CN115300428B

CN115300428B - Centella asiatica extract and preparation method and application thereof

Info

Publication number: CN115300428B
Application number: CN202211002860.XA
Authority: CN
Inventors: 李晓敏; 任红红; 袁方; 李明坤; 黄楚雯; 林志东; 欧景麟
Original assignee: Perfect Guangdong Commodity Co Ltd
Current assignee: Perfect Guangdong Commodity Co Ltd
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2023-07-28
Anticipated expiration: 2042-08-19
Also published as: CN115300428A

Abstract

The invention discloses a centella asiatica extract and a preparation method and application thereof, and relates to the technical field of extracts. The invention provides a preparation method of centella asiatica extract, which comprises the following steps: (1) pretreating centella asiatica to obtain centella asiatica powder; (2) Uniformly stirring the centella asiatica powder and the ionic liquid to obtain a pretreatment liquid; the ionic liquid comprises choline hydroxide and arginine, wherein the molar ratio of the choline hydroxide to the arginine is choline hydroxide: arginine= (1-2.5): 1; (3) Washing and centrifuging the pretreatment liquid, concentrating by rotary evaporation, and regulating the pH value to obtain the centella asiatica extract. According to the invention, the centella asiatica is extracted by taking the specific ionic liquid as the extracting agent, a special processing auxiliary process is not needed, compared with the existing product, the prepared centella asiatica extract is higher in yield, the content ratio of active ingredients is large, and the using effect is remarkably improved; the ionic liquid is safe and nontoxic, high in stability and less in use limit.

Description

Centella asiatica extract and preparation method and application thereof

Technical Field

The invention relates to the technical field of extracts, in particular to a centella asiatica extract and a preparation method and application thereof.

Background

Centella asiatica is a dry whole herb of centella asiatica (Centella asiatica (L.) in Urban) of Umbelliferae, and is widely distributed in the south of Yangtze river basin, and has abundant resources, and can be collected throughout the year, and can be used as medicine; the Chinese medicinal composition is applied to the medicine field of many countries for thousands of years, and is mainly used for damp-heat jaundice, heatstroke diarrhea, carbuncle, sore and injuries from falls, etc., wherein the Chinese medicinal composition is firstly recorded in Shennong Ben Cao Jing (Shennong's herbal Jing), is classified as a Chinese medicinal composition, has cold nature and bitter taste, is pungent in flavor, has the effects of clearing heat and promoting diuresis, and removing toxin and detumescence. The chemical components in centella asiatica comprise main effective components such as triterpenes and glycosides thereof, polyacetylenes, volatile oil, flavonoids, alkaloids, amino acids and the like. Modern researches have proved that centella asiatica extract can effectively promote skin injury and local collagen anabolism, resist inflammation and play an important role in tissue repair after skin injury.

The main method for preparing centella extract in China comprises the steps of adsorbing an obtained product prepared by solvent extraction by macroporous resin, eluting with ethanol water in different proportions, collecting target components, and refining the target components to obtain a high-purity centella triterpene monomer product. At present, the centella asiatica extract in the prior art has more types of extracting agents, most of the extracting agents are organic solvents (such as ethanol and the like), and more organic solvents are used in the washing process, so that the centella asiatica extract is not environment-friendly. And the direct application to cosmetics causes organic solvent residues, which cannot exert the effect of centella asiatica extract well.

Disclosure of Invention

Based on the above, the present invention aims to overcome the above-mentioned shortcomings of the prior art and provide a centella asiatica extract, and a preparation method and application thereof. According to the invention, the centella asiatica is extracted by taking the specific ionic liquid as the extracting agent, a special processing auxiliary process is not needed, and compared with the existing product, the prepared centella asiatica extract is higher in yield, the content ratio of active ingredients is large, and the using effect is remarkably improved. The ionic liquid is safe and nontoxic, high in stability and less in use limit.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a preparation method of centella asiatica extract comprises the following steps:

(1) Pretreating centella asiatica to obtain centella asiatica powder;

(2) Uniformly stirring the centella asiatica powder and the ionic liquid to obtain a pretreatment liquid; the ionic liquid comprises choline hydroxide and arginine, wherein the molar ratio of the choline hydroxide to the arginine is choline hydroxide: arginine= (1-2.5): 1;

(3) Washing and centrifuging the pretreatment liquid, concentrating by rotary evaporation, and regulating the pH value to obtain the centella asiatica extract.

Ionic Liquids (ILs) are widely paid attention to due to high thermal stability, low saturated vapor pressure and wide liquid temperature range, and when the Ionic Liquids (ILs) are extracted as solvents, the Ionic Liquids (ILs) have the advantages of being more environment-friendly, higher in extraction efficiency and the like compared with the traditional organic solvents. As an extraction solvent, ILs can adjust physicochemical properties by a combination of various cations and anions to efficiently extract different target compounds. The invention provides a novel preparation method for extracting centella asiatica by using ionic liquid through the ionic liquid of the combination of specific choline hydroxide and arginine, and the prepared centella asiatica extract has higher yield and more remarkable effect.

After a great deal of experimental investigation, the inventor finds that the yield of the prepared centella asiatica extract is higher only when a specific ionic liquid is selected, the ionic liquid keeps good stability at the high temperature of 48+/-2 ℃ and the low temperature of-18+/-2 ℃ in a circulating state, and the effect is more remarkable. The molar ratio of choline hydroxide to arginine in the ionic liquid is in a specific range of the invention, so that the ionic liquid has certain fluidity, and the ionic liquid is in a homogeneous transparent state, so that the yield of the prepared centella asiatica extract can be higher.

Preferably, in the step (1), the specific process of the pretreatment is as follows: cleaning herba Centellae, and pulverizing to obtain herba Centellae powder.

Preferably, in the step (2), the stirring time is 4-6h, and the molar ratio of the choline hydroxide to the arginine is choline hydroxide: arginine = 2.5:1.

after a lot of experimental investigation, the inventor finds that the molar ratio of the choline hydroxide to the arginine is choline hydroxide: arginine = 2.5: in the step 1, the prepared ionic liquid has the best fluidity, is in a homogeneous and transparent state, and is favorable for higher yield of centella asiatica extract prepared by the ionic liquid.

Preferably, in the step (2), the weight ratio of the centella powder to the ionic liquid is: ionic liquid = 1: (5-20). Further preferably, the weight ratio of centella asiatica powder to the ionic liquid is: ionic liquid = 1:10.

the inventor finds that the specific ionic liquid can realize high-efficiency extraction of centella asiatica active ingredients after a large number of experimental researches. The ionic liquid can efficiently extract asiaticoside and madecassoside in centella asiatica because anions in the ionic liquid are connected with H on asiaticoside and madecassoside molecules, so that strong intermolecular interaction is formed. The cations bind to O on asiaticoside and madecassoside molecules, forming stronger intermolecular hydrogen bonds. This process disrupts the original intermolecular interactions of asiaticoside and madecassoside molecules in centella asiatica, thereby dissolving asiaticoside and madecassoside in centella asiatica. With the above specific amount, the yield of the extract is higher, and the effect of the obtained extract is better.

Preferably, in the step (3), at least one of acetone and water is used as a detergent for washing, and the pH value is adjusted to be 1-2. Further preferably, in the step (3), washing is performed with water.

In the process of extracting centella asiatica by adopting the ionic liquid, the conventional organic solvent can be selected as the detergent, but more importantly, the centella asiatica is extracted by adopting the specific ionic liquid, and the prepared centella asiatica extract can achieve high yield by only using water as the detergent. And the inventor finds that when the specific ionic liquid is adopted to extract centella, the yield is obviously higher than that of the centella extracted by adopting a conventional organic solvent.

The method provided by the invention is environment-friendly and saves to the greatest extent by following the principle of small amount and repeated washing in the extraction process. The washing is fully and completely carried out, otherwise, the obtained final product presents pasty state, the moisture can not be completely removed, and the final product has certain viscosity after being dried, which is not beneficial to the subsequent operation.

Preferably, the preparation method of the ionic liquid comprises the following steps:

(a) Dissolving choline hydroxide and arginine in water, heating and stirring to obtain an ionic liquid solution;

(b) And (3) spin-steaming the ionic liquid solution to obtain the ionic liquid.

Preferably, in the step (a), the temperature of heating and stirring is 75-85 ℃, and the time of heating and stirring is 12-18 hours; in the step (b), the temperature of rotary steaming is 80-90 ℃.

Preferably, in the step (a), the ratio of the amount of arginine substance to the volume of water is arginine: water = 1mol: (400-600) mL.

In addition, the invention provides the centella asiatica extract prepared by the preparation method of the centella asiatica extract.

Further, the invention provides application of the centella asiatica extract in cosmetics.

The invention also provides a cosmetic comprising the centella asiatica extract.

The invention provides toning lotion which comprises the following components in parts by weight, based on 100 parts by weight: 0.1-0.3 part of centella asiatica extract, 0.2-0.4 part of phenoxyethanol, 0.5-1 part of 1, 2-hexanediol, 2-10 parts of butanediol, 0.1-0.5 part of xanthan gum, 2-10 parts of glycerin, 0.2-0.5 part of Xinxian ketone, 0.02-0.05 part of EDTA disodium and the balance of purified water.

The invention provides a cosmetic emulsion, which comprises the following components in parts by weight, based on 100 parts by weight: 0.1-0.3 part of centella asiatica extract, 0.4-0.8 part of phenoxyethanol, 0.3-0.5 part of arginine, 0.5-1 part of 1, 2-hexanediol, 2-5 parts of polydimethylsiloxane, 0.5-2 parts of cetostearyl alcohol, 3-8 parts of caprylic triglyceride, 2-5 parts of glycerol stearate, 0.1-0.5 part of xanthan gum, 3-10 parts of 1, 3-butanediol, 0.3-0.5 part of acrylic ester, 0.02-0.05 part of EDTA disodium and the balance of purified water.

The invention provides a face cream which comprises the following components in parts by weight, based on 100 parts by weight: 0.1-0.3 part of centella asiatica extract, 0.4-0.8 part of phenoxyethanol, 0.5-1 part of 1,2 hexanediol, 0.25-0.5 part of arginine, 0.2-0.5 part of vitamin E, 2-3 parts of polydimethylsiloxane, 2-6 parts of tocopherol, 1-5 parts of canola oil, 3-6 parts of caprylic triglyceride, 0.5-2 parts of cetostearyl alcohol, 0.5-1 part of behenyl alcohol, 0.5-1 part of synthetic beeswax, 2-3 parts of olive oil emulsified wax, 1-3 parts of glycerol stearate, 0.25-0.5 part of acrylic ester, 4-10 parts of glycerol, 0.02-0.05 part of EDTA disodium and the balance of purified water.

Compared with the prior art, the invention has the beneficial effects that: (1) The ionic liquid prepared from choline hydroxide and arginine is adopted in the research, and active ingredients such as asiaticoside, madecassoside and the like are extracted from centella asiatica. (2) The ionic liquid can efficiently extract asiaticoside and madecassoside in centella asiatica because anions in the ionic liquid are connected with H on asiaticoside and madecassoside molecules, so that strong intermolecular interaction is formed. The cations bind to O on asiaticoside and madecassoside molecules, forming stronger intermolecular hydrogen bonds. This process disrupts the original intermolecular interactions of asiaticoside and madecassoside molecules in centella asiatica, thereby dissolving asiaticoside and madecassoside in centella asiatica. (3) In the process of extracting centella asiatica by adopting the ionic liquid, the conventional organic solvent can be selected as the detergent, but more importantly, the centella asiatica is extracted by adopting the specific ionic liquid, and the prepared centella asiatica extract can achieve high yield by only using water as the detergent. And the inventor finds that when the specific ionic liquid is adopted to extract centella, the yield is obviously higher than that of the centella extracted by adopting a conventional organic solvent.

Drawings

FIG. 1 is a graph of the anti-inflammatory test results of zebra fish.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available. Examples 1 to 8 and comparative examples 1 to 15

Example 1

The invention relates to a preparation method of centella asiatica extract, which comprises the following steps:

(1) Cleaning herba Centellae, and pulverizing to obtain herba Centellae powder;

(2) Uniformly stirring 1g of centella asiatica powder and 10g of ionic liquid for 5 hours to obtain pretreatment liquid;

(3) Washing and centrifuging the pretreatment liquid, concentrating by rotary evaporation, adjusting the pH value to 1-2 (adjusting by hydrochloric acid) to obtain a precipitate, fully washing the precipitate, and drying to obtain the centella asiatica extract; wherein the detergent used for washing is water.

The preparation method of the ionic liquid comprises the following steps:

(a) Dissolving 2.5mol of choline hydroxide and 1mol of arginine in 500mL of water, heating and stirring, wherein the temperature of heating and stirring is 80 ℃, and the time of heating and stirring is 15 hours, so as to obtain an ionic liquid solution;

(b) Spin-steaming the ionic liquid solution to obtain the ionic liquid; wherein the temperature of rotary steaming is 85 ℃.

Example 2

The difference between this example and example 1 is that in the step (3), the washing agent used for washing is different and the ionic liquid is the same; specifically, the preparation method of the centella asiatica extract comprises the following steps:

(1) Cleaning herba Centellae, and pulverizing to obtain herba Centellae powder;

(3) Washing and centrifuging the pretreatment liquid, concentrating by rotary evaporation, adjusting the pH value to 1-2 (adjusting by hydrochloric acid) to obtain a precipitate, fully washing the precipitate, and drying to obtain the centella asiatica extract; wherein the detergent used for washing is a mixture of acetone and water, and the volume ratio of the acetone to the water is that: water=1:1.

Example 3

The difference between this example and example 1 is only that the amounts of centella asiatica powder and ionic liquid used in step (2) are different, and the amounts of detergent used in step (3) are different; specifically, the preparation method of the centella asiatica extract comprises the following steps:

(1) Cleaning herba Centellae, and pulverizing to obtain herba Centellae powder;

(2) Uniformly stirring 20g of centella asiatica powder and 200g of ionic liquid for 5 hours to obtain a pretreatment liquid;

Example 4

The difference between this example and example 2 is only that the amounts of centella asiatica powder and ionic liquid used in step (2) are different, and the amounts of detergent used in step (3) are different; specifically, the preparation method of the centella asiatica extract comprises the following steps:

(1) Cleaning herba Centellae, and pulverizing to obtain herba Centellae powder;

Example 5

The difference between this example and example 1 is only that in the above-mentioned step (2), the amount of the ionic liquid added was 5g.

Example 6

The difference between this example and example 1 is only that in the above-mentioned step (2), the amount of the ionic liquid added was 20g.

Example 7

The difference between this example and example 1 is only that in the preparation method of the ionic liquid, the molar ratio of choline hydroxide to arginine is choline hydroxide: arginine=2:1.

Example 8

The difference between this example and example 1 is only that in the preparation method of the ionic liquid, the molar ratio of choline hydroxide to arginine is choline hydroxide: arginine=1:1.

Comparative examples 1 to 14

Comparative examples 1 to 14 differ from example 1 only in the raw materials for preparing the ionic liquid in the preparation method of the ionic liquid, specifically, the raw materials for preparing the ionic liquid in comparative examples 1 to 14 differ from example 1, and are specifically shown in table 1.

TABLE 1

Comparative example 15

Comparative example 15 is a conventional centella asiatica extraction method, the conventional method is alcohol extraction, specifically, a preparation method of centella asiatica extract comprises the following steps:

(1) Cleaning herba Centellae, and pulverizing to obtain herba Centellae powder;

(2) Uniformly stirring 1g of centella asiatica powder and 10g of 70% ethanol for 5 hours to obtain a pretreatment liquid;

(3) Washing and centrifuging the pretreatment liquid, concentrating by rotary evaporation, and freeze-drying to obtain the centella asiatica extract; wherein the detergent used for washing is water.

Application example 1

The toning lotion comprises the following components in parts by weight based on 100 parts by weight: 0.2 part of centella asiatica extract, 0.2 part of phenoxyethanol, 0.5 part of 1, 2-hexanediol, 5 parts of butanediol, 0.3 part of xanthan gum, 5 parts of glycerol, 0.3 part of Xinxianone, 0.02 part of EDTA disodium and the balance of purified water.

Application example 2

The invention provides a cosmetic emulsion, which comprises the following components in parts by weight, based on 100 parts by weight: 0.1 part of centella asiatica extract, 0.4 part of phenoxyethanol, 0.3 part of arginine, 1,2 parts of hexanediol, 2 parts of polydimethylsiloxane, 1 part of cetostearyl alcohol, 5 parts of caprylic triglyceride, 3 parts of glycerol stearate, 0.3 part of xanthan gum, 5 parts of 1, 3-butanediol, 0.3 part of acrylic ester, 0.03 part of EDTA disodium and the balance of purified water.

Application example 3

The invention provides a face cream which comprises the following components in parts by weight, based on 100 parts by weight: 0.3 part of centella asiatica extract, 0.5 part of phenoxyethanol, 0.8 part of 1,2 hexanediol, 0.3 part of arginine, 0.3 part of vitamin E, 2 parts of polydimethylsiloxane, 2 parts of tocopherol, 2 parts of canola oil, 3 parts of caprylic triglyceride, 1 part of cetostearyl alcohol, 1 part of behenyl alcohol, 0.8 part of synthetic beeswax, 2 parts of olive oil emulsifying wax, 2 parts of glyceryl stearate, 0.3 part of acrylic ester, 6 parts of glycerin, 0.03 part of EDTA disodium and the balance of purified water.

Effect verification

Test example 1 stability test

The test process comprises the following steps: the stability of the ionic liquids prepared in examples 1, 7-8 and comparative examples 1-14 was tested, and the conditions of the samples were observed after 3 cycles, in which the ionic liquids were tested at a high temperature of 48.+ -. 2 ℃ (constant temperature oven) and at a low temperature of-18.+ -. 2 ℃ (refrigerator), and were cycled at-18 ℃ and at a normal temperature of-48 ℃ (high and low temperature alternating oven).

Test results: as shown in table 2.

TABLE 2

As shown in the table above, the ionic liquid prepared by the embodiment of the invention has good stability at the high temperature of 48+/-2 ℃ and the low temperature of-18+/-2 ℃ in the circulating state. The ionic liquid prepared by the comparative example has poor stability, has certain requirements on the use environment, and is difficult to be widely used.

Test example 2 yield test

The test process comprises the following steps: the ionic liquids prepared in examples 1 to 4 and comparative example 15 were subjected to yield calculation, yield = centella asiatica extract/centella asiatica powder x 100%.

Test results: as shown in table 3.

TABLE 3 Table 3

As shown in the table results, the centella asiatica is extracted by adopting the specific ionic liquid, the prepared centella asiatica extract can reach more than 10% yield only by using water as a detergent, and the extraction process is environment-friendly and simple to operate. Although common organic solvent detergents can be used for extracting centella asiatica by adopting the specific ionic liquid, the yield is only about 8.7 percent from the viewpoint of the yield, which is slightly lower than that of the ionic liquid extraction by using water only as the detergent.

Therefore, in the process of extracting centella asiatica by adopting the ionic liquid, the conventional organic solvent can be selected as the detergent, but more importantly, the centella asiatica is extracted by adopting the specific ionic liquid, and the detergent can achieve high yield by only using water. In addition, the content of active substances extracted by adopting an ionic liquid extraction mode is obviously higher than that of the conventional centella asiatica extraction mode adopted in comparative example 15.

Test example 3 DPPH radical scavenging test

The test process comprises the following steps: 1, 1-diphenylhydrazine-2-trinitrophenylhydrazine (DPPH) is a stable long-life free radical, and the ethanol solution thereof turns dark purple and has strong absorption near 517 nm. When a free radical scavenger exists, the light absorption of the DPPH ethanol solution is weakened due to the single electron pairing. The extent of discoloration of the DPPH ethanol solution is linearly related to the number of electrons it receives, and thus the ability of the test sample to scavenge free radicals, i.e., the magnitude of antioxidant activity, can be evaluated.

Test standard for DPPH radical scavenging experiments: shanghai household chemicals industry society standard T/SHRH 006-2018: cosmetic-free radical scavenging assay.

In the DPPH free radical scavenging test process, centella asiatica extract is dissolved by absolute ethyl alcohol, so that a sample solution used in the DPPH free radical scavenging test process is obtained.

Test results: as shown in table 4.

TABLE 4 Table 4

Performance of	DPPH radical scavenging Rate (%)
		Example 1 (1 mg/mL)	82.82
Example 1 (0.1 mg/mL)	41.85
		Comparative example 15 (1 mg/mL)	56.69

As is clear from the above table, the centella asiatica extract prepared in example 1 has a better effect on DPPH clearance at 1 mg/mL. In the process of extracting centella asiatica by adopting the ionic liquid, the detergent can be washed by adopting a conventional organic solvent, or only water can be used, and the clearance rate of the centella asiatica extract prepared by using only water to DPPH free radicals is higher when the concentration of the centella asiatica extract is 1 mg/mL.

Test example 4 tyrosinase activity inhibition test

The test process comprises the following steps: the detection mechanism is as follows: melanin synthesis is affected by tyrosinase, which catalyzes the hydroxylation of L-tyrosine to dopa and the oxidation of dopa to dopaquinone, which forms the final reaction product melanin. The test substance with tyrosinase activity inhibition effect can slow down the conversion of L-tyrosine into dopaquinone by tyrosinase, and the inhibition effect of the test substance on tyrosinase activity is evaluated according to the change of absorbance by measuring the absorbance of dopaquinone at 475 nm.

The specific experimental steps of tyrosinase activity inhibition are as follows:

(1) Sequentially adding an L-tyrosine solution, a sample solution, a PBS solution and a solvent (PBS buffer solution) into a 96-well plate according to the addition amount of the reagent in the table 5, and fully and uniformly mixing;

(2) After incubating for 10min at 37 ℃, sequentially adding 20uL of tyrosinase solution;

(3) Uniformly mixing at 37 ℃ for reaction for 5min, then placing the mixture into an enzyme-labeled instrument, and measuring at 475nm wavelength;

the tyrosinase activity inhibition calculation formula is inhibition ratio (%) = [1- (Td-Tc)/(Tb-Ta) ]. Times.100%.

TABLE 5

Test results: as shown in table 6.

TABLE 6

Performance of	Tyrosinase activity inhibition rate (%)
		Example 1 (1 mg/mL)	87.5
Example 1 (0.1 mg/mL)	70.8
		Example 2 (1 mg/mL)	49.23
Example 2 (0.1 mg/mL)	62.5
		Comparative example 15	19.13

As is clear from the table above, the centella asiatica extract prepared in examples 1-2 has a higher inhibition rate of tyrosinase activity at 1mg/mL and 0.1mg/mL, which is significantly better than centella asiatica extract prepared in conventional extraction method of comparative example 15.

Test example 5 antibacterial test

Test standard: the inhibition rate of propionibacterium acnes was tested, QB/T2738-2012.3.

Test results: as shown in table 7.

TABLE 7

As is clear from the above table, the centella asiatica extract prepared in examples 1-2 has a good inhibition rate against Propionibacterium acnes. The centella asiatica extract prepared in example 2 had less inhibition of propionibacterium acnes than the centella asiatica extract prepared in example 1 in a short period of time. In addition, the centella asiatica extract prepared by adopting the ionic liquid extraction method has obviously higher inhibition effect on propionibacterium acnes than the conventional centella asiatica extraction method adopted in comparative example 15.

Test example 6 zebra fish whitening test

The test process comprises the following steps: the experimental model selects an embryo of AB line wild zebra fish 24 hours (24 hpf) after fertilization. Zebra fish are divided into a plurality of groups, including normal control group and test group. The normal control group is not treated, the test sample group uses the centella asiatica extract prepared in the embodiment 1-2, after incubation for a period of time, the head of the zebra fish is photographed, and the melanin signal intensity of the head of the zebra fish is analyzed by using advanced image processing software.

Test results: as shown in table 8.

TABLE 8

Note that: in contrast to the blank, P < 0.05, P < 0.01, and P < 0.0001.

As can be seen from the table, in the process of extracting centella asiatica by using the ionic liquid in examples 1-2, the centella asiatica is extracted by using the specific ionic liquid of the present invention, and the prepared centella asiatica extract has a certain whitening effect when the dilution ratio is 50.

Test example 7 anti-inflammatory test of zebra fish

The test process comprises the following steps: the centella asiatica extract is diluted 1:1 into a diluted solution of 1mg/ml by the stock solution and the embryo culture solution, and then diluted into a series of concentration gradients, wherein the dilution concentration multiples are 20 times and 40 times.

(1) LYZ zebra fish embryos with normal development of 24hpf were randomly picked under a stereo microscope using plastic droppers for the experiment.

(2) Pre-test: the normal 24hpf transgenic zebra fish is randomly selected in 96-well plates, 15 zebra fish are arranged in each well, 1 compound well is arranged in each group, buffer water in the 96-well plates is removed under the condition of not damaging the zebra fish, 100 mu L of test substance solutions with different concentrations are added, a blank control group is added with buffer water, and a solvent control group is required to be arranged for non-water-soluble samples.

Incubated in a biochemical incubator at 28.5.+ -. 0.5 ℃ to 72hpf. The case of zebra fish was observed under an optical microscope. The concentration group at which no death of the 72hpf zebra fish occurred was determined as the maximum tolerated concentration of the sample.

(4) Formal experiments: and determining the concentration range of the test object in the formal experiment according to the result of the preliminary experiment, wherein the highest concentration in the experiment is not higher than the maximum tolerance concentration of the sample. According to the experimental requirement, the concentration of the test object is diluted in an equal ratio, and the dilution multiple is not less than 10 times.

According to experimental requirements, a sufficient number of 72hpf transgenic LYZ zebra fish that develop normally were pre-screened and randomly distributed into 24 well plates, 15 per well, with 3 wells set at each concentration. The buffer water in the 24-well plate was removed without damaging the young fish, and then 1mL of the sample solution to be tested with different concentrations was rapidly added to each well of the test subject test group, and 1mL of the embryo culture solution was added to the blank control group, the model group, and the positive control group. Cover the plate and incubate in a biochemical incubator at 28.5.+ -. 0.5 ℃ for 120min.

After 120min incubation, the solution in the 24 well plate was removed without damaging the zebra fish, 1mL of embryo culture solution was rapidly added to the blank, 1mL of 100 μm SDS was added to the model and test subjects, and 1mL of 5 μm dexamethasone was added to the positive control. Cover the plate and incubate in a biochemical incubator at 28.5.+ -. 0.5 ℃ for 120min.

(5) And (3) observing and photographing: at least 12 zebra fish are randomly selected from the zebra fish juvenile fish with normal phenotype and behavior after incubation, are fixed by 4% methyl cellulose, are observed and photographed under a fluorescence microscope, one side of the zebra fish faces downwards when photographing, the body is kept horizontal, photographing results of all the zebra fish are required to be completed under the same instrument and environment, and the body positions of the zebra fish are required to be kept consistent.

(6) Image analysis: after photographing is completed, the obtained zebra fish pictures are analyzed by using Image analysis software, and Image J Image analysis software is recommended. And selecting an inflammation part, and carrying out green fluorescence area statistics by Image J software, so as to count the number of inflammatory cells.

(7) Statistical analysis: the number of inflammatory cells of zebra fish was counted, and the Mean (Mean) and standard deviation (Standard Deviation, SD) of each group of experiments were calculated and the statistical analysis results were expressed as mean±sd. Statistical analysis of the data was performed using statistical analysis software (e.g., graphPad Prism 8) using the model group as a standard, and the number of inflammatory cells was compared for each experimental group, as compared to the model group,/indicated no significant difference, P < 0.05, P < 0.01, and P < 0.001.

Test results: as shown in fig. 1.

As can be seen from fig. 1, the anti-inflammatory effect of centella asiatica extract of example 2 diluted 40-fold is very remarkable, and the anti-inflammatory effect of centella asiatica extract diluted 20-fold is remarkable, indicating that the anti-inflammatory effect of centella asiatica extract prepared in example 2 is optimal. The centella asiatica extract prepared in example 1 also has a good anti-inflammatory effect relative to the model group.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A preparation method of centella asiatica extract is characterized by comprising the following steps:

(1) Pretreating centella asiatica to obtain centella asiatica powder;

(3) Washing and centrifuging the pretreatment liquid, concentrating by rotary evaporation, and washing and drying the precipitate obtained after the pH value is adjusted to be 1-2 to obtain the centella asiatica extract; the washing is performed with at least one of acetone and water as a detergent.

2. The method of preparing centella asiatica extract according to claim 1, wherein in the step (2), the stirring time is 4 to 6 hours, and the molar ratio of choline hydroxide to arginine is choline hydroxide: arginine = 2.5:1.

3. the method of preparing centella asiatica extract according to claim 1, wherein in the step (2), the weight ratio of centella asiatica powder to ionic liquid is: ionic liquid = 1: (5-20).

4. The method for preparing centella asiatica extract according to claim 3, wherein the weight ratio of centella asiatica powder to the ionic liquid is: ionic liquid = 1:10.

5. the method for preparing centella asiatica extract according to claim 1, wherein in the step (3), washing with water is performed.

6. The method for preparing centella asiatica extract according to claim 1, wherein the method for preparing the ionic liquid comprises the steps of:

(b) And (3) spin-steaming the ionic liquid solution to obtain the ionic liquid.

7. The method of preparing centella asiatica extract according to claim 6, wherein in the step (a), the temperature of heating and stirring is 75-85 ℃, and the time of heating and stirring is 12-18 hours; in the step (b), the temperature of rotary steaming is 80-90 ℃.

8. The method of preparing centella asiatica extract according to claim 6, wherein in the step (a), the ratio of the amount of arginine substance to the volume of water is arginine: water = 1mol: (400-600) mL.

9. A centella asiatica extract prepared by the method of preparing a centella asiatica extract according to any one of claims 1-8.

10. Use of the centella asiatica extract according to claim 9 in cosmetics.

11. A cosmetic comprising the centella asiatica extract of claim 9.

12. The toning lotion is characterized by comprising the following components in parts by weight based on 100 parts by weight: the centella asiatica extract of claim 9, 0.1-0.3 parts, phenoxyethanol 0.2-0.4 parts, 1, 2-hexanediol 0.5-1 parts, butylene glycol 2-10 parts, xanthan gum 0.1-0.5 parts, glycerin 2-10 parts, umarone 0.2-0.5 parts, disodium EDTA 0.02-0.05 parts, and the balance of purified water.

13. A cosmetic emulsion, characterized by comprising the following components in parts by weight, based on 100 parts by weight as a total: the centella asiatica extract of claim 9, 0.1-0.3 parts, phenoxyethanol 0.4-0.8 parts, arginine 0.3-0.5 parts, 1, 2-hexanediol 0.5-1 parts, polydimethylsiloxane 2-5 parts, cetostearyl alcohol 0.5-2 parts, caprylic triglyceride 3-8 parts, glyceryl stearate 2-5 parts, xanthan gum 0.1-0.5 parts, 1, 3-butylene glycol 3-10 parts, acrylic ester 0.3-0.5 parts, disodium EDTA 0.02-0.05 parts, and the balance of purified water.

14. A face cream characterized by comprising the following components in parts by weight, based on 100 parts by weight as a total: the centella asiatica extract of claim 9, 0.1-0.3 parts, phenoxyethanol 0.4-0.8 parts, 1, 2-hexanediol 0.5-1 parts, arginine 0.25-0.5 parts, vitamin E0.2-0.5 parts, polydimethylsiloxane 2-3 parts, tocopherol 2-6 parts, canola oil 1-5 parts, caprylic triglyceride 3-6 parts, cetostearyl alcohol 0.5-2 parts, behenyl alcohol 0.5-1 parts, synthetic beeswax 0.5-1 part, olive oil emulsifying wax 2-3 parts, glyceryl stearate 1-3 parts, acrylic ester 0.25-0.5 parts, glycerin 4-10 parts, disodium EDTA 0.02-0.05 parts, and the balance of purified water.