, then adding the extraction solution to the solid and repeating the extraction step 1-2 times, combining the supernatants to obtain a Centella asiatica extract;

[0011] (4) The Centella asiatica extract obtained in step (3) is concentrated, precipitated with alcohol, and then the ethanol is removed under reduced pressure and dried to obtain total Centella asiatica glycosides.

[0012] In some embodiments, the specific degreasing agent includes sodium alginate derivatives, alkyl glycosides, fatty alcohol polyethers and cocamidopropyl betaine in a mass ratio of 1:(1-2):(1-2):(0.5-1); the volume ratio of the mass of Centella asiatica to the specific degreasing agent is 1g:(5-10)mL.

[0013] Preferably, the specific degreasing agent comprises sodium alginate derivative, alkyl glycoside, fatty alcohol polyether and cocamidopropyl betaine in a mass ratio of 1:1.5:1.5:0.75.

[0014] In some embodiments, the preparation steps of the sodium alginate derivative are:

[0015] Sodium alginate is completely dissolved in pure water, hydrogen peroxide and ascorbic acid are added under an inert gas atmosphere, and the mixture is stirred for 20-30 minutes. Chlorogenic acid is then added, and the mixture is stirred at 35-50°C for 20-24 hours. The reactant is dialyzed and then freeze-dried in a vacuum to obtain a sodium alginate derivative.

[0016] In some embodiments, the molar ratio of sodium alginate to chlorogenic acid is 1:(1.2-1.3).

[0017] Preferably, the molar ratio of sodium alginate to chlorogenic acid is 1:1.25.

[0018] In some embodiments, the mixed functional enzyme comprises cellulase, xylanase and pectinase in a mass ratio of (2-3):(1-2):(2-3); the added amount of the mixed functional enzyme is 0.025-0.05 wt% of Centella asiatica powder.

[0019] Preferably, the enzyme activities of the cellulase, xylanase and pectinase are 100,000 U / g.

[0020] Preferably, the mixed functional enzyme comprises cellulase, xylanase and pectinase in a mass ratio of 2.5:1.5:2.5; and the added amount of the mixed functional enzyme is 0.035 wt% of the Centella asiatica powder.

[0021] In some embodiments, the extraction solution consists of hydrogen peroxide, modified β-cyclodextrin and pure water; the content of hydrogen peroxide is 1-3wt% of the extraction solution, and the content of modified β-cyclodextrin is 1-3wt% of the extraction solution; the ratio of the extraction solution to the enzymatically hydrolyzed Centella asiatica solid is (10-15)mL:1g.

[0022] Preferably, the content of hydrogen peroxide is 2 wt% of the extraction solution, and the content of modified β-cyclodextrin is 2 wt% of the extraction solution; the ratio of the extraction solution to the enzymatically hydrolyzed Centella asiatica solid is 12.5 mL:1 g.

[0023] In some embodiments, the modified β-cyclodextrin is prepared by:

[0024] S1: The recrystallized β-cyclodextrin was completely dissolved in DMF at 0-4°C, sodium hydride was added, and the mixture was reacted at 0-4°C for 1.5-2.5 hours. Then, a DMF solution of p-toluenesulfonyl chloride was added dropwise, and the mixture was reacted at 20-30°C for 3-4 hours. The mixture was concentrated under reduced pressure and purified to obtain solid 1;

[0025] S2: dissolving the solid 1 from step S1 in deionized water, adding sodium azide, reacting at 55-65°C under inert gas for 18-22 hours, concentrating under reduced pressure, and purifying to obtain solid 2;

[0026] S3: dissolving the solid 2 from step S2 in DMF, adding triphenylphosphine, reacting at 85-95°C for 16-20h, concentrating under reduced pressure, and purifying to obtain solid 3;

[0027] S4: Boc-L-histidine was dissolved in DMF, and N,N-diisopropylethylamine and HATU were added thereto. The mixture was stirred under the protection of an inert gas for 1-2 hours, and then the solid 3 from step S3 was added. The mixture was stirred and reacted at 20-30°C for 3-4 hours. The reaction solution was added to acetone and purified to obtain solid 4. The solid 4 was then dissolved in trifluoroacetic acid, stirred and reacted at 20-30°C for 1-2 hours, and then concentrated under reduced pressure and purified to obtain modified β-cyclodextrin.

[0028] In some embodiments, the steps of recrystallizing β-cyclodextrin are as follows: dissolving β-cyclodextrin in 90°C hot water to form a saturated solution, performing hot filtration, cooling the filtrate to 20-30°C, and then cooling it at 0-4°C for 4-6 hours, filtering, placing solid phosphorus pentoxide next to the obtained solid for drying, replacing the phosphorus pentoxide every 1-2 hours until the phosphorus pentoxide no longer absorbs water, and obtaining recrystallized β-cyclodextrin.

[0029] In some embodiments, the mass ratio of solid 1 to sodium azide in step S2 is 5:(1-1.5).

[0030] Preferably, in step S2, the mass ratio of solid 1 to sodium azide is 5:1.25.

[0031] In some embodiments, the mass ratio of solid 2 to triphenylphosphine in step S3 is (2-2.2):1.

[0032] Preferably, in step S3, the mass ratio of solid 2 to triphenylphosphine is 2.1:1.

[0033] In some embodiments, the mass ratio of solid 3 to Boc-L-histidine in step S4 is (4-4.5):1.

[0034] Preferably, in step S4, the mass ratio of solid 3 to Boc-L-histidine is 4.25:1.

[0035] The present invention firstly degreases Centella asiatica powder by using a specific degreasing agent, then enzymatically hydrolyzes the Centella asiatica powder by using a specific enzyme to improve the dissolution rate of total asiatica glycosides therein, and then uses an extraction solution to extract the active ingredients of total asiatica glycosides, thereby improving the final extraction rate and purity of total asiatica glycosides.

[0036] The present invention constructs a mixed functional enzyme by combining cellulase, xylanase, and pectinase in a certain ratio. The three enzymes can produce a synergistic effect, decomposing macromolecules such as cellulose and polysaccharides in Centella asiatica and destroying cell walls, thereby improving the dissolution of the active ingredients in Centella asiatica. β-cyclodextrin can increase the stability, solubility, and bioavailability of substances to a certain extent. These properties of β-cyclodextrin are utilized to extract pharmaceutical ingredients. The present invention is directed to the extraction process of Centella asiatica by grafting histidine onto β-cyclodextrin to obtain a modified β-cyclodextrin. This modified β-cyclodextrin can selectively form inclusion complexes with active ingredients in Centella asiatica, such as asiaticoside and madecassoside, thereby improving the extraction efficiency and stability of total asiaticoside, as well as the purity of the total asiaticoside product. Histidine-modified β-cyclodextrin is a β-cyclodextrin derivative obtained by introducing a histidine group into the β-cyclodextrin molecule. The obtained modified β-cyclodextrin has good solubility and stability. At the same time, the modified β-cyclodextrin has a strong recognition ability for asiaticoside and hydroxy-madecassoside. It can form a strong binding ability with asiaticoside and hydroxy-madecassoside through the non-covalent interaction formed between molecules, thereby separating most of the total asiaticoside in Centella asiatica and reducing the dissolution of impurities.

[0037] Furthermore, the present invention modifies sodium alginate with chlorogenic acid to obtain a sodium alginate derivative. The applicant unexpectedly discovered that compounding this sodium alginate derivative with an alkyl glycoside, a fatty alcohol polyether, and cocamidopropyl betaine in a certain proportion to form a degreasing agent showed good wettability during the degreasing process of Centella asiatica, forming a stable degreasing system with the Centella asiatica and improving degreasing efficiency. This may be because, on the one hand, the sodium alginate derivative has a certain surface activity, which can reduce the surface tension of water and assist in the removal of oil from Centella asiatica; on the other hand, it can also serve as a thickener or stabilizer in the degreasing agent, enhancing the viscosity and stability of the formula. When blended with an alkyl glycoside, a fatty alcohol polyether, and cocamidopropyl betaine, it may form a degreasing agent with good biocompatibility and degreasing efficiency through physical mixing or chemical crosslinking.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The extraction and purification process of the total Centella asiatica glycosides in the present invention is simple and easy to operate, and the prepared total Centella asiatica glycosides have high purity and good biosafety.

[0040] 2. The present invention grafts histidine onto β-cyclodextrin to obtain a modified β-cyclodextrin, which can selectively form inclusion complexes with active ingredients such as asiaticoside and madecassoside in Centella asiatica, thereby improving the extraction efficiency and stability of total asiaticosides and the purity of the total asiaticosides product.

[0041] 3. The present invention modifies sodium alginate by chlorogenic acid to obtain a sodium alginate derivative, which is compounded with alkyl glycoside, fatty alcohol polyether, and cocamidopropyl betaine to form a degreasing agent. The degreasing agent has good wettability and can form a stable degreasing system with Centella asiatica, thereby improving the degreasing efficiency. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value within any stated value or stated range and any other stated value or intermediate value within the range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. Various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention, as will be apparent to those skilled in the art. Other embodiments obtained from the present invention description will be apparent to those skilled in the art. This application description and examples are exemplary only.

[0044] The terms “include,” “including,” “have,” or “contain” used in this document are open-ended terms, meaning including but not limited to.

[0045] It should be noted that the post-processing steps such as "rotary evaporation", "recrystallization", "concentration under reduced pressure", and "stirring" appearing in the following examples are routine operations for those skilled in the art and can be selected according to actual operations.

[0046] The fatty alcohol polyether used in the present invention is fatty alcohol polyether LF-901 purchased from Jinan Yunuo Chemical Co., Ltd.; the cocamidopropyl betaine used is cocamidopropyl betaine (CAB-35) purchased from Suzhou Minghua Sugar Alcohol Co., Ltd.; the enzyme activities of the cellulase, xylanase and pectinase used are all 100,000 U / g; and the alkyl glycoside used is APG0810.

[0047] Preparation Example 1

[0048] The preparation steps of sodium alginate derivatives are:

[0049] Take 0.1 mol of sodium alginate and completely dissolve it in 2 L of pure water. Add 3.5 mmol of hydrogen peroxide and 1 mmol of ascorbic acid under a nitrogen atmosphere, stir for 25 minutes, then add 0.125 mol of chlorogenic acid. After stirring at 45 ° C, the reaction is stirred for 22 hours. The reactant is poured into a dialysis bag (MWCO = 3500 Da) and dialyzed for 40 hours. The deionized water is replaced every 8 hours. After the dialysis is completed, vacuum freeze-drying is performed to obtain a sodium alginate derivative.

[0050] Preparation Example 2

[0051] The preparation steps of the sodium alginate derivative were the same as those in Preparation Example 1, except that 0.1 mol of chlorogenic acid was used.

[0052] Preparation Example 3

[0053] The preparation steps of the sodium alginate derivative were the same as those in Preparation Example 1, except that 0.14 mol of chlorogenic acid was used.

[0054] Preparation Example 4

[0055] The steps for recrystallizing β-cyclodextrin used in Preparation Example 5-8 are as follows:

[0056] Dissolve β-cyclodextrin in 90°C hot water to form a saturated solution, then perform hot filtration. Cool the filtrate to 25°C and then cool it at 0°C for 4 hours, filter it, place solid phosphorus pentoxide next to the obtained solid, and dry it at 80°C. Replace the phosphorus pentoxide every 1-2 hours until the phosphorus pentoxide no longer absorbs water, to obtain recrystallized β-cyclodextrin.

[0057] Preparation Example 5

[0058] The preparation steps of modified β-cyclodextrin are:

[0059] S1: 19 g of recrystallized β-cyclodextrin was completely dissolved in 250 mL of DMF at 0°C, 1.1 g of sodium hydride was added, and the mixture was reacted at 0°C for 2 h. Then, 100 mL of a DMF solution containing 5.3 g of p-toluenesulfonyl chloride was added dropwise. The mixture was reacted at 25°C for 3.5 h and concentrated under reduced pressure to 20 mL. The concentrated solution was added dropwise to 200 mL of acetone, stirred for 1.5 h, and filtered. The resulting filter cake was dissolved in 100 mL of 50°C deionized water, filtered, purified by reverse silica gel column chromatography, and rotary evaporated to obtain solid 1;

[0060] S2: 10 g of the solid 1 obtained in step S1 was dissolved in 300 mL of deionized water, and 2.5 g of sodium azide was added. The mixture was reacted at 60°C under nitrogen for 20 h, and then concentrated under reduced pressure to 20 mL. The concentrate was cooled to 25°C, added dropwise to 400 mL of acetone, stirred for 1 h, and filtered. The resulting filter cake was dissolved in 10 mL of deionized water, purified by reverse silica gel column chromatography, and rotary evaporated to obtain solid 2.

[0061] S3: 8.4 g of the solid 2 from step S2 was dissolved in 30 mL of DMF, 4 g of triphenylphosphine was added, and the mixture was reacted at 90°C for 18 h. The mixture was then concentrated under reduced pressure to 10 mL, added dropwise to 200 mL of acetone, stirred for 1 h, and filtered. The resulting filter cake was dissolved in 100 mL of deionized water, purified by reverse silica gel column chromatography, and rotary evaporated to obtain solid 3;

[0062] S4: 1 g of Boc-L-histidine was dissolved in 75 mL of DMF, and 2.75 mL of N,N-diisopropylethylamine and 4.05 g of HATU were added thereto. The mixture was stirred under nitrogen for 1.5 h, and then 4.25 g of solid 3 from step S3 was added. The mixture was stirred and reacted at 25 ° C for 3.5 h. The reaction solution was added to 500 mL of acetone, stirred for 1 h, and filtered. The filter cake was dissolved with 10 mL of deionized water, purified by reverse silica gel column, and rotary evaporated to obtain solid 4. Then 2 g of solid 4 was dissolved in 5 mL of trifluoroacetic acid, stirred and reacted at 25 ° C for 1.5 h, and concentrated under reduced pressure to remove trifluoroacetic acid. The obtained solid was added to 2 mL of saturated sodium bicarbonate aqueous solution, and 50 mL of acetone was added dropwise. The mixture was stirred for 1 h, filtered, and dried at 50 ° C to constant weight to obtain modified β-cyclodextrin.

[0063] Preparation Example 6

[0064] The preparation steps of the modified β-cyclodextrin are the same as those in Preparation Example 5, except that the amount of solid 1 used in step S2 is 12 g.

[0065] Preparation Example 7

[0066] The preparation steps of the modified β-cyclodextrin are the same as those in Preparation Example 5, except that the amount of solid 2 used in step S3 is 10 g.

[0067] Preparation Example 8

[0068] The preparation steps of the modified β-cyclodextrin are the same as those in Preparation Example 5, except that the amount of solid 3 used in step S4 is 4 g.

[0069] Example 1

[0070] An efficient extraction and purification process for total glycosides of Centella asiatica comprises the following preparation steps:

[0071] (1) 150 g of dried Centella asiatica was crushed and passed through a 20-mesh sieve. 750 mL of a specific degreasing agent was added to the powder. The mixture was stirred at 300 rpm for 1 h, centrifuged, washed, and dried to collect the Centella asiatica powder.

[0072] (2) adding 0.025 g of the mixed functional enzyme and 500 mL of pure water to 100 g of the Centella asiatica powder of step (1), adjusting the pH to 5, performing enzymatic hydrolysis at 45° C. for 4 h, inactivating the enzyme at 100° C. for 20 min, concentrating and drying to constant weight, and obtaining an enzymatically hydrolyzed Centella asiatica solid;

[0073] (3) adding 1 L of the extraction solution to 100 g of the enzymatically hydrolyzed Centella asiatica solid obtained in step (2), heating and stirring at 60° C. for 1 h, and filtering to obtain a supernatant and a solid, then adding 1 L of the extraction solution to the solid and repeating the extraction step once, combining the two supernatants to obtain a Centella asiatica extract;

[0074] (4) The Centella asiatica extract obtained in step (3) is concentrated, precipitated with alcohol, and then the ethanol is removed under reduced pressure, and dried at 60° C. to a constant weight to obtain total Centella asiatica glycosides.

[0075] The specific degreasing agent used is composed of sodium alginate derivative, alkyl glycoside, fatty alcohol polyether and cocamidopropyl betaine in a mass ratio of 1:1:1:0.5;

[0076] The mixed functional enzyme used was composed of cellulase, xylanase, and pectinase in a mass ratio of 2:1:2;

[0077] The extraction solution used contains 1 wt% hydrogen peroxide, 1 wt% modified β-cyclodextrin, and the balance is water;

[0078] The sodium alginate derivative used was obtained from Preparation Example 1; the modified β-cyclodextrin used was obtained from Preparation Example 5.

[0079] Example 2

[0080] An efficient extraction and purification process for total glycosides of Centella asiatica comprises the following preparation steps:

[0081] (1) 150 g of dried Centella asiatica was crushed and passed through an 80-mesh sieve. 1.5 L of a specific degreasing agent was added to the powder. The mixture was stirred at 300 rpm for 1 h, centrifuged, washed, and dried to collect the Centella asiatica powder.

[0082] (2) adding 0.05 g of the mixed functional enzyme and 500 mL of pure water to 100 g of the Centella asiatica powder of step (1), adjusting the pH to 6.5, performing enzymatic hydrolysis at 55° C. for 2 h, inactivating the enzyme at 100° C. for 20 min, concentrating and drying to constant weight, and obtaining an enzymatically hydrolyzed Centella asiatica solid;

[0083] (3) adding 1.5 L of the extraction solution to 100 g of the enzymatically hydrolyzed Centella asiatica solid obtained in step (2), heating and stirring at 60° C. for 1 h, and filtering to obtain a supernatant and a solid, then adding 10 L of the extraction solution to the solid and repeating the extraction step twice, combining the three supernatants to obtain a Centella asiatica extract;

[0084] (4) The Centella asiatica extract obtained in step (3) is concentrated, precipitated with alcohol, and then the ethanol is removed under reduced pressure, and dried at 60° C. to a constant weight to obtain total Centella asiatica glycosides.

[0085] The specific degreasing agent used is composed of sodium alginate derivative, alkyl glycoside, fatty alcohol polyether and cocamidopropyl betaine in a mass ratio of 1:2:2:1;

[0086] The mixed functional enzyme used was composed of cellulase, xylanase, and pectinase in a mass ratio of 3:2:3;

[0087] The extraction solution used contained 3 wt% hydrogen peroxide, 3 wt% modified β-cyclodextrin, and the balance water;

[0088] The sodium alginate derivative used was obtained from Preparation Example 1; the modified β-cyclodextrin used was obtained from Preparation Example 5.

[0089] Example 3

[0090] An efficient extraction and purification process for total glycosides of Centella asiatica comprises the following preparation steps:

[0091] (1) 150 g of dried Centella asiatica was crushed and passed through a 60-mesh sieve. 1.25 L of a specific degreasing agent was added to the powder. The mixture was stirred at 300 rpm for 1 h, centrifuged, washed, and dried to collect the Centella asiatica powder.

[0092] (2) adding 0.035 g of the mixed functional enzyme and 500 mL of pure water to 100 g of the Centella asiatica powder of step (1), adjusting the pH to 6, performing enzymatic hydrolysis at 50° C. for 3 h, inactivating the enzyme at 100° C. for 20 min, concentrating and drying to constant weight, and obtaining an enzymatically hydrolyzed Centella asiatica solid;

[0093] (3) adding 1.25 L of the extraction solution to 100 g of the enzymatically hydrolyzed Centella asiatica solid obtained in step (2), heating and stirring at 60° C. for 1 h, and filtering to obtain a supernatant and a solid, then adding 1.25 L of the extraction solution to the solid and repeating the extraction step once, combining the two supernatants to obtain a Centella asiatica extract;

[0094] (4) The Centella asiatica extract obtained in step (3) is concentrated, precipitated with alcohol, and then the ethanol is removed under reduced pressure, and dried at 60° C. to a constant weight to obtain total Centella asiatica glycosides.

[0095] The specific degreasing agent used is composed of sodium alginate derivative, alkyl glycoside, fatty alcohol polyether and cocamidopropyl betaine in a mass ratio of 1:1.5:1.5:0.75;

[0096] The mixed functional enzyme used was composed of cellulase, xylanase, and pectinase in a mass ratio of 2.5:1.5:2.5;

[0097] The extraction solution used contained 2 wt% hydrogen peroxide, 2 wt% modified β-cyclodextrin, and the balance water;

[0098] The sodium alginate derivative used was obtained from Preparation Example 1; the modified β-cyclodextrin used was obtained from Preparation Example 5.

[0099] Example 4

[0100] An efficient extraction and purification process for total glycosides of Centella asiatica is disclosed. The specific implementation method is the same as that of Example 3, except that the sodium alginate derivative used is obtained from Preparation Example 2.

[0101] Example 5

[0102] An efficient extraction and purification process for total glycosides of Centella asiatica is disclosed. The specific implementation method is the same as that of Example 3, except that the sodium alginate derivative used is obtained from Preparation Example 3.

[0103] Example 6

[0104] An efficient extraction and purification process for total glycosides of Centella asiatica, the specific implementation method is the same as that of Example 3, except that the modified β-cyclodextrin used is obtained from Preparation Example 6.

[0105] Example 7

[0106] An efficient extraction and purification process for total glycosides of Centella asiatica, the specific implementation method is the same as that of Example 3, except that the modified β-cyclodextrin used is obtained from Preparation Example 7.

[0107] Example 8

[0108] An efficient extraction and purification process for total glycosides of Centella asiatica, the specific implementation method is the same as that of Example 3, except that the modified β-cyclodextrin used is obtained from Preparation Example 8.

[0109] Comparative Example 1

[0110] An efficient extraction and purification process for total glycosides of Centella asiatica is disclosed, the specific implementation method of which is the same as that of Example 3, except that an equal volume of ethyl acetate is used instead of the specific degreasing agent.

[0111] Comparative Example 2

[0112] An efficient extraction and purification process for total glycosides of Centella asiatica, the specific implementation method is the same as that of Example 3, except that an equal mass of β-cyclodextrin is used instead of modified β-cyclodextrin.

[0113] Comparative Example 3

[0114] An efficient extraction and purification process for total glycosides of Centella asiatica comprises the following preparation steps:

[0115] (1) 150 g of dried Centella asiatica was crushed and passed through a 60-mesh sieve. 1.25 L of a specific degreasing agent was added to the powder. The mixture was stirred at 300 rpm for 1 h, centrifuged, washed, and dried to collect the Centella asiatica powder.

[0116] (2) adding 1.25 L of the extraction solution to 100 g of the Centella asiatica powder obtained in step (1), heating and stirring at 60° C. for 1 h, and filtering to obtain a supernatant and a solid. Then, adding 1.25 L of the extraction solution to the solid and repeating the extraction step once, combining the two supernatants to obtain a Centella asiatica extract;

[0117] (3) The Centella asiatica extract obtained in step (2) is concentrated, precipitated with alcohol, and then the ethanol is removed under reduced pressure, and dried at 60° C. to a constant weight to obtain total Centella asiatica glycosides.

[0118] The specific degreasing agent used is composed of sodium alginate derivative, alkyl glycoside, fatty alcohol polyether and cocamidopropyl betaine in a mass ratio of 1:1.5:1.5:0.75;

[0119] The extraction solution used contained 2 wt% hydrogen peroxide, 2 wt% modified β-cyclodextrin, and the balance water;

[0120] The sodium alginate derivative used was obtained from Preparation Example 1; the modified β-cyclodextrin used was obtained from Preparation Example 5.

[0121] Example 9

[0122] An efficient extraction and purification process for total glycosides of Centella asiatica, the specific implementation method is the same as that of Example 3, except that the specific degreasing agent used is composed of alkyl glycoside, fatty alcohol polyether and cocoamidopropyl betaine in a mass ratio of 1.5:1.5:0.75.

[0123] Performance test of total glycosides of Centella asiatica

[0124] 1. Purity analysis

[0125] The purity of the total glycosides of Centella asiatica obtained in each embodiment and comparative example was tested by high performance liquid chromatography. The chromatographic conditions referred to the Centella asiatica content determination method in the 2020 edition of the "Chinese Pharmacopoeia" (page 296). The specific analysis and test results are shown in Table 1.

[0126] Table 1

[0127]

[0128] As can be seen from Table 1, the purity of the total Centella asiatica glycosides prepared in Examples 1-3 is relatively high, reaching more than 95.8%, and the contents of asiatica glycoside and hydroxy-madecassoside are relatively high. Compared with Example 3, the preparation conditions of the sodium alginate derivatives used in Examples 4 and 5 are changed, the performance of the prepared sodium alginate derivatives are changed, the lipid removal efficiency in Centella asiatica is reduced, and the purity of the total Centella asiatica glycosides is reduced; the preparation conditions of the modified β-cyclodextrin used in Examples 6-8 are changed, the synthesis rate of the modified β-cyclodextrin obtained is reduced, and the structure may have changed, resulting in the recognition and complexation effect of the modified β-cyclodextrin on asiatica glycoside and hydroxy-madecassoside being reduced, affecting the purity of the total Centella asiatica glycosides and the contents of asiatica glycoside and hydroxy-madecassoside; in Comparative Example 1, ethyl acetate is used as a degreasing agent, and ethyl acetate has a poor effect on asiatica glycoside and hydroxy-madecassoside. The glycosides are destroyed to a certain extent, so that the purity of the total glycosides of Centella asiatica and the content of asiaticoside and hydroxymadecassoside are reduced; in Comparative Example 2, unmodified β-cyclodextrin is used instead of modified β-cyclodextrin, and the extraction effect of unmodified β-cyclodextrin on asiaticoside and hydroxymadecassoside is worse than that of modified β-cyclodextrin, so that the purity of the total glycosides of Centella asiatica and the content of asiaticoside and hydroxymadecassoside are reduced; in Comparative Example 3, no enzyme is used for enzymatic hydrolysis, so that the purity of the total glycosides of Centella asiatica and the dissolution rate and content of asiaticoside and hydroxymadecassoside are reduced; the specific degreasing agent used in Example 9 does not add sodium alginate derivatives, so that the purity of the total glycosides of Centella asiatica and the content and purity of asiaticoside and hydroxymadecassoside are reduced.

[0129] 2. Biosafety Analysis

[0130] Based on the above purity and content analysis data, the total Centella asiatica glycosides in Example 3 were selected for biosafety analysis.

[0131] The present invention conducted a biosafety test on the total Centella asiatica glycosides obtained in Example 3 in strict accordance with the experimental procedures described in the entry-exit inspection and quarantine industry standard SN / T 2329-2009, "Chicken Embryo Chorioallantoic Membrane Test for Eye Irritation / Corrosion of Cosmetics." The total Centella asiatica glycosides obtained in Example 3 were prepared with purified water to form 5, 30, and 50 wt% total Centella asiatica glycoside solutions. A 0.9 wt% concentration of physiological saline was used as a blank control group. Irritation score calculations were performed using the formula of the irritation scoring method. Specific test results are shown in Table 2.

[0132] Table 2

[0133]

[0134] As shown in Table 2, the total Centella asiatica glycosides prepared in Example 3 are mild and non-irritating, and have good biosafety.

[0135] The above is only a preferred embodiment of the present invention and does not limit the present application in any form. Although the present application is disclosed as above with a preferred embodiment, it is not intended to limit the present application. Any technician familiar with this profession, without departing from the scope of the technical solution of the present application, makes some changes or modifications using the technical content disclosed above, which are equivalent to equivalent implementation cases. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. An efficient extraction and purification process for total glycosides of Centella asiatica, characterized in that: The method comprises the following preparation steps: (1) grinding the dried Centella asiatica, adding a specific degreasing agent thereto, stirring, centrifuging, drying, and collecting the Centella asiatica powder; (2) adding the mixed functional enzyme and pure water to the Centella asiatica powder of step (1), adjusting the pH value to 5-6.5, performing enzymatic hydrolysis at 45-55° C. for 2-4 hours, inactivating the enzyme, concentrating and drying to obtain an enzymatically hydrolyzed Centella asiatica solid; (3) adding an extraction solution to the enzymatically hydrolyzed Centella asiatica solid of step (2), heating and stirring, and separating to obtain a supernatant and a solid, then adding the extraction solution to the solid and repeating the extraction step 1-2 times, combining the supernatants to obtain a Centella asiatica extract; (4) The Centella asiatica extract obtained in step (3) is concentrated, precipitated with alcohol, and then the ethanol is removed under reduced pressure and dried to obtain total Centella asiatica glycosides.

2. The efficient extraction and purification process according to claim 1, wherein The specific degreasing agent includes a sodium alginate derivative, an alkyl glycoside, a fatty alcohol polyether and cocamidopropyl betaine in a mass ratio of 1:(1-2):(1-2):(0.5-1); the volume ratio of the mass of the Centella asiatica to the specific degreasing agent is 1g:(5-10)mL.

3. The efficient extraction and purification process according to claim 1, wherein The preparation steps of the sodium alginate derivative are: Sodium alginate is completely dissolved in pure water, hydrogen peroxide and ascorbic acid are added under an inert gas atmosphere, and the mixture is stirred for 20-30 minutes. Chlorogenic acid is then added, and the mixture is stirred at 35-50°C for 20-24 hours. The reactant is dialyzed and then freeze-dried in a vacuum to obtain a sodium alginate derivative.

4. The efficient extraction and purification process according to claim 3, characterized in that: The molar ratio of sodium alginate to chlorogenic acid is 1:(1.2-1.3).

5. The efficient extraction and purification process according to claim 1, characterized in that: The mixed functional enzyme comprises cellulase, xylanase and pectinase in a mass ratio of (2-3):(1-2):(2-3); the added amount of the mixed functional enzyme is 0.025-0.05 wt% of the Centella asiatica powder.

6. The efficient extraction and purification process according to claim 1, characterized in that: The extraction solution consists of hydrogen peroxide, modified β-cyclodextrin and pure water; the content of the hydrogen peroxide is 1-3wt% of the extraction solution, and the content of the modified β-cyclodextrin is 1-3wt% of the extraction solution; the ratio of the extraction solution to the enzymatically hydrolyzed Centella asiatica solid is (10-15)mL:1g.

7. The efficient extraction and purification process according to claim 6, characterized in that: The preparation steps of the modified β-cyclodextrin are: S1: The recrystallized β-cyclodextrin was completely dissolved in DMF at 0-4°C, sodium hydride was added, and the mixture was reacted at 0-4°C for 1.5-2.5 hours. Then, a DMF solution of p-toluenesulfonyl chloride was added dropwise, and the mixture was reacted at 20-30°C for 3-4 hours. The mixture was concentrated under reduced pressure and purified to obtain solid 1; S2: dissolving the solid 1 from step S1 in deionized water, adding sodium azide, reacting at 55-65°C under inert gas for 18-22 hours, concentrating under reduced pressure, and purifying to obtain solid 2; S3: dissolving the solid 2 from step S2 in DMF, adding triphenylphosphine, reacting at 85-95°C for 16-20h, concentrating under reduced pressure, and purifying to obtain solid 3; S4: Boc-L-histidine was dissolved in DMF, and N,N-diisopropylethylamine and HATU were added thereto. The mixture was stirred under the protection of an inert gas for 1-2 hours, and then the solid 3 from step S3 was added. The mixture was stirred and reacted at 20-30°C for 3-4 hours. The reaction solution was added to acetone and purified to obtain solid 4. The solid 4 was then dissolved in trifluoroacetic acid, stirred and reacted at 20-30°C for 1-2 hours, and then concentrated under reduced pressure and purified to obtain modified β-cyclodextrin.

8. The efficient extraction and purification process according to claim 7, characterized in that: The mass ratio of solid 1 to sodium azide in step S2 is 5:(1-1.5).

9. The efficient extraction and purification process according to claim 7, characterized in that: The mass ratio of solid 2 to triphenylphosphine in step S3 is (2-2.2):

1.

10. The efficient extraction and purification process according to claim 7, characterized in that: The mass ratio of solid 3 to Boc-L-histidine in step S4 is (4-4.5):1.

Images