Extraction method of asiaticoside, plant extract composition containing asiaticoside and application of plant extract composition

Abstract

The invention discloses an extraction method of asiaticoside, a plant extract composition containing asiaticoside and application of the plant extract composition. The extraction method comprises the following steps: S1, drying raw materials; s2, extracting a high-voltage pulse electric field; s3, clarifying the feed liquid; s4, performing enrichment; s5, decoloring; and S6, crystallizing and drying. The extraction method disclosed by the invention is stable in process and suitable for industrial large-scale production, and has a good market prospect, the plant extract composition containing asiaticoside disclosed by the invention takes asiaticoside as a main component, and at the same time, the plant extract composition containing asiaticoside disclosed by the invention is synergistic with the components such as the lasiospermum brachypodum extract, the hirsutella laterospora extract, the sesbania grandiflora flower extract and the fatty alcohol, so that the plant extract composition containing asiaticoside is prepared. The traditional Chinese medicine composition can play a good anti-oxidation effect, can effectively play respective effects when being applied to a formula, relieves the sensitive phenomenon of the skin from multiple dimensions, and effectively relieves the sensitive skin.

Description

Technical Field

[0001] The invention discloses an asiaticoside extraction method and a plant extract composition containing asiaticoside and application thereof, and belongs to the technical field of cosmetic raw material preparation. Background Art

[0002] Cellular activities that maintain the integrity of skin cells, as well as all immune mechanisms, involve a series of chemical reactions that generate free radicals and reactive oxygen species (ROS). Endogenous and exogenous antioxidant mechanisms work by neutralizing these bioactive molecules, and an imbalance in this neutralization can have a variety of consequences: Excess free radicals and reactive oxygen species (ROS) are associated with the etiology of various skin diseases.

[0003] Antioxidants can produce antioxidant effects by transferring electrons, providing hydrogen, reducing peroxides, quenching singlet oxygen and superoxide, and chelating metal ions. At present, most chemically synthesized antioxidants are highly toxic, and some are even carcinogenic, which makes people doubt and reject synthetic antioxidants, and there are great limitations in clinical application. Finding safe, low-toxic and effective antioxidants from abundant natural products has become the focus of research. Plant ingredients are an important component of natural antioxidant active ingredients, among which phenols and flavonoids dominate. The structural characteristics of most polyphenols and flavonoids are the presence of phenolic hydroxyl groups or unsaturated conjugated double bonds, which have strong antioxidant activity. Centella asiatica has this type of chemical structure, and its chemical molecule has 5 phenolic hydroxyl groups and a strong conjugated double bond structure. The inventors found through the study of the cosmetic antioxidant efficacy model that Centella asiatica has a strong antioxidant ability and can be used as a natural antioxidant in cosmetics.

[0004] In view of the above problems, it is necessary to develop an extraction method that is stable and suitable for industrial large-scale production to obtain asiatica glycoside and improve the bioavailability of asiatica glycoside. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned technical defects and develop an extraction method that is stable in process and suitable for industrial large-scale production to obtain Centella asiatica.

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

[0007] A method for extracting asiatica glycoside comprises the following steps:

[0008] S1. Drying the raw material: drying the whole Centella asiatica plant under natural conditions to obtain a dried Centella asiatica plant raw material;

[0009] S2, extraction: the Centella asiatica root powder pretreated in S2 is mixed with an extraction solvent, and subjected to high-voltage pulse electric field extraction, after the extraction is completed, a first filtrate and a first filter residue are obtained by filter pressing, the first filter residue is mixed with the extraction solvent for extraction, and a second filtrate and a second filter residue are obtained by filter pressing, the second filter residue is mixed with the extraction solvent for extraction, and a third filtrate and a third filter residue are obtained by filter pressing, and the first filtrate, the second filtrate and the third filtrate are combined to obtain a Centella asiatica extract;

[0010] S3, clarification of feed liquid: the extract obtained in S3 is concentrated under reduced pressure to recover ethanol to obtain a concentrated solution, the concentrated solution is sent to a horizontal spiral sedimentation centrifuge to remove water-insoluble matter, the clear liquid is collected, the centrifugal residue is discarded, the clear liquid is filtered through a microfiltration membrane, and after filtering to a certain volume, it is top-washed with water, the permeate and the water washing liquid are combined and collected to obtain a macroporous adsorption resin loading solution for standby use, and the retained residue is discarded;

[0011] S4, enrichment: the sample solution obtained in S3 is passed through a macroporous adsorption resin column at a certain flow rate, and the loading is stopped when Centella asiatica glycoside can be detected in the effluent, the loaded macroporous adsorption resin is washed with water to remove impurities in the resin gap, and then eluted with an elution solvent, the eluate is concentrated to dryness and then dissolved in 95% ethanol, filtered, the alcohol-insoluble matter is discarded and the alcohol-soluble matter is collected, and after concentrating to dryness, the total Centella asiatica glycosides are obtained, which are light yellow in color;

[0012] S5, decolorization: dissolving the total Centella asiatica glycosides obtained in S4 in water and passing through an anion exchange resin column, washing with water after the column is completed, eluting with ethanol after the water washing, combining and collecting the anion exchange column effluent, the water washing liquid and the ethanol eluate, and concentrating to dryness to obtain decolorized total Centella asiatica glycosides;

[0013] S6, crystallization and drying: dissolving the total Centella asiatica glycoside obtained in S5 with a solvent, adding seed crystals and leaving them for crystallization, filtering and washing the crystals after the crystallization is completed, and drying the washed crystals under certain conditions to obtain asiatica glycoside crystalline powder.

[0014] Preferably, the solid-liquid ratio of Centella asiatica to the extraction solvent in S2 is 1:3-1:20 (g / mL); the intensity of the high-voltage pulse electric field is 15-25 kV / cm, the extraction temperature is 0-5°C, the extraction solvent is 30-95% ethanol water (v / v), and the extraction time is 0.5-1.5h.

[0015] Preferably, the rotation speed of the horizontal spiral decanter centrifuge in S3 is 10000-20000 rpm / min.

[0016] Preferably, the pore size of the microfiltration membrane in S3 is 10-500 nm, the filtration to a certain volume means that the intercepted liquid volume is 1%-10% of the feed liquid volume before microfiltration, and the amount of the circulating top wash water is 20%-80% of the feed liquid volume before microfiltration.

[0017] Preferably, the S4 macroporous adsorption resin is a non-polar or weakly polar macroporous adsorption resin, the upper column flow rate is 1-4BV / h, the elution solvent is ethanol water with a concentration of 30%-95%, the elution volume and elution flow rate of the ethanol water elution are 2-5BV and 2-4BV / h respectively, and the concentration temperature of the eluent is 30-50°C.

[0018] Preferably, after the S4 eluate is concentrated and dried, the amount of ethanol used for alcohol dissolution is 5-20 times (mL / g), the dissolution temperature is room temperature, and the concentration and drying temperature of the alcohol solution is 30-50°C.

[0019] Preferably, the filler of the anion exchange resin column in S5 is a strong alkaline anion exchange resin or a weak alkaline anion exchange resin with styrene divinylbenzene as the skeleton, the column flow rate is 0.5-2BV / h, the column end point is that the effluent begins to turn from colorless to yellow, the water wash flow rate is 1-3BV / h, the water wash volume is 0.5-1.5BV, the alcohol wash flow rate is 1-3BV / h, and the alcohol wash volume is 2-4BV.

[0020] Preferably, the crystallization solvent in S6 is 10%-90% (v / v) methanol water, the amount of seed crystal added is 0.1%-10% of the mass of asiatica glycoside in the decolorized total asiatica glycosides, the crystallization temperature is 0-10°C, the crystallization time is 8-24h, and the crystal washing solvent is ice water.

[0021] Preferably, the crystal drying method in S6 is vacuum low-temperature drying, vacuum freeze drying or spray drying.

[0022] The present invention also discloses a plant extract composition containing asiaticoside obtained by any of the above-mentioned extraction methods, which is composed of the following components in parts by weight: 0.5-5 parts of asiaticoside, 2-10 parts of short-stalked wild sesame extract, 2-10 parts of chrysanthemum pubescens extract, 2-10 parts of sesame large flower extract, and 200-500 parts of fatty alcohol.

[0023] The invention also discloses the application of the plant extract composition. The composition is applied to the preparation of cosmetics. The cosmetics are skin external preparations prepared by using the effective ingredients as active components and adding conventional pharmaceutical or cosmetic auxiliary materials or auxiliary ingredients.

[0024] Preferably, the cosmetics are one or more of lotion, essence, cream, mask and gel.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. Using high-voltage pulse electric field for low-temperature extraction can reduce the infiltration of impurities such as chlorophyll and oil caused by temperature increase, and reduce the difficulty of subsequent processing. At the same time, the high-voltage pulse electric field can make the solvent fully penetrate into the plant raw materials, so that Centella asiatica glycoside can be fully leached.

[0027] 2. The plant extract composition containing high-purity Centella asiatica disclosed in the present invention has Centella asiatica as a main component and synergistically uses 4 other components to exert good anti-inflammatory and soothing effects. When applied to formulas, it has good soothing and anti-allergic abilities, and can fight skin inflammation and repair damaged skin parts.

[0028] 3. The plant extract composition containing high-purity Centella asiatica disclosed in the present invention has better anti-inflammatory and soothing effects than using Centella asiatica alone. Due to the synergistic effect among the components, the efficacy and activity of Centella asiatica can be maximized.

[0029] 4. The plant extract composition containing high-purity Centella asiatica disclosed in the present invention has better anti-inflammatory and soothing effects than using Centella asiatica alone. This can reduce the amount of Centella asiatica added during use while ensuring the efficacy, and can greatly reduce the cost of using Centella asiatica as an effective ingredient. DETAILED DESCRIPTION

[0030] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0032] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0033] In the examples, unless otherwise specified, all the means used are conventional means in the art.

[0034] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion.

[0035] For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0036] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] The reagents and instruments used in the examples and comparative examples of the present invention are all commercially available products.

[0038] The following is a further detailed description of the claims of the present invention in conjunction with specific implementation methods, but does not constitute any limitation to the present invention. Any limited modifications made by anyone within the scope of protection of the claims of the present invention are still within the scope of protection of the claims of the present invention.

[0039] Preparation Example of Centella asiatica glycoside:

[0040] S1. Raw material drying: The whole Centella asiatica plant is air-dried under natural conditions to obtain a dried Centella asiatica plant raw material.

[0041] S2. Extraction: 10 kg of the Centella asiatica plant material pretreated in S2 was mixed with 60 L of 50% ethanol (v / v), and subjected to high-voltage pulse electric field extraction at a temperature of 5°C and a field strength of 20 kV / cm for 30 min. After the extraction was completed, the first filter residue was filtered to obtain a first filtrate and a first filter residue. The first filter residue was mixed with 40 L of extraction solvent, extracted for 30 min under the conditions of the first extraction, and filtered to obtain a second filtrate and a second filter residue. The second filter residue was mixed with 40 L of extraction solvent, extracted for 30 min under the conditions of the first extraction, and filtered to obtain a third filtrate and a third filter residue. The first filtrate, the second filtrate and the third filtrate were combined to obtain a Centella asiatica extract, and the third filter residue was discarded.

[0042] S3, clarification of feed liquid: The extract obtained from S3 is concentrated under reduced pressure at 50°C, ethanol is recovered to obtain a concentrated solution, the concentrated solution is sent to a horizontal spiral sedimentation centrifuge to remove water-insoluble matter, the clear liquid is collected, and the centrifugal residue is discarded. The clear liquid is filtered through a microfiltration membrane with a retention pore size of 200nm for circulation, and after filtering to 5% of the volume of the feed liquid, it is top-washed with deionized water for circulation, and the permeate and the water washing liquid are combined and collected to obtain a macroporous adsorption resin loading solution for use, and the retained residue is discarded.

[0043] S4, enrichment: the sample solution obtained in S3 is passed through a D101 macroporous adsorption resin column at a flow rate of 1BV / h, and the loading is stopped when asiaticoside can be detected in the effluent. The loaded macroporous adsorption resin is washed with water for 1BV and then eluted with 50% ethanol for 3BV. The 50% ethanol eluate is concentrated to dryness and then dissolved in 95% ethanol, filtered, the alcohol insoluble matter is discarded and the alcohol soluble matter is collected. The alcohol soluble matter is concentrated to dryness to obtain total Centella asiatica glycosides. HPLC method is used to measure the sum of the contents of asiatica glycoside and hydroxymadecassoside in total Centella asiatica glycosides to be 64.82%, and the color is light yellow.

[0044] S5. Decolorization: The total Centella asiatica glycosides obtained in S4 were prepared into a 15% solution with water, and the aqueous solution was passed through a D301 anion exchange resin column at a flow rate of 1 BV / h (washed with 3% hydrochloric acid for 3 times the column volume to convert it to the chlorine type, and the column bed was washed with water until the pH of the effluent was 5-6 for standby use). When the color of the effluent turned yellow, the loading was stopped. After the loading was completed, 1 BV of water was used to wash out the Centella asiatica glycoside in the gaps between the resins. After the washing was completed, 2 BV of 50% ethanol was used to elute the Centella asiatica glycoside adsorbed on the resin due to intermolecular forces. The effluent from the anion exchange column, the water washing liquid and the ethanol eluate were collected and concentrated to dryness at 50°C to obtain decolorized total Centella asiatica glycosides in white color. The total content of asiatica glycoside and madecassoside in the total Centella asiatica glycosides was measured by HPLC to be 82.12%.

[0045] S6, crystallization and drying: the total glycosides of Centella asiatica obtained in S5 were heated and dissolved with 50% methanol water, and 1% of the weight of asiatica glycoside in the sample was added, and then crystallized at 4°C for 16 hours. After the crystallization was completed, the crystals were filtered and top-washed with ice water until the top-washing liquid was colorless. The wet crystals after the top washing were vacuum-dried at 45°C and -0.9MPa to constant weight, and the crystals were crushed to obtain 125.91g of asiatica glycoside crystalline powder. HPLC measured that the content of asiatica glycoside in the crystalline powder was 91.78%, and the total recovery rate of asiatica glycoside in the extraction process was 86.32%.

[0046] Example 1

[0047] A plant extract composition containing high-purity Centella asiatica is prepared by the following method: 1 g of Centella asiatica, 5 g of Sesame brevis extract, 5 g of Cynaphalium spondias extract and 6 g of Sesbania grandiflora extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0048] Example 2

[0049] A plant extract composition containing high-purity Centella asiatica is prepared by the following method: 1 g of Centella asiatica, 8 g of Sesame brevis extract, 6 g of Cynaphalium spondias extract and 5 g of Sesbania grandiflora flower extract are weighed and added into 420 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0050] Example 3

[0051] A plant extract composition containing high-purity Centella asiatica is prepared by the following method: 1.5 g of Centella asiatica, 5 g of Sesame brevis extract, 6 g of Cynaphalium spondias extract and 6 g of Sesbania grandiflora extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0052] Example 4

[0053] A plant extract composition containing high-purity Centella asiatica is prepared by the following method: 1 g of Centella asiatica, 5 g of Sesame brevis extract, 6 g of Cyperus rotundus extract and 5 g of Sesbania grandiflora extract are weighed and added into 420 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0054] Example 5

[0055] A plant extract composition containing high-purity Centella asiatica is prepared by the following method: 1.2 g of Centella asiatica, 8 g of Sesame brevis extract, 5 g of Cynaphalium spondias extract and 6 g of Sesbania grandiflora extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0056] Comparative Example 1

[0057] A plant extract composition containing high-purity Centella asiatica is prepared by the following method: 1.5 g of Centella asiatica, 5 g of Trichosanthes distichous extract and 6 g of Sesbania grandiflora flower extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0058] Comparative Example 2

[0059] A plant extract composition containing high-purity Centella asiatica is prepared by the following method: 1.5 g of Centella asiatica, 5 g of Sesame sphaerocephala extract and 5 g of Sesbania grandiflora flower extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0060] Comparative Example 3

[0061] A plant extract composition containing high-purity Centella asiatica is prepared by weighing 1.5 g of Centella asiatica, 8 g of Sesame brevis extract and 6 g of Cyperus rotundus extract, adding them into 400 parts of hexyldecanol, stirring and dissolving to obtain the composition.

[0062] Comparative Example 4

[0063] A plant extract composition containing high-purity asiatica glycoside is prepared by the following method: 1.5 g of asiatica glycoside and 6 g of sesbania grandiflora flower extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0064] Comparative Example 5

[0065] A plant extract composition containing high-purity Centella asiatica is prepared by the following method: 1.5 g of Centella asiatica and 5 g of a Trichosanthes distichous extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0066] In order to better illustrate the advantages of the present invention, the solubility and antioxidant tests of the composition with plant extract efficacy provided by the present invention are given below.

[0067] Test 1. Hyaluronidase inhibition test

[0068] This effect example verifies the inhibitory effect of the soothing and anti-inflammatory composition obtained in Examples 1-5 and Comparative Examples 1-5 on hyaluronidase. 1 part of the soothing and anti-inflammatory composition obtained in Examples 1-5 and Comparative Examples 1-5 is mixed with 3 parts of water to obtain the corresponding soothing and anti-inflammatory composition aqueous solution;

[0069] Specifically, the hyaluronidase inhibition experiment includes the following steps:

[0070] 1. Pre-prepare buffers and reagents:

[0071] (1) Acetate buffer solution (pH = 5.6): (1) Measure 1.155 mL of glacial acetic acid and dilute it to 100 mL with ultrapure water. After mixing, take 4.8 mL of it as solution A; (2) Weigh 2.72 g of sodium acetate crystals and dissolve it in ultrapure water, make up to 100 mL with ultrapure water, and after mixing, take 45.2 mL of it as solution B; (3) Mix solutions A and B, make up to 100 mL with ultrapure water, and mix; accurately measure the pH value and adjust it to 5.6 with solution A or B;

[0072] (2) Hyaluronidase solution (1250u / mL): weigh 0.01g of hyaluronidase into a beaker and add 4mL of acetate buffer solution to dissolve;

[0073] (3) Sodium hyaluronate solution (0.5 mg / mL): weigh 0.01 g of sodium hyaluronate into a beaker and add 20 mL of acetate buffer solution to dissolve;

[0074] (4) Ehrlich reagent: weigh 0.8 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol, and mix well;

[0075] (5) Sodium carbonate solution (1.0 mol / L): weigh 5.3 g of sodium carbonate, dissolve it in ultrapure water and make up to 50 mL;

[0076] (6) Acetylacetone solution: Dissolve 3.5 mL of acetylacetone in 50 mL of 1.0 mol / L sodium carbonate solution. Prepare the solution as needed.

[0077] (7) Calcium chloride solution (0.25 mmol / L): weigh 2.77 g of calcium chloride, dissolve it in ultrapure water and make up to 100 mL;

[0078] (8) Sodium hydroxide solution (0.4 mol / L): weigh 1.6 g of calcium chloride, dissolve it in ultrapure water and make up to 100 mL;

[0079] (9) Preparation of sample solution: dilute the soothing anti-inflammatory composition aqueous solution prepared in each example and comparative example with a buffer solution to two concentrations of 1% and 2% respectively;

[0080] 2. Determination of hyaluronidase activity inhibition rate: Select the sample solution and perform the following operations to obtain the absorbance value of the sample group:

[0081] (1) Take 0.1 mL of calcium chloride solution and 0.5 mL of hyaluronidase solution and incubate at 37°C for 20 min;

[0082] (2) Add 0.5 mL of each sample solution and continue to incubate at 37°C for 20 min;

[0083] (3) Add 0.5 mL of sodium hyaluronate solution, incubate at 37°C for 30 min, and place at room temperature for 5 min;

[0084] (4) Add 0.1 mL of sodium hydroxide solution and 0.5 mL of acetylacetone solution, heat in a boiling water bath for 15 min, and immediately cool with ice water for 5 min;

[0085] (5) Add 1.0 mL of Ehrlich reagent and dilute with 3.0 mL of anhydrous ethanol. Leave at room temperature for 20 min to develop color, and then measure the absorbance using a spectrophotometer.

[0086] 3. Result analysis: Hyaluronidase inhibition rate (%) = [(AB) - (CD)] / (AB) × 100%

[0087] In the formula: A is the absorbance value of the blank group, which means that the sample solution is replaced by acetate buffer solution; B is the absorbance value of the blank control group, which means that the sample solution and hyaluronidase solution are replaced by acetate buffer solution; C is the absorbance value of the sample group; D is the absorbance value of the sample control group, which means that the hyaluronidase solution is replaced by acetate buffer solution.

[0088] The test was performed three times in parallel and the average value was taken. The results are shown in Table 1 (the concentration was 0.5%).

[0089] Table 1. Hyaluronidase inhibition test results of Examples 1-5 and Comparative Examples 1-5

[0090]

[0091]

[0092] It can be seen from Table 1 that the soothing and anti-inflammatory composition prepared by the technical solution of the present invention has an excellent hyaluronidase inhibition effect. When the concentration is 1%, the inhibition rate of hyaluronidase is above 86.17%. It can be seen from Examples 1-5 and Comparative Examples 1-5 that when any one of the added components or any two components in the anti-inflammatory and soothing composition are reduced, the effect of the obtained composition shows a significant downward trend.

[0093] Experiment 2: Macrophage Inflammation Model Experiment

[0094] 1. Test purpose and principle

[0095] After IL-6 specifically binds to the IL-6 antibody coated on the ELISA plate, it binds to the anti-IL-6 antibody labeled with a substrate. After the substrate is catalyzed by the enzyme, a colored product is generated. The IL-6 content is positively correlated with the color depth of the colored product. The optical density (OD value) is measured at a wavelength of 450nm using an ELISA reader to calculate the IL-6 content.

[0096] In this experiment, LPS was used to induce allergic reactions in RAW264.7 cells, releasing inflammatory factors (IL-6). The soothing effect of the samples was evaluated by detecting their inhibitory effects on IL-6 content.

[0097] 2. Materials and Equipment

[0098] (1) Experimental reagents and materials

[0099] Mouse macrophage RAW264.7 cell line, DMEM, fetal bovine serum, trypsin-EDTA, penicillin / streptomycin double antibody, DPBS, Escherichia coli lipopolysaccharide (LPS), dexamethasone sodium phosphate, MTT, DMSO, IL-6 Elisa kit, and pure water.

[0100] (2) Main instruments and equipment

[0101] CO2 incubator, laminar flow clean cabinet (biosafety standard), constant temperature water bath, inverted phase contrast microscope, electronic balance, microplate reader, cell counter, vortex mixer.

[0102] 3. Experimental Methods

[0103] (1) Cytotoxicity assay

[0104] Adjust the concentration of RAW264.7 cell suspension and add it to 96-well cell plates. Culture overnight at 37°C and 5% CO2 for detection. Dilute the test samples with cell culture medium to different concentrations and add them to the cell plates. Culture them at 37°C and 5% CO2 for 20 to 48 hours. Discard the culture medium, wash with PBS 1-2 times, add MTT working solution and continue to culture. After the culture is completed, discard the culture medium, add DMSO, shake and mix, and measure the absorbance at 570nm (with 630nm as the background). Calculate the cytotoxicity based on the results, and screen the sample concentration without obvious cytotoxicity for the next experiment based on the cell morphology.

[0105] (2) Inflammatory factor IL-6 test

[0106] Adjust the concentration of RAW264.7 cell suspension and add it to a 96-well cell plate. Culture it overnight at 37°C and 5% CO2 for detection. The test sample was diluted to a non-cytotoxic concentration with a cell culture medium containing LPS and then added. The culture medium containing LPS was used as a negative control (NC), and the cell culture medium without LPS was used as a blank control (BC). The positive control diluted with the cell culture medium containing LPS was used as a positive control (PC). After adding the sample, the cells were cultured for 20 to 24 hours. After the action ended, the supernatant was taken and detected according to the operating procedures of the IL-6 Elisa kit.

[0107] 4. Result calculation and judgment

[0108] The calculation formula for the down-regulation rate of the inflammatory factor IL-6 is: down-regulation rate = (1-T / NC) × 100%, where: T is the average value of the inflammatory factor IL-6 in the experimental group; NC is the average value of the inflammatory factor IL-6 in the negative control group.

[0109] The data were statistically analyzed and the P value was calculated. P < 0.05 indicated a significant difference, otherwise there was no statistical difference. When the inflammatory factor IL-6 content in the negative control group was much higher than that in the blank control group, and there was a significant difference between the two (P < 0.05), the test was established. When the inflammatory factor IL-6 content in the sample group was lower than that in the negative control group, and there was a significant difference between the two (P < 0.05), it can be determined that the sample has a soothing effect.

[0110] 5. Experimental Results

[0111] (1) Cytotoxicity results

[0112] The samples were diluted to different concentrations and cytotoxicity experiments were carried out on mouse macrophages. The cytotoxicity test results showed that the cell viability of the sample at a concentration of 5% was 97.55%, with no obvious cytotoxicity. Therefore, a concentration of 5.00% was selected for the next test. The results are shown in Table 2.

[0113] Table 2. Cytotoxicity test results of samples

[0114]

[0115] (2) Test results of inflammatory factor IL-6

[0116] The sample was diluted to the required concentration, and dexamethasone sodium phosphate (test concentration was 2 mg / mL) was used as a positive control to conduct an inflammatory factor IL-6 test on macrophages. According to the test results, the content of inflammatory factor IL-6 in the negative control group was much higher than that in the blank control group, and there was a significant difference between the two (P < 0.05), indicating that the test was established. At a concentration of 5.00%, the sample had a downregulation rate of IL-6 inflammatory factors caused by exogenous stimulation of 31.51%, which was significantly different from the negative control group (P < 0.05), which was stronger than the positive control test results. The test results are shown in Table 3.

[0117] Table 3. IL-6 inflammatory factor test results

[0118]

[0119] Note: If P>0.05, it means there is no significant difference, recorded as "ns"; if P<0.05, it means there is a significant difference, and 0.01<P≤0.05, recorded as "*"; 0.001<P≤0.01, recorded as "**"; 0.0001<P≤0.001, recorded as "***"; P≤0.0001, recorded as "****"

[0120] 6. Experimental Conclusion

[0121] At a concentration of 5.00%, the sample had a downregulation rate of 31.51% on IL-6 inflammatory factors caused by exogenous stimulation, which was significantly different from the negative control group (P < 0.05). This showed that dihydroquercetin could significantly inhibit the release of inflammatory factor IL-6 and had a good soothing effect.

[0122] Experiment 3: Zebrafish embryo tail fin repair promotion experiment

[0123] 1. Experimental Principle

[0124] Wound healing in mammals involves multiple steps, including blood coagulation, inflammation, epidermal formation, angiogenesis, and granulation tissue formation and maturation. The main steps (except for fibrin clot formation) and principles of wound healing in zebrafish are the same as those in mammals. After the zebrafish tail fin is injured, both the bones and the tail fin will regenerate. Skin cells produce Sonichedgehog and FGF to promote cell proliferation, thereby regulating the regeneration process. In terms of wound healing, zebrafish embryos have similar cellular and molecular regulatory mechanisms as adult zebrafish. Therefore, small individual zebrafish embryos were selected to facilitate photography under a microscope and to measure the level of repair of the zebrafish embryo tail fin. By testing and comparing the changes in the repair level of the zebrafish embryo tail fin in the sample treatment group and the model control group, the repair promotion rate was calculated to evaluate the repair promotion effect of the raw material or product.

[0125] 2. Main reagents and consumables

[0126] (1) Water complies with the provisions of GB / T6682. (2) Solvents such as methanol, dimethyl sulfoxide, and ethanol are of analytical grade. (3) Water for broodstock breeding: Weigh 4g of sea salt and dissolve it in 10L of water to prepare the water. The salinity is 0.25-0.50‰, the conductivity is 500-800μS / cm, the dissolved oxygen is 80% saturated, the pH value is 6.5-8.5, and the hardness is 30-300mg / L (calculated as calcium carbonate). (4) Artemia salina eggs: Artemia salina dormant eggs need to be kept dry and dark and refrigerated. Weigh about 15g of sea salt and dissolve it in 1L of water to prepare water for hatching artemia eggs. The density should not exceed 4g / L artemia eggs. (5) Fish embryo culture medium: Weigh 2940mg of anhydrous calcium chloride, 1233mg of magnesium sulfate heptahydrate, 630mg of sodium bicarbonate, and 55mg of potassium chloride and dissolve them in 10L of water to prepare the water. The pH value is 6.5-8.5. All chemicals were of analytical grade. (6) Tricaine solution: Weigh 200 mg of tricaine and dissolve it in 48.95 mL of water. Store at 4°C. It is effective within 30 days. (7) Rehmannia glutinosa extract solution: Weigh 0.5 g of Rehmannia glutinosa extract and dissolve it in 5 mL of distilled water to prepare the positive control solution. Store at 4°C. It is effective within 1 month. The working solution is prepared by diluting the positive control solution 100 times with fish embryo culture medium. Prepare it fresh before use.

[0127] 3. Preparation of test substances

[0128] Water-soluble test product: The plant extract composition containing asiaticaside can be directly dissolved in fish embryo culture medium to prepare a test solution.

[0129] 4. Test steps

[0130] Pick healthy 3-day-post-fertilization (DPF) zebrafish embryos.

[0131] (1) Test grouping: The test needs to set up a model control group (fish embryo culture medium), a positive control group (Rehmannia glutinosa extract solution) and a test substance group (test substance).

[0132] (2) Model control group setting: 24 fish embryos with caudal fin removed were randomly selected and transferred to a 96-well plate, with each well containing one fish embryo and 0.2 mL of fish embryo culture medium.

[0133] (3) Positive control group setting: 24 fish embryos with caudal fins removed were randomly selected and transferred to a 96-well plate, with each well containing one fish embryo and 0.2 mL of Rehmannia glutinosa extract solution (for the preparation method, see 3. Preparation of test substances).

[0134] (4) Test substance treatment: 24 fish embryos with caudal fins removed were randomly selected and transferred to a 96-well plate, with each well containing one fish embryo and 0.2 mL of the test substance solution. The plates were placed in a 28±1°C incubator for 48±1 h.

[0135] (5) Microscopic analysis of samples: Fish embryos were photographed under a stereomicroscope using uniform photographic parameters.

[0136] (6) Data and result calculation: Use analysis software such as ImageJ to open the photos and measure the regenerated tail fin of each fish embryo.

[0137] 5. Calculation of results

[0138] Calculate the repair promotion rate: Repair promotion rate = (CT) / C × 100%, where: T is the repair level of the embryonic tail fin of the fish in the test substance treatment group; C is the repair level of the embryonic tail fin of the fish in the model control group; perform a two-tailed T test on the embryonic tail fin repair level of the fish in the test substance treatment group and the model control group to obtain the p value.

[0139] 6. Test validity verification:

[0140] (1) Test judgment: The mortality rate of each test group after exposure is lower than 10% (LC10), otherwise the results of the corresponding test group are invalid.

[0141] (2) Test requirements: A positive control group must be set up for each batch of tests, and the tail fin repair level of the positive control group must be significantly (P < 0.05) higher than that of the model control group.

[0142] 7. Result Report

[0143] (1) Repair promotion rate: The repair promotion rate of the zebrafish embryo tail fin at the corresponding test concentration.

[0144] (2) Evaluation: Evaluate the ability of the test substance to promote repair, and analyze whether the test values ​​between the test substance treatment group and the model control group are statistically different (P<0.05). The expression of the test substance's repair promotion rate should include the test concentration.

[0145] 8. Interpretation of the relevance of results

[0146] When the repair promotion rate is positive and statistically significant (P<0.05), the test results can be used as supporting evidence that the test substance has the effect of promoting repair, but it cannot replace human efficacy testing.

[0147] 9. Test Results

[0148] See Table 4.

[0149] Table 4. Effect of Centella asiatica on repairing tail fin of zebrafish embryos

[0150]

[0151] Notes:

[0152] (1) Injury model: Zebrafish embryos with caudal fin removed were used as an injury model.

[0153] (2) Promotion rate of zebrafish embryo tail fin repair = [(zebrafish embryo tail fin repair level in sample treatment group - zebrafish embryo tail fin repair level in model control group) * 100 / zebrafish embryo tail fin repair level in model control group] %.

[0154] (3) The P value is obtained by T-Test analysis of the tail fin repair level of zebrafish embryos in the sample treatment group and the model control group. P < 0.05 is generally considered to be statistically significant.

[0155] (4) Based on the statistical differences in the promotion rates and promotion rates of the samples on the zebrafish embryonic tail fin repair, the repair effects of the test samples were evaluated and divided into significant (the promotion rate was positive and P < 0.05) and insignificant (the promotion rate was negative or P = 0.05).

[0156] In summary, the present invention scientifically combines Centella asiatica, Sesame brachypetalum extract, Cyperus rotundus extract, Sesbania grandiflora flower extract and fatty alcohol, and the composition has good anti-inflammatory and soothing effects, which can not only improve the soothing and anti-allergic ability of the skin, but also has anti-inflammatory effects on the skin and can repair damaged skin parts.

[0157] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for extracting asiatica glycoside, characterized in that: The following steps are involved: S1. Drying the raw material: drying the whole Centella asiatica plant under natural conditions to obtain a dried Centella asiatica plant raw material; S2, extraction: the Centella asiatica root powder pretreated in S2 is mixed with an extraction solvent, and subjected to high-voltage pulse electric field extraction, after the extraction is completed, a first filtrate and a first filter residue are obtained by filter pressing, the first filter residue is mixed with the extraction solvent for extraction, and a second filtrate and a second filter residue are obtained by filter pressing, the second filter residue is mixed with the extraction solvent for extraction, and a third filtrate and a third filter residue are obtained by filter pressing, and the first filtrate, the second filtrate and the third filtrate are combined to obtain a Centella asiatica extract; S3, clarification of feed liquid: the extract obtained in S3 is concentrated under reduced pressure to recover ethanol to obtain a concentrated solution, the concentrated solution is sent to a horizontal spiral sedimentation centrifuge to remove water-insoluble matter, the clear liquid is collected, the centrifugal residue is discarded, the clear liquid is filtered through a microfiltration membrane, and after filtering to a certain volume, it is top-washed with water, the permeate and the water washing liquid are combined and collected to obtain a macroporous adsorption resin loading solution for standby use, and the retained residue is discarded; S4, enrichment: the sample solution obtained in S3 is passed through a macroporous adsorption resin column at a certain flow rate, and the loading is stopped when Centella asiatica glycoside can be detected in the effluent, the loaded macroporous adsorption resin is washed with water to remove impurities in the resin gap, and then eluted with an elution solvent, the eluate is concentrated to dryness and then dissolved in 95% ethanol, filtered, the alcohol-insoluble matter is discarded and the alcohol-soluble matter is collected, and after concentrating to dryness, the total Centella asiatica glycosides are obtained, which are light yellow in color; S5, decolorization: dissolving the total Centella asiatica glycosides obtained in S4 in water and passing through an anion exchange resin column, washing with water after the column is completed, eluting with ethanol after the water washing, combining and collecting the anion exchange column effluent, the water washing liquid and the ethanol eluate, and concentrating to dryness to obtain decolorized total Centella asiatica glycosides; S6, crystallization and drying: dissolving the total Centella asiatica glycoside obtained in S5 with a solvent, adding seed crystals and leaving them for crystallization, filtering and washing the crystals after the crystallization is completed, and drying the washed crystals under certain conditions to obtain asiatica glycoside crystalline powder.

2. The extraction method according to claim 1, characterized in that The solid-liquid ratio of Centella asiatica to the extraction solvent in S2 is 1:3-1:20 (g / mL); the intensity of the high-voltage pulse electric field is 15-25 kV / cm, the extraction temperature is 0-5°C, the extraction solvent is 30-95% ethanol water (v / v), and the extraction time is 0.5-1.5h.

3. The extraction method according to claim 1, characterized in that The rotation speed of the horizontal spiral sedimentation centrifuge in S3 is 10000-20000rpm / min.

4. The extraction method according to claim 1, characterized in that The pore size of the microfiltration membrane in S3 is 10-500nm, the filtration to a certain volume means that the intercepted liquid volume is 1%-10% of the volume of the feed liquid before microfiltration, and the amount of the circulating top wash water is 20%-80% of the volume of the feed liquid before microfiltration.

5. The extraction method according to claim 1, characterized in that The macroporous adsorption resin in S4 is a non-polar or weakly polar macroporous adsorption resin, the upper column flow rate is 1-4BV / h, the elution solvent is ethanol water with a concentration of 30%-95%, the elution volume and elution flow rate of the ethanol water elution are 2-5BV and 2-4BV / h respectively, and the concentration temperature of the eluent is 30-50°C.

6. The extraction method according to claim 1, characterized in that After the S4 eluate is concentrated and dried, the amount of ethanol used for alcohol dissolution is 5-20 times (mL / g), the dissolution temperature is room temperature, and the concentration and drying temperature of the alcohol solution is 30-50°C.

7. The extraction method according to claim 1, characterized in that The filler of the anion exchange resin column in S5 is a strong alkaline anion exchange resin or a weak alkaline anion exchange resin with styrene divinylbenzene as the skeleton, the column flow rate is 0.5-2BV / h, the column end point is that the effluent begins to turn from colorless to yellow, the water wash flow rate is 1-3BV / h, the water wash volume is 0.5-1.5BV, the alcohol wash flow rate is 1-3BV / h, and the alcohol wash volume is 2-4BV.

8. The extraction method according to claim 1, characterized in that The crystallization solvent in S6 is 10%-90% (v / v) methanol water, the amount of seed crystal added is 0.1%-10% of the mass of asiatica glycoside in the decolorized total asiatica glycosides, the crystallization temperature is 0-10°C, the crystallization time is 8-24h, and the crystal washing solvent is ice water.

9. A plant extract composition containing asiaticoside obtained by the extraction method according to any one of claims 1 to 8, characterized in that: The invention is composed of the following ingredients in parts by weight: 0.5-5 parts of asiatica glycoside, 2-10 parts of short-stalked wild sesame extract, 2-10 parts of chrysanthemum pubescens extract, 2-10 parts of sesbania grandiflora flower extract and 200-500 parts of fatty alcohol.

10. An application of the plant extract composition according to claim 9, characterized in that: The composition is used in the preparation of cosmetics, which are external skin preparations prepared by using the effective ingredients as active components and adding conventional pharmaceutical or cosmetic auxiliary materials or auxiliary ingredients.

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