Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide a composition capable of improving acne marks and scars and a preparation method thereof.
The invention aims to provide a composition for improving acne marks and scars, which is prepared from the following components in part by weight:
inoculating yeast with yeast culture medium containing Geranium tenuipilum extract as fermentation substrate, and fermenting to obtain primary fermentation active substance;
mixing the obtained primary fermentation active substance with Xylaria nigripes culture medium to obtain a secondary fermentation substrate, inoculating Xylaria nigripes, and fermenting.
Furthermore, the geranium wilfordii extract is obtained by extracting with an organic solvent as an extraction solvent. In the present invention, the organic solvent is ethanol or methanol. Further, the organic solvent is an ethanol solution with the volume fraction of 50-70%.
Further, when a 50-70% ethanol solution is used as an extraction solvent, the preparation method of the geranium wilfordii extract comprises the following specific steps: cleaning herba Erodii seu Geranii, drying, and pulverizing into fine powder; according to the weight ratio of 1g: adding 50-70% ethanol solution into 10-20 ml of the mixture, performing reflux extraction for 1-3 times, combining the extracting solutions, removing ethanol under reduced pressure, and concentrating to obtain the product. Further, the volume percentage concentration of the ethanol solution is preferably 55%, and the ethanol solution is preferably extracted for 2 times; the ratio of the feed to the liquid is preferably 1g:10ml, 1g:12ml, 1g:15ml, 1g:16ml, 1g:18ml, 1g:20ml, more preferably 1g:15 ml.
Further, the preparation method of the composition comprises the following steps:
s1, mixing the geranium wilfordii extract with a yeast culture medium, and sterilizing to obtain a primary fermentation substrate; inoculating yeast to the primary fermentation substrate, fermenting, taking fermentation liquor after fermentation is finished, sterilizing, standing and cooling to obtain a primary fermentation active substance;
and S2, mixing the obtained primary fermentation active substance with a Xylaria nigripes culture medium, sterilizing to obtain a secondary fermentation substrate, inoculating the Xylaria nigripes into the fermentation substrate for fermentation, taking the fermentation liquid after the fermentation is finished, concentrating, adding ethyl acetate 1-3 times of the fermentation liquid for extraction, taking the extraction supernatant, removing the solvent, concentrating, and filtering to obtain the Xylaria nigripes.
Further, the adding volume of the geranium wilfordii extract is 10-25% of the volume of the yeast culture medium. Preferably, the geranium wilfordii extract is added in a volume of 10%, 12%, 16%, 18%, 22%, 23% or 25% of the volume of the yeast medium, most preferably 22%.
Further, in step S1, the fermentation parameters are: the pH value is 4.3-5.8; the stirring speed is 100-200 rpm; the ventilation volume is 1-4 VVM; the fermentation temperature is 25-32 ℃; the fermentation time is 2-5 days. Preferably, the fermentation temperature is 28 ℃, 25 ℃ or 30 ℃, more preferably 28 ℃; the fermentation time is preferably 2 days, 3 days, 4 days or 5 days, more preferably 4 days.
Further, the yeast is Candida lamblia (Candida lambda) CGMCC 2.1763.
Further, the yeast culture medium adopted in the invention is a conventional commercial yeast culture medium, specifically, the yeast culture medium can adopt a potato glucose culture medium, and the composition of the potato glucose culture medium is as follows: each 100ml of water contains 20g of potato, 2g of glucose and 1.5-2 g of agar.
Further, in step S2, the fermentation parameters are: the fermentation temperature is 28-30 ℃; fermenting for 2-3 days; the pH value is 5.0-6.3; the ventilation volume is 1-4 VVM; the stirring speed is 120-200 rpm. Preferably, the fermentation temperature is preferably 28 ℃ or 30 ℃, more preferably 30 ℃; the fermentation time is preferably 2 days or 3 days, more preferably 3 days.
Further, the volume of the primary fermentation active substance is 12-20% of the volume of the Xylaria nigripes culture medium. The addition volume is preferably 12%, 15%, 18% or 20%, more preferably 15%.
Further, the Xylaria nigripes culture medium comprises the following components: per 1000ml of water contained: 80g of glucose, 2g of magnesium sulfate, 3g of glutamic acid, 2g of triammonium citrate, 4mg of zinc sulfate and 5mg of ferrous sulfate heptahydrate.
The invention also aims to provide the application of the composition in preparing cosmetics for improving the acne marks and scars.
Further, the cosmetic is essence water, essence milk, a mask or a cream. The composition of the invention can be directly added into cosmetics, and the addition amount is preferably 10-30%, more preferably 10-25%, and more preferably 15%, 18%, 20%, 21%, 22% or 25%. The specific addition amount can be determined according to the use and cost of the cosmetic.
The invention has the following beneficial effects:
(1) according to the invention, the alcohol extract of the geranium wilfordii is sequentially fermented by adopting saccharomycetes and Xylaria nigripes, and tests show that the obtained fermented active substance shows new performance, can effectively inhibit the excessive proliferation of fibroblasts, prevents the excessive synthesis of collagen, and realizes the effect of repairing acne marks and scars, and tests prove that after the geranium wilfordii is continuously used for 28 days, the facial brightness is increased by more than 28 percent compared with that before the geranium wilfordii is used; after the extract is continuously used for 56 days, 90 percent of acne marks on the face of a subject are eliminated, and the extract has no effect on the fine geranium alcohol extract and has remarkable advantages compared with single-strain fermentation.
(2) According to the invention, the organic solvent is adopted to extract the active substances obtained by secondary fermentation, and the obtained fermentation product can provide sufficient nutrients for skin, promote cell regeneration, moisten skin, and is more beneficial to realizing the purpose of repairing the acne marks and scars, so that the acne marks and scars are further effectively repaired, and the irritation is lower.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Yeast medium composition: each 100ml of water contains 20g of potato, 2g of glucose and 1.5-2 g of agar.
The Xylaria nigripes culture medium comprises the following components: per 1000ml of water contained: 80g of glucose, 2g of magnesium sulfate, 3g of glutamic acid, 2g of triammonium citrate, 4mg of zinc sulfate and 5mg of ferrous sulfate heptahydrate.
Example 1 preparation of an alcohol extract of Geranium wilfordii
Cleaning herba Erodii seu Geranii, drying, and pulverizing into fine powder; according to the weight ratio of 1g: adding 55% ethanol solution into 15ml of the raw materials, reflux-extracting for 2 times, mixing extractive solutions, removing ethanol under reduced pressure, and concentrating.
Example 2 preparation of composition for improving acne marks and scars
S1, mixing the geranium wilfordii extract with a yeast culture medium according to the addition volume of the geranium wilfordii extract being 22% of the volume of the yeast culture medium, and sterilizing at 121 ℃ to obtain a primary fermentation substrate;
s2, inoculating Candida lamblia (Candida lambda-lambda) CGMCC2.1763 which is subjected to shaking culture in advance to the primary fermentation substrate to ensure that the concentration of yeast in the substrate is 8 multiplied by 106cfu/ml, fermenting, taking the fermentation liquor after the fermentation is finished, sterilizing, standing and cooling to obtain a primary fermentation active substance, wherein the fermentation parameters are as follows: the pH value is 4.3-5.8; the stirring speed is 100-200 rpm; the ventilation volume is 1-4 VVM; the fermentation temperature is 28 ℃; the fermentation time is 4 days;
s3, mixing the obtained primary fermentation active substance and the Xylaria nigripes culture medium according to the condition that the adding volume of the primary fermentation active substance is 15% of the volume of the Xylaria nigripes culture medium, and sterilizing at 121 ℃ to obtain a secondary fermentation substrate;
s4, inoculating Xylaria nigripes cultured in a shaking table in advance to the fermentation substrate for fermentation, so that the concentration of the Xylaria nigripes in the substrate is 6 multiplied by 106cfu/ml, after fermentation, taking the fermentation liquor, concentrating, adding ethyl acetate 1.5 times of the fermentation liquor for extraction for 2 times, combining the extracted supernatants, removing the solvent, concentrating, and filtering by an ultrafiltration membrane to obtain clear and bright fermented products; the fermentation parameters are as follows: the fermentation temperature is 30 ℃; fermenting for 3 days; the pH value is 5.0-6.3; the ventilation volume is 1-4 VVM; the stirring speed is 120-200 rpm.
Example 3 preparation of composition for improving acne marks and scars
S1, mixing the geranium wilfordii extract with a yeast culture medium according to the addition volume of the geranium wilfordii extract being 16% of the volume of the yeast culture medium, and sterilizing at 121 ℃ to obtain a primary fermentation substrate;
s2, inoculating Candida lamblia (Candida lambda-lambda) CGMCC2.1763 which is subjected to shaking culture in advance to the primary fermentation substrate to ensure that the concentration of yeast in the substrate is 8 multiplied by 106cfu/ml, fermenting, taking the fermentation liquor after the fermentation is finished, sterilizing, standing and cooling to obtain a primary fermentation active substance, wherein the fermentation parameters are as follows: the pH value is 4.3-5.8; the stirring speed is 100-200 rpm; the ventilation volume is 1-4 VVM; the fermentation temperature is 30 ℃; the fermentation time is 3 days;
s3, mixing the obtained primary fermentation active substance with the Xylaria nigripes culture medium according to the condition that the adding volume of the primary fermentation active substance is 18 percent of the volume of the Xylaria nigripes culture medium, and sterilizing at 121 ℃ to obtain a secondary fermentation substrate;
s4, inoculating Xylaria nigripes cultured in a shaking table in advance to the fermentation substrate for fermentation, so that the concentration of the Xylaria nigripes in the substrate is 6 multiplied by 106cfu/ml, after fermentation, taking fermentation liquor, concentrating, adding ethyl acetate 2 times of the fermentation liquor for extraction for 2 times, combining extraction supernatants, removing solvent, concentrating, and filtering with ultrafiltration membrane to obtain clear and transparent fermented product; the fermentation parameters are as follows: the fermentation temperature is 28 ℃; fermenting for 2 days; pH valueThe value is 5.0 to 6.3; the ventilation volume is 1-4 VVM; the stirring speed is 120-200 rpm.
Example 4 preparation of composition for improving acne marks and scars
S1, mixing the geranium wilfordii extract with a yeast culture medium according to the addition volume of the geranium wilfordii extract being 25% of the volume of the yeast culture medium, and sterilizing at 121 ℃ to obtain a primary fermentation substrate;
s2, inoculating Candida lamblia (Candida lambda-lambda) CGMCC2.1763 which is subjected to shaking culture in advance to the primary fermentation substrate to ensure that the concentration of yeast in the substrate is 8 multiplied by 106cfu/ml, fermenting, taking the fermentation liquor after the fermentation is finished, sterilizing, standing and cooling to obtain a primary fermentation active substance, wherein the fermentation parameters are as follows: the pH value is 4.3-5.8; the stirring speed is 100-200 rpm; the ventilation volume is 1-4 VVM; the fermentation temperature is 25 ℃; the fermentation time is 2 days;
s3, mixing the obtained primary fermentation active substance and the Xylaria nigripes culture medium according to the condition that the adding volume of the primary fermentation active substance is 20% of the volume of the Xylaria nigripes culture medium, and sterilizing at 121 ℃ to obtain a secondary fermentation substrate;
s4, inoculating Xylaria nigripes cultured in a shaking table in advance to the fermentation substrate for fermentation, so that the concentration of the Xylaria nigripes in the substrate is 6 multiplied by 106cfu/ml, after fermentation, taking fermentation liquor, concentrating, adding ethyl acetate in an amount which is 3 times that of the fermentation liquor for extraction for 2 times, combining extraction supernatants, removing a solvent, concentrating, and filtering by an ultrafiltration membrane to obtain a clear and transparent fermented product; the fermentation parameters are as follows: the fermentation temperature is 28 ℃; fermenting for 3 days; the pH value is 5.0-6.3; the ventilation volume is 1-4 VVM; the stirring speed is 120-200 rpm.
Experimental examples 5-7 formula and dosage (mass percent%) of essence for improving acne marks and scars
Composition (I)
|
Example 5
|
Example 6
|
Example 7
|
Composition for improving acne mark and scar
|
Example 2, 22%
|
Example 3, 18%
|
Example 4, 20%
|
Butanediol
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3%
|
3%
|
3%
|
1, 2-pentanediol
|
1%
|
1%
|
1%
|
Sodium benzoate
|
0.05%
|
0.05%
|
0.05%
|
Sorbic acid
|
0.01%
|
0.01%
|
0.01%
|
Deionized water
|
Balance of
|
Balance of
|
Balance of |
The preparation method comprises the following steps:
adding deionized water into a stirring pot, heating to 40 deg.C, sequentially adding composition for improving acne and scar, 1.2-pentanediol, and butanediol, stirring, and keeping the temperature for 20 min; cooling to 35 deg.C, adding sodium benzoate and sorbic acid, stirring, inspecting, discharging, and bottling.
Comparative example 1 differs from example 5 in that the preparation process of the composition omits the primary fermentation process, and the specific steps are as follows:
s1, adding the geranium wilfordii alcohol extract prepared according to the example 1, wherein the volume of the geranium wilfordii alcohol extract is 15% of the volume of the Xylaria nigripes culture medium, mixing the geranium wilfordii alcohol extract with the Xylaria nigripes culture medium, and sterilizing at 121 ℃ to obtain a fermentation substrate;
s2, inoculating Xylaria nigripes cultured in a shaking table in advance to the fermentation substrate for fermentation, so that the concentration of the Xylaria nigripes in the substrate is 6 multiplied by 106cfu/ml, after fermentation, taking the fermentation liquor, concentrating, adding ethyl acetate 1.5 times of the fermentation liquor for extraction for 2 times, combining the extracted supernatants, removing the solvent, concentrating, and filtering by an ultrafiltration membrane to obtain clear and bright fermented products; the fermentation parameters are as follows: the fermentation temperature is 30 ℃; fermenting for 3 days; the pH value is 5.0-6.3; the ventilation volume is 1-4 VVM; the stirring speed is 120-200 rpm.
Comparative example 2 differs from example 5 in that the preparation process of the composition omits the secondary fermentation process, and the specific steps are as follows:
s1, mixing the geranium wilfordii extract with a yeast culture medium according to the addition volume of the geranium wilfordii extract being 22% of the volume of the yeast culture medium, and sterilizing at 121 ℃ to obtain a primary fermentation substrate;
s2, inoculating Candida lamblia (Candida lambda-lambda) CGMCC2.1763 which is subjected to shaking culture in advance to the primary fermentation substrate to ensure that the concentration of yeast in the substrate is 8 multiplied by 106cfu/ml, fermenting, collecting fermentation liquid, sterilizing, and standingCooling to obtain primary fermentation active substances, wherein the fermentation parameters are as follows: the pH value is 4.3-5.8; the stirring speed is 100-200 rpm; the ventilation volume is 1-4 VVM; the fermentation temperature is 28 ℃; the fermentation time was 4 days.
Comparative example 3 differs from example 5 in that in the preparation of the composition, in step S4, an equal amount of n-butanol was added instead of ethyl acetate for extraction, and the remaining parameters and operation were as in example 5.
Comparative example 4 differs from example 5 in that in the primary fermentation process, yeast is replaced by lactobacillus plantarum, which is fermented under the following conditions: the temperature is 30-35 ℃, the rotating speed is 120-200 rpm, the pH is 5-5.5, the fermentation time is 4 days, and the other parameters are the same as those in the embodiment 5.
Test example one, patch test
Taking the skin care essence water prepared in the embodiment 5-7, inviting 30 subjects (each test sample uses the same batch of subjects) to accord with the volunteer selection standard of the subjects; a closed test is adopted, 2ml of a test object is placed in a patch applicator, a hypoallergenic adhesive tape is applied to the arm curve side of a test object, the test area is at least 5 x 5cm, the test object is stretched to the arm when the test object is applied, the test object is applied from the lower part to the upper part, the adhesive tape is slightly pressed by the palm to discharge air, the test object is tightly attached to the skin for 24 hours, a blank control group does not use a test sample, the test sample is respectively interpreted according to the following interpretation standards 48 hours after the patch applicator is removed, test results are counted, and the test results are shown in the following table 1.
Interpretation criteria: (one) negative reaction; (+ -suspicious reaction: only mild erythema; (+) weak positive: erythema, infiltrates, and possibly small amounts of papules; strong positive (++): erythema, infiltrates, papules, blisters; (+++) very positive: erythema, infiltrates, papules, blisters, bulla.
TABLE 1 Patch test results
Group of
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Number of negative cases/negative rate
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Number of positive cases/Positive Rate
|
Example 5
|
30/100%
|
0/0
|
Example 6
|
30/100%
|
0/0
|
Example 7
|
30/100%
|
0/0 |
Test example two, acne mark repair and scar test
1.1 Experimental sample: the essence liquids of examples 5 to 7 and comparative examples 1 to 4.
1.2 Experimental methods: selecting 84 subjects with obvious acne on face and acne marks and scars after evaluation, wherein the average age is 28 +/-2 years, the subjects are randomly divided into 7 groups, and each group comprises 12 persons; after cleaning the face in the morning and evening, taking 1 yuan coin-sized essence water, smearing and beating, massaging the essence liquid by using a warm palm until the essence liquid is absorbed by the skin, continuously using the essence liquid for 56 days, photographing and recording the face conditions of a subject before and after use on the 56 th day through a skin face analyzer VISA and face analysis software Image-Pro Plus, analyzing the face acne mark elimination quantity or fading degree of the subject before and after use, judging the effect of the essence liquid on repairing the acne marks and scars according to the following judgment standard, counting the number of examples, and showing the test result in table 2; simultaneously, the minoxidil analysis tester CN2600D was used to test L values of the facial skin of the subject before and after use on day 28, the brightness increase value was calculated, and the change of the facial skin color of the subject was judged, and the test results are shown in table 3.
And (3) judging standard:
excellent: the pox printed on 80% or more of the original parts with the pox prints are eliminated; good: eliminating or only leaving the acne marks with light color at 60-80% of the original parts with the acne marks; preferably: eliminating 40-60% of the acne marks on the original parts with the acne marks or only leaving the acne marks with light color; poor: the pox marks on the original parts with the pox marks are eliminated or left with obvious colors below 40 percent.
Brightness increase ratio (L value after use-L value before use)/L value before use × 100%
TABLE 2 post-treatment of acne marks and scar repair in the subjects 56 days
Sample (I)
|
Excellence/rate
|
Good/rate
|
Better rate/rate
|
Poor/rate
|
Example 5
|
12/100%
|
0/0
|
0/0
|
0/0
|
Example 6
|
11/91.67%
|
1/8.33%
|
0/0
|
0/0
|
Example 7
|
11/91.67%
|
1/8.33%
|
0/0
|
0/0
|
Comparative example 1
|
0/0
|
0/0
|
5/41.67%
|
7/58.33%
|
Comparative example 2
|
0/0
|
2/16.67%
|
3/25.0%
|
7/58.33%
|
Comparative example 3
|
5/41.67%
|
3/25.0%
|
1/8.33%
|
3/25.0%
|
Comparative example 4
|
7/58.33%
|
1/8.33%
|
4/33.33%
|
0/0 |
TABLE 3 changes in the skin tone of the subjects' face after 28 days of use
Sample (I)
|
Using the brightness increase ratio (%)
|
Example 5
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32.56%
|
Example 6
|
30.42%
|
Example 7
|
28.73%
|
Comparative example 1
|
9.62%
|
Comparative example 2
|
11.75%
|
Comparative example 3
|
16.35%
|
Comparative example 4
|
20.29% |
As can be seen from tables 2 and 3, the fermentation product obtained by adopting the composite bacteria fermentation and the organic solvent extraction has more excellent effect of repairing the acne marks and scars, and 90% of the acne marks on the face of a subject are eliminated after the continuous use for 56 days; the facial brightness was increased by 28% or more after 28 days of use compared to before use, with the best results from example 5.
Analysis of other groups shows that the fermentation product obtained by independently adopting the saccharomycetes or the Xylaria nigripes does not have good functions of repairing the acne marks and brightening the skin color; if the ethyl acetate is replaced by the n-butanol for extraction, the performance of the fermentation product is greatly influenced.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.