CN115120541A

CN115120541A - Centella asiatica extract and preparation method and application thereof

Info

Publication number: CN115120541A
Application number: CN202210849858.XA
Authority: CN
Inventors: 刘露; 贺青
Original assignee: Baocui Biotechnology Co ltd
Current assignee: Baocui Biotechnology Co ltd
Priority date: 2022-07-19
Filing date: 2022-07-19
Publication date: 2022-09-30
Anticipated expiration: 2042-07-19
Also published as: CN115120541B

Abstract

The invention discloses a centella extract, a preparation method and application thereof, and relates to the technical field of extracts. The preparation method of the centella extract comprises the following steps: (1) cutting the whole dry centella asiatica into pieces, and performing reflux extraction under the condition that the material-liquid ratio is 1 (15-30) to obtain a filtrate, wherein the extraction solvent is water; (2) concentrating the filtrate to obtain a concentrated solution, wherein the volume of the filtrate is 5-15 times of the volume of the concentrated solution; (3) adding a water solution of polyhydric alcohol into the concentrated solution, and performing reflux extraction for 30-80 min under the conditions of 0.1-1 Pa and 30-60 ℃ to obtain the centella asiatica extract; the polyalcohol is at least one of glycerol, butanediol and propylene glycol; and adding the water solution of the polyhydric alcohol into the concentrated solution to obtain a mixed solution, wherein the mass percent of the polyhydric alcohol in the mixed solution is 20-60%. The asiatic centella extract obtained by the method has higher contents of asiaticoside and asiatic centella total flavone, and has good skin repairing effect.

Description

Centella asiatica extract and preparation method and application thereof

Technical Field

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

Background

The increase of working pressure and the irregularity of daily work and rest can make the skin state unbalance, and the skin barrier is impaired, leads to skin to receive external injury more, causes skin roughness, produces the wrinkle, loses elasticity.

Centella asiatica is a plant of the Umbelliferae family, and is recorded in Chinese pharmacopoeia: centella asiatica has the effects of clearing heat, promoting diuresis, detoxifying and reducing swelling. Centella asiatica contains various functional components, such as asiaticoside, asiatic acid, asiatic flavone, etc. Wherein asiaticoside is used for treating trauma, surgical wound, burn, etc., asiatic acid is used for improving depression, resisting cancer, preventing and treating myocardial infarction, etc., and asiatic flavone has good antioxidation effect.

Most of the existing centella extraction processes on the market only aim at asiaticoside or asiatic flavone, and the processes do not disclose the simultaneous extraction of the asiaticoside and the asiatic flavone; and the single component extracted by the existing process has very limited effect on repairing damaged skin.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the centella extract, the preparation method and the application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for preparing a centella asiatica extract, comprising the steps of:

(1) chopping the whole-plant hay of centella asiatica, and performing reflux extraction under the condition that the material-liquid ratio of the whole-plant hay of centella asiatica to an extraction solvent is 1 (15-30) to obtain a filtrate, wherein the extraction solvent is water;

(2) concentrating the filtrate to obtain a concentrated solution, wherein the volume of the filtrate is 5-15 times of the volume of the concentrated solution;

(3) adding a water solution of polyhydric alcohol into the concentrated solution, and performing reflux extraction for 30-80 min under the conditions of 0.1-1 Pa and 30-60 ℃ to obtain the centella asiatica extract; the polyalcohol is at least one of glycerol, butanediol and propylene glycol; and adding the water solution of the polyhydric alcohol into the concentrated solution to obtain a mixed solution, wherein the mass percent of the polyhydric alcohol in the mixed solution is 20-60%.

The invention firstly uses water as an extraction solvent for extraction, then concentrates the water, and then adds the polyol for extraction, organic solvents such as ethyl acetate, chloroform, n-butanol and the like are not added, recrystallization is not needed, the preparation process is green and simple, the operation is easy, the energy is saved, and no stimulation is caused to the skin. In addition, the extracted asiaticoside and asiatic total flavone have high content and high skin repairing effect.

Preferably, in the step (1), the reflux extraction is carried out twice, each extraction is carried out for 40-50 min, the extraction temperature is 50-70 ℃, and the pressure is 0.08-0.12 MPa. The two extractions can ensure that the effective components in the centella are fully extracted, if the extraction times are increased, components which are ineffective or even harmful to the repairing effect exist in the extract, and the production cost is also increased.

Preferably, in the step (3), after the extraction is finished, the centella asiatica extract is obtained by filtering and removing impurities through a 0.5 μm membrane, then adding a preservative, and then filtering and removing bacteria and removing impurities through a 0.22 μm membrane.

In addition, the invention also discloses a centella extract prepared by the method and application of the centella extract in cosmetics.

Compared with the prior art, the invention has the beneficial effects that:

the centella extract prepared by the two-step reflux and hydroalcoholic critical extraction process has high content of total asiaticoside and total asiaticflavone, does not irritate the skin, and has an excellent effect of repairing damaged skin.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

The commercially available asiaticoside product and the commercially available asiatic acid product used in the comparative examples contained 98 wt.% asiaticoside and 98 wt.% asiatic acid, respectively, and were purchased from natural pharmaceutical companies, Guangxiang.

Example 1

In an embodiment of the present invention, the preparation method of the centella asiatica extract comprises the following steps:

(1) chopping the whole dry plant of centella asiatica, adding water into the chopped dry plant of centella asiatica according to a material-liquid ratio of 1:20, performing reflux extraction twice, performing extraction for 45min each time at the extraction temperature of 60 ℃ and the extraction pressure of 0.1MPa, and combining the filtrates of the two times;

(2) concentrating the combined filtrate obtained in the step (1) to obtain a concentrated solution, wherein the volume of the filtrate is 10 times of that of the concentrated solution;

(3) adding an aqueous solution of glycerol into the concentrated solution to obtain a mixed solution, wherein the concentration of the glycerol in the mixed solution is 50 wt.%, performing reflux extraction for 50min under the conditions of 0.5Pa and 50 ℃, then performing filtration and impurity removal by using a 0.5-micrometer membrane, adding p-hydroxyacetophenone, the addition amount of the p-hydroxyacetophenone is 0.5 wt.% of the filtrate, and then performing filtration by using a 0.22-micrometer membrane to obtain the centella asiatica extract.

Example 2

An example of the centella asiatica extract according to the present invention, the method for preparing the centella asiatica extract according to this example is different from that of example 1 in that: in the step (1), the material-liquid ratio is 1: 15; in the step (2), the volume of the filtrate is 5 times of that of the concentrated solution; in the step (3), the concentration of the glycerol is 20 wt.%, and the reflux extraction is carried out for 30min at 0.1Pa and 30 ℃.

Example 3

An example of the extract of centella asiatica according to the present invention, the preparation method of the extract of centella asiatica according to this example is different from that of example 1 in that: in the step (1), the material-liquid ratio is 1: 25; in the step (2), the volume of the filtrate is 12 times of the volume of the concentrated solution; in the step (3), the concentration of the glycerol is 30 wt.%, and the reflux extraction is carried out for 60min at the temperature of 40 ℃ and under the pressure of 0.4 Pa.

Example 4

An example of the extract of centella asiatica according to the present invention, the preparation method of the extract of centella asiatica according to this example is different from that of example 1 in that: in the step (1), the ratio of material to liquid is 1: 30; in the step (2), the volume of the filtrate is 15 times of the volume of the concentrated solution; in the step (3), the concentration of the glycerol is 60 wt.%, and the reflux extraction is carried out for 80min at 1Pa and 60 ℃.

Example 5

An example of the centella asiatica extract according to the present invention, the method for preparing the centella asiatica extract according to this example is different from that of example 1 in that: in the step (2), the volume of the filtrate is 8 times of the volume of the concentrated solution; in the step (3), propylene glycol is used for replacing glycerol, the concentration is unchanged, and reflux extraction is carried out for 70min at the temperature of 40 ℃ under the pressure of 0.2 Pa.

Example 6

An example of the extract of centella asiatica according to the present invention, the preparation method of the extract of centella asiatica according to this example is different from that of example 1 in that: in the step (3), butanediol is used for replacing glycerol, the concentration is unchanged, and reflux extraction is carried out for 40min at the temperature of 50 ℃ and under the pressure of 0.7 Pa.

Comparative example 1

A centella asiatica extract, which is prepared by a method different from that of example 1, wherein: in the step (1), the ratio of material to liquid is 1: 10; in the step (3), the concentration of glycerin is 10 wt.%.

Comparative example 2

A centella asiatica extract, which is prepared by a method different from that of example 1, wherein: in the step (2), the volume of the filtrate is 4 times of the volume of the concentrated solution; in the step (3), the concentration of glycerin is 70 wt.%.

Comparative example 3

A centella asiatica extract, which is prepared by a method different from that of example 1, wherein: in the step (1), the material-liquid ratio is 1: 35; in the step (2), the volume of the filtrate is 20 times of the volume of the concentrated solution.

Comparative example 4

A centella asiatica extract, which is prepared by a method different from that of example 1, wherein: in the step (3), reflux extraction is carried out for 50min at the temperature of 70 ℃ under the pressure of 0.05 Pa.

Comparative example 5

A centella asiatica extract, which is prepared by a method different from that of example 1, wherein: in the step (3), reflux extraction is carried out for 50min at the temperature of 25 ℃ under the pressure of 1.1 Pa.

Comparative example 6

A centella asiatica extract, which is prepared by a method different from that of example 1, wherein: in the step (3), reflux extraction is carried out for 25 min.

Comparative example 7

A centella asiatica extract, which is prepared by a method different from that of example 1, wherein: in the step (3), reflux extraction is carried out for 90 min.

Comparative example 8

A centella asiatica extract, which is prepared by a method different from that of example 1, wherein: in the step (3), ethanol is used for replacing glycerol, and the concentration is unchanged.

Comparative example 9

A centella asiatica extract is prepared into a solution with the content of total asiaticosides of 0.25 wt.% by using a commercially available asiaticoside product and a commercially available asiatic acid product according to the mass ratio of 3:1, wherein the solvent is the same aqueous solution of glycerin as the centella asiatica extract described in example 1, and the concentration of the glycerin is the same as the concentration of the glycerin in the centella asiatica extract described in example 1.

Comparative example 10

A centella asiatica extract is prepared from a commercially available asiaticoside product to form a solution with asiaticoside content of 0.25 wt.%, wherein the solvent is the same aqueous solution of glycerin as the centella asiatica extract described in example 1, and the concentration of glycerin is the same as the concentration of glycerin in the centella asiatica extract described in example 1.

Comparative example 11

A centella asiatica extract is prepared from a commercially available centella asiatica product to obtain a solution with a centella asiatica content of 0.25 wt.%, wherein the solvent is an aqueous solution of glycerol identical to that of the centella asiatica extract described in example 1, and the concentration of glycerol is identical to that of glycerol in the centella asiatica extract described in example 1.

Comparative example 12

A centella asiatica extract is prepared by extracting with ethanol-water, separating and purifying with HP2MGL resin to obtain solid centella asiatica total flavonoids (with the purity of 80 wt.%), and preparing a solution with the concentration of 0.25 wt.%, wherein the solvent is the same aqueous solution of glycerin as the centella asiatica extract in example 1, and the concentration of glycerin is the same as the concentration of glycerin in the centella asiatica extract in example 1.

Comparative example 13

A centella asiatica extract is prepared from a commercially available asiaticoside product, a commercially available asiatic acid product and the total asiaticoside solid with the purity of 80 wt.% prepared in the proportion 12 into a solution with the total asiaticoside content of 0.25 wt.% and the total asiaticoside content of 0.25 wt.%, wherein the mass ratio of the asiaticoside to the asiatic acid is 1:1, the solvent is the same aqueous solution of glycerin as the centella asiatica extract in example 1, and the concentration of the glycerin is the same as the concentration of the glycerin in the centella asiatica extract in example 1.

Comparative example 14

A centella asiatica extract is prepared into a solution with the content of total asiaticoside in the centella asiatica of 0.5 wt% by using a commercially available asiaticoside product and a commercially available asiatic acid product according to the mass ratio of 3:1, wherein the solvent is an aqueous solution of glycerol which is the same as that of the centella asiatica extract in example 1, and the concentration of the glycerol is the same as that of the glycerol in the centella asiatica extract in example 1.

Comparative example 15

Centella asiatica extract, which is prepared from the centella asiatica total flavone solid with the purity of 80 wt% prepared in comparative example 12 to prepare a solution with the content of the centella asiatica total flavone of 0.5 wt%, wherein the solvent is the same aqueous solution of glycerin as the centella asiatica extract described in example 1, and the concentration of the glycerin is the same as the concentration of the glycerin in the centella asiatica extract described in example 1.

Performance test

And (3) measuring the content of total asiaticoside: testing the content of asiaticoside and asiatic acid by using an HPLC method to obtain the total asiaticoside content;

and (3) measuring the content of the asiatic centella total flavonoids: measuring the content of total flavonoids in herba Centellae by ultraviolet spectrophotometry with rutin as standard;

the test results are shown in table 1.

TABLE 1

Item	Centella asiatica total glycosides wt. -%)	Centella asiatica glycosides wt. -%)	Centella asiatica acid wt. -%)	Centella asiatica total flavone wt. -%)
					Example 1	0.25	0.171	0.083	0.25％
Example 2	0.20	0.144	0.061	0.20
					Example 3	0.23	0.161	0.072	0.24
Example 4	0.20	0.134	0.068	0.21
					Example 5	0.21	0.143	0.069	0.23
Example 6	0.22	0.152	0.68	0.20
					Comparative example 1	0.10	0.078	0.032	0.12
Comparative example 2	0.11	0.069	0.047	0.15
					Comparative example 3	0.15	0.085	0.068	0.17
Comparative example 4	0.14	0.087	0.058	0.17
					Comparative example 5	0..10	0.061	0.042	0.13
Comparative example 6	0.12	0.073	0.051	0.16
					Comparative example 7	0.17	0.111	0.062	0.18
Comparative example 8	0.18	0.12	0.06	0.19

As can be seen from Table 1, the content of total asiaticoside and total asiaticoside in the centella asiatica extracts of examples 1 to 6 is significantly higher than that in comparative examples 1 to 8, and the results show that the preparation conditions have a great influence on the content of total asiaticoside and total asiaticoside in the products.

The efficacy of the centella asiatica extracts of examples 1 to 6 and comparative examples 1 to 15 in repairing were verified:

(1) effect on NIH/3T3 cell proliferation

The sample to be tested is prepared into a 5mg/mL stock solution by adopting a PBS buffer solution. The stock solution was diluted with serum-free medium to 6 final concentrations before each plate spotting: 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, which is ready for use.

NIH/3T3 cells growing in logarithmic phase were selected, digested, centrifuged, counted, and diluted with DMEM medium containing 10 wt.% Fetal Bovine Serum (FBS)To 3 x 10 per ml ⁴ Individual concentrations were seeded in 96-well plates using a row gun at 100 μ L per well. Culturing for 4-5 h in a constant temperature incubator at 37 ℃, wherein CO is contained in the incubator ₂ The concentration of (2) was 5 vol.% (ensuring that cells were adherent), the medium in the 96-well plate was aspirated, the medium was washed twice with PBS buffer and then the medium containing samples of different concentrations was added, and the control group used equal amounts of PBS buffer instead of samples. Each group was treated 6 times and cultured for 48 hours and then measured by methylene blue method.

After culturing for 48h, the medium in the 96-well plate is aspirated, washed twice with PBS buffer, replaced with 50. mu.L of methylene blue dye solution, and placed in CO ₂ The content is 5 vol.%, and the culture is continued for 60min in a constant-temperature incubator at 37 ℃. The methylene blue dye solution is removed by suction, and the wells are washed gently with distilled water for 6 times, and the number of washing times is ensured to be the same for each well. After the distilled water in the well plate was drained, 100. mu.L of methylene blue washing solution was added to each well, and the mixture was placed on a shaking plate at room temperature and shaken until the blue crystals were completely dissolved. The absorbance (OD value) at 570nm of each well was measured using an enzyme-linked immunosorbent assay. The proliferation rate was calculated according to the following formula:

(2) effect on migration of NIH/3T3 cells

The influence on cell migration under the concentration condition corresponding to the highest proliferation promoting rate of cells is selected by taking the number of cells entering the scratch at different times as an index.

NIH/3T3 cells were administered at 4 x 10 per ml ⁵ The individual concentrations were plated in 24-well plates at 500. mu.L per well in DMEM medium containing 10 wt.% FBS in CO ₂ The cells were incubated overnight at 37 ℃ in an incubator at a concentration of 5 vol.% to form a monolayer of cells. After the cell coverage reached 80%, the wound model of the cultured cells was made by scoring the monolayer longitudinally as vertically as possible using a 10 μ L tip against a ruler (sterilized). After scratching, the culture solution is aspirated, the scratched cells are removed by carefully washing with PBS buffer solution for 3 times, and the PBS buffer solution is slowly added after the cells adhere to the wall so as to prevent the scattered monolayer wall-attached cells from influencing the test photographing result.

After cleaning, randomly dividing the sample into a control group, a blank group and a sample group for sample adding, wherein each group comprises 8 multiple wells; the blank group was prepared without adding the sample or the buffer solution, and the control group was prepared by adding the buffer solution in the same volume as the sample group. At this point photographs were taken under an inverted fluorescence microscope (100) as 0h results. Put in CO ₂ The cells were cultured in a 37 ℃ incubator at a concentration of 5 vol.%, and the number of cells entering the scratch was calculated for 24h after the wound was formed using Image-Pro Plus version6.0(IPP) Image analysis software. The numbers of cells entering the wound of the blank group and the control group are 400 and 500 respectively.

The test results are shown in table 2.

TABLE 2

Item	NIH/3T3 cell proliferation Rate (%)	Number of cells entering the wound
			Example 1	162.5	4867
Example 2	150.3	4423
			Example 3	160.8	4670
Example 4	153.1	4487
			Example 5	159.2	4561
Example 6	158.3	4509
			Comparative example 1	112.3	3009
Comparative example 2	114.6	3172
			Comparative example 3	119.3	3341
Comparative example 4	118.1	3298
			Comparative example 5	115.3	3109
Comparative example 6	117,4	3212
			Comparative example 7	120.4	3456
Comparative example 8	102.7	2980
			Comparative example 9	145.6	3897
Comparative example 10	135.8	3768
			Comparative example 11	130.8	3690
Comparative example 12	110.45	2876
			Comparative example 13	150.2	4190
Comparative example 14	149.8	4210
			Comparative example 15	120.8	3190

As shown in Table 2, the proliferation rates of NIH/3T3 cells cultured with the centella asiatica extracts of examples 1-6 were all over 150%, which is significantly higher than those of comparative examples 1-8. In addition, the test results of the comparative example 1 and the comparative examples 9 to 15 show that the asiaticoside and the asiatictotal flavone have a synergistic effect, and the combination of the asiaticoside and the asiatictotal flavone can greatly improve the skin repairing effect of the asiatic centella extract.

(3) Human skin repair experiment

1) Preparing a repairing gel: and mixing a carbomer aqueous solution with the carbomer content of 1 wt.% with the centella asiatica extracts in example 1 and comparative examples 9-15 respectively to prepare a carbomer gel containing the centella asiatica extract of 1 wt.%.

2) According to sensitive skin Huaxi questionnaire of Li professor in Huaxi hospital, 26-30 years old moderate sensitive skin persons 80 and 10 persons with questionnaire total score of 24-32 are selected, each group uses a repairing gel once in the morning and evening, and is used continuously for 28 days, after the subjects wash the face on the 0 th day and the 28 th day, after standing for 30min under the conditions of ambient temperature of 21 +/-1 ℃ and humidity of 50 +/-10%, the subjects sequentially use Probe of Tewameter TM300 and Mexameter MX18 instruments to test the face TEWL value, hemoglobin and erythema values, and take the values of 6 places on the face, take the average value, use the data of the 0 th day as the reference, compare the data of the 28 th day with the data thereof, and reflect the change condition of the data before and after use. The average value of the change of the index data of 10 persons is taken as the final result. The test results are shown in table 3.

TABLE 3

Item	TEWL change condition	Improvement of skin redness	Amelioration of hemoglobin	Improvement of erythema value
					Example 1	27.45％	18.15％	11.36％	12.12％
Comparative example 9	17.41％	15.15％	8.14％	9.07％
					Comparative example 10	15.14％	13.07％	8.04％	8.75％
Comparative example 11	13.73％	12.41％	7.98％	7.45％
					Comparative example 12	9.42％	9.04％	4.85％	5.29％
Comparative example 13	19.85％	16.45％	8.78％	10.17％
					Comparative example 14	18.79％	16.78％	9.03％	11.21％
Comparative example 15	10.43％	9.67％	5.05％	5.98％

As can be seen from Table 3, the improvement conditions of the repairing gel containing the comparative examples 9-15 are worse than those of the repairing gel in the example 1 after the repairing gel is used for 28 days, and the result shows that the centella asiatica extract prepared by the process in the example 1 has a good skin repairing effect, and the asiaticoside and the asiaticflavone have a synergistic effect in the skin repairing effect.

Table 4 shows the subjective evaluation results of the subjects after 28 days of using the gel containing example 1 and comparative examples 9 to 15, and the score from 0 to 10 represents that the repairing effect is gradually increased.

TABLE 4

Item	The individual considers whether effective improvement of skin sensitivity condition is achieved
		Example 1	8.5
Comparative example 9	5.2
		Comparative example 10	4.1
Comparative example 11	4.0
		Comparative example 12	3.9
Comparative example 13	6.7
		Comparative example 14	7.2
Comparative example 15	4.5

As can be seen from Table 4, the gel containing example 1 has a better repairing efficacy, significantly better than the gel prepared in the comparative example.

From the test results, the centella asiatica extract prepared by the method of the invention has good effect of repairing damaged skin, and the test proves that: the asiaticoside and the asiatic flavone can synergistically improve the repairing effect of the asiatic centella extract.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

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

2. The method for preparing the centella asiatica extract according to claim 1, wherein the step (1) comprises reflux-extracting twice, each time for 40-50 min, at 50-70 ℃ and under 0.08-0.12 MPa.

3. The method for preparing an extract of centella asiatica according to claim 1, wherein in step (3), after the extraction is completed, the extract is filtered through a 0.5 μm membrane to remove bacteria and impurities, then a preservative is added, and then the extract is filtered through a 0.22 μm membrane to remove impurities, thereby obtaining the extract of centella asiatica.

4. An extract of centella asiatica prepared by the method according to any one of claims 1 to 3.

5. Use of an extract of centella asiatica according to claim 4 in cosmetics.