CN111329799B - Preparation method and application of nonirritating gynura divaricata active substance - Google Patents

Preparation method and application of nonirritating gynura divaricata active substance Download PDF

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CN111329799B
CN111329799B CN202010098151.0A CN202010098151A CN111329799B CN 111329799 B CN111329799 B CN 111329799B CN 202010098151 A CN202010098151 A CN 202010098151A CN 111329799 B CN111329799 B CN 111329799B
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fermentation
gynura divaricata
irritation
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陈广文
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Beijing guosutang Biotechnology Co.,Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/70—Biological properties of the composition as a whole
    • A61K2800/72—Hypo-allergenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine

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Abstract

The invention relates to a preparation method and application of a nonirritating gynura divaricata active substance, wherein the preparation method of the active substance comprises the following steps: s1, preparing the gynura divaricata alcohol extract: taking roots or stems of gynura divaricata as a raw material, cleaning, crushing, taking 65-85% ethanol solution as an extraction solvent, wherein the material-liquid ratio is 1: 5-10, performing heating reflux, removing ethanol, and concentrating to prepare an ethanol extract; s2, fermentation: inoculating the Xylaria nigripes subjected to secondary culture into a 30L fermentation tank by using the inoculation amount of 10%, adding the alcohol extract into the fermentation tank, and fermenting at the temperature of 25-30 ℃ for 40-80 h to obtain fermentation filtrate; s3, filtering the fermentation filtrate by an ultrafiltration membrane to obtain the fermentation active substance. The invention adopts the Xylaria nigripes biological fermentation gynura divaricata alcohol extract to obviously reduce the irritation to the skin, the irritation strength is reduced from irritation to nonirritant, the active matter is endowed with better moisturizing performance after the Xylaria nigripes fermentation, and the Xylaria nigripes alcohol extract can be used as an active ingredient with whitening, moisturizing and nonirritant to be applied to the preparation of cosmetics.

Description

Preparation method and application of nonirritating gynura divaricata active substance
Technical Field
The invention relates to the technical field of cosmetics, in particular to a preparation method of a nonirritating gynura divaricata active substance and application of the nonirritating gynura divaricata active substance in preparing a nonirritating cosmetic and/or skin care product with whitening and moisturizing effects.
Background
Gynura divaricata (Gynura divaricata DC) is a plant of genus Gynura of Compositae, also called Gynura divaricata, and hypoglycemic grass, etc., and is widely used for treating hyperlipidemia and diabetes, for example, see Chinese patent application CN102631391A, CN104644734A and CN 110279873A.
At present, the overground part of the gynura divaricata is most applied, the overground part extract of the gynura divaricata contains a plurality of components, and the known chemical components of the overground part extract of the gynura divaricata mainly contain alkaloids, flavonoids, triterpenes, sterols, nucleosides, phenolic acids, esters and the like. The content of volatile oil of different medicinal parts of gynura divaricata is analyzed by scholars, and a plurality of chemical components are identified, wherein the content of caryophyllene, myrcene D and the like in the volatile oil of root, stem and leaf parts is the highest. At present, the inventor tries to form an anti-aging mask (CN108703936A) by using the alcohol extract of gynura divaricata, peony pistil flavone, loquat leaf flavone, pearl powder, avocado oil and rice washing water, however, the anti-aging function is the effect achieved by the synergy of all the components, the biological activity of the gynura divaricata alcohol extract cannot be determined, and at present, the use of related extracts of gynura divaricata for whitening is not seen.
Disclosure of Invention
Based on the above, the invention aims to provide a preparation method of a nonirritating gynura divaricata active substance and application of the nonirritating gynura divaricata active substance in preparation of whitening, moisturizing and nonirritating cosmetics and/or skin care products.
In the process of developing a new whitening active ingredient, the inventor of the invention unexpectedly finds that the gynura divaricata aqueous extract and the alcohol extract both show better whitening activity (the tyrosinase inhibition rate of the extract of 0.1mg/mL reaches more than 50%), and particularly the alcohol extract (the tyrosine inhibition rate of the alcohol extract of 0.1mg/mL 80% is 64.48%). But in the skin irritation test, both the alcohol extract and the water extract are found to generate larger irritation to the skin (the alcohol extract is larger than the water extract), and multiple skin irritation tests show that the irritation strength of the two extracts is medium irritation, and the irritation is increased along with the increase of the ethanol solution.
Therefore, the difficulty in developing the gynura divaricata extract whitening products mainly lies in how to reduce the irritation of the gynura divaricata extract to the skin. The inventor of the invention finds that the irritability of the gynura divaricata extract can be caused by the fact that caryophyllene existing in the gynura divaricata extract is relatively irritant to the skin after being oxidized, and easily causes allergy, so that the gynura divaricata extract is relatively irritant to the skin. The alcohol extract contains more caryophyllene than the water extract, so the alcohol extract has larger irritation. Further research shows that the caryophyllene is easy to be allergic probably because double bonds of the caryophyllene are easy to be oxidized, and the formed peroxide is unstable in nature and causes irritation to the skin.
Therefore, under the above background, the present invention proposes the following technical solutions: a method for preparing a nonirritating gynura divaricata active substance comprises the following steps:
s1, preparing the gynura divaricata alcohol extract: taking roots or stems of gynura divaricata as a raw material, cleaning, crushing, taking 65-85% ethanol solution as an extraction solvent, wherein the material-liquid ratio is 1: 5-10, performing heating reflux, removing ethanol, and concentrating to prepare an ethanol extract;
s2, fermentation: inoculating the Xylaria nigripes subjected to secondary culture into a 30L fermentation tank by using the inoculation amount of 10%, adding an alcohol extract into the fermentation tank, and fermenting at the temperature of 25-30 ℃ for 40-80 h to obtain a fermentation filtrate;
s3, filtering the fermentation filtrate by an ultrafiltration membrane to obtain the nonirritating active substance.
Further, in the step S1, the concentration of the ethanol solution is 80%; and/or the feed-liquid ratio is 1: 8; and/or the extraction temperature is 60-85 ℃; and/or the crushing means that the roots or stems of the gynura divaricata are crushed into powder with the granularity of 30-60 meshes.
Further, in the step S1, the extraction temperature is 70 ℃; and/or pulverizing root or stem of gynura divaricata into powder with particle size of 40 meshes.
Further, the fermentation medium adopted in the fermentation process of step S2 is composed of: 60-80 g/L of glucose, 5-10 g/L of peptone, 1-3 g/L of citric acid, 1-3 mg/L of magnesium sulfate and 1-2 mg/L of zinc sulfate, and the pH value is 5.0-6.0.
Further, the adding amount of the alcohol extract in the step S2 is 1-15%.
Further, the fermentation temperature in the step S2 is 25 ℃, and the fermentation time is 60 h.
Further, in the fermentation process of the step S2, the stirring speed is 100-200 rpm, the ventilation volume is 1-4 vvm, and the tank pressure is 0.03-0.05 MPa.
The invention also provides application of the active substance in preparation of non-irritant cosmetics and/or skin care products with whitening and moisturizing effects.
In addition, the invention provides a skin care and/or cosmetic product comprising a nonirritating active according to any one of claims 1 to 8.
In order to reduce the irritation of the gynura divaricata alcohol extract, the invention adopts Xylaria nigripes (KI) Sacc ] which belongs to the Ascomycotina, Xylaria nigripes (KI) and Xylaria nigripes (KI) belongs to the family of Xylaria, Xylaria. In fermentation, it is well known that the selection of species is of critical importance, and the advantages of Xylaria nigripes over other species are: after fermentation, the gynura divaricata alcohol extract has greatly reduced irritation (the irritation strength is reduced from irritation to nonirritant), and the active matter has excellent moisture retention, which is an advantage that other strains do not have. It is further speculated that the reason for the reduction of the irritation of the alcohol extract of gynura divaricata may be that the caryophyllene with sensitization property finally forms an oxidation product with more stable property and low sensitization in the fermentation process, or the Xylaria nigripes generates a substance for resisting the irritation of the alcohol extract of gynura divaricata in the complex fermentation process, so that the irritation of the active substance to the skin is greatly reduced, and experiments show that the Schizophyllum communa fermentation does not have the effect of greatly reducing the irritation, and the reduction effect is speculated to be related to the strain type, so the higher probability is that the Xylaria nigripes reduces the irritation of the alcohol extract of gynura divaricata. At present, no example of fermentation of the alcohol extract of gynura divaricata by adopting Xylaria nigripes is seen.
Therefore, the invention has the following beneficial effects:
the invention adopts Xylaria nigripes to ferment the gynura divaricata alcohol extract, obviously reduces the irritation to the skin, reduces the irritation strength from the irritation to the nonirritant, endows the active substance with better moisturizing performance after the Xylaria nigripes is fermented, and can be used as an active ingredient with whitening, moisturizing and nonirritant to be applied to the preparation of cosmetics.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments of examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.
In the examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
EXAMPLE one preparation of Alcoholic extract of Gynura divaricata
Taking the root of gynura divaricata as a raw material, cleaning, pulverizing into powder with the particle size of 40 meshes, adding 80% ethanol solution with the concentration of 8 times of the total amount of the powder, heating and refluxing for 2 times, extracting for 1.5h each time at the extraction temperature of 70 ℃, filtering, combining filtrates, removing ethanol under reduced pressure, and concentrating to obtain the gynura divaricata extract.
Extracting with 75% and 65% ethanol solution to obtain 75% ethanol extract and 65% ethanol extract, respectively.
EXAMPLE two, aqueous extract preparation of gynura divaricata
Taking the root of gynura divaricata as a raw material, cleaning, crushing into powder of 40 meshes, adding hot water with the temperature of 80 ℃ which is 8 times of the total amount of the coarse powder, leaching for 2 times, 6 hours each time, filtering, combining the filtrates, and concentrating to obtain the gynura divaricata extract.
EXAMPLE III comparison of whitening Activity and irritation of Alcoholic extract and aqueous extract of Gynura divaricata
3.1 comparison of whitening Activity
Diluting each group of samples into multi-concentration samples by using a disodium hydrogen phosphate-citric acid buffer solution (pH6.8, 0.05mol/L), putting each concentration sample into a glass test tube, and respectively adding a tyrosinase solution; reacting in 30 ℃ water bath for 10min, sequentially adding levodopa solution into each group of glass test tubes, reacting in 30 ℃ water bath for 10min, and measuring the OD value at 475nm, wherein the experimental system is shown in Table 1.
Figure BDA0002385904230000041
In the formula: t is the light absorption value of the sample tube, namely the light absorption value of the solution after the sample reacts with tyrosinase; t is0The background light absorption value of the sample is obtained; c is the average value of the light absorption values of the enzyme reaction tubes for 3 times, namely the light absorption value of the reaction of tyrosinase and dopa when no sample is added; c0The background light absorption value of the solvent is shown.
TABLE 1 Experimental System design
T-sample tube T0Background of sample C-DPPH tube C0Background of solvent
Sample liquid (mL) 1.0 1.0 - -
Buffer solution (mL) - 0.5 1.0 1.5
Tyrosinase enzyme solution (mL) 0.5 - 0.5 -
Levodopa solution (mL) 2.0 2.0 2.0 2.0
TABLE 2 tyrosinase activity inhibition results produced by each sample
Figure BDA0002385904230000042
Figure BDA0002385904230000051
Note: compared with the corresponding concentration of the gynura divaricata 80% alcohol extract,*P≤0.05，**P≤0.01。
as can be seen from the above table, the tyrosinase inhibition effect of the gynura divaricata 80% alcohol extract in each extract is strongest, and the tyrosinase inhibition rate of the gynura divaricata 80% alcohol extract with the concentration of 0.1mg/mL is 64.48%, so that the gynura divaricata has a better tyrosinase activity inhibition effect, and the gynura divaricata alcohol extract is prompted to have a certain whitening function. Compared with alcohol extract, the water extract has weaker inhibiting ability to tyrosinase activity.
3.2 comparison of skin irritation
Test animals: the New Zealand white rabbits are fed in a single cage, and the weight of the New Zealand white rabbits is 2.0-3.0 kg.
The test method comprises the following steps: 2mL of test sample (the concentration is 100 mug/mL) is taken from each group, the test sample is ensured to be in good contact with the skin, the back hairs on two sides of the spinal column of the test animal are cut off 24 hours before the test, the epidermis is not damaged, the hair removal range is 3cm multiplied by 3cm, the smearing area is 2.5 multiplied by 2.5cm, the test sample is smeared on one side, the control is made on one side, the test sample is smeared for 1 time every day, and the smearing is continuously carried out for 14 days. The hair is sheared before each smearing from day 2, the residual test substance is removed by warm water, and the result is observed after 1 h.
Test scoring: the test results were scored according to the reaction scoring criteria in the multiple skin irritation test method in the cosmetic hygiene code (2007 edition) (table 3), the average score per animal per day was settled according to the following formula, and the skin irritation intensity was judged according to table 4, and the test results are shown in table 5 below.
Figure BDA0002385904230000052
TABLE 3 skin irritation response score
Figure BDA0002385904230000053
Figure BDA0002385904230000061
Table 4 skin irritation intensity grading
Integral mean value Strength of
0～＜0.5 Has no irritation
0.5～＜2.0 Light irritation
2.0～＜6.0 Middle irritation
6.0～8.0 Strong irritation
TABLE 5 multiple skin irritation test results
Figure BDA0002385904230000062
Figure BDA0002385904230000071
As can be seen from table 5 above, the 80% alcohol extract, 65% alcohol extract, 75% alcohol extract and water extract of gynura divaricata in the multiple skin irritation experiments respectively have the average value of 3.88, 3.59, 3.25 and 2.18 per animal per day, the irritation strength is evaluated as medium irritation, and the irritation of the gynura divaricata alcohol extract to the skin is found to be increased along with the increase of the ethanol concentration.
EXAMPLE four Effect of strain type on active whitening, irritation and moisturizing Properties
4.1 fermentation parameter determination
The influence of each parameter on the related performance of the active substances in the fermentation process is examined, the specific parameter settings are shown in the following table 6, and the fermentation active substances are prepared according to the parameters shown in the following table 6.
TABLE 6 fermentation parameters
Figure BDA0002385904230000072
The culture medium adopted in the fermentation of Xylaria nigripes comprises the following components: glucose 70g/L, peptone 6g/L, citric acid 1.5g/L, magnesium sulfate 1.5mg/L and zinc sulfate 1.5mg/L, pH 5.5.
The culture medium adopted in the Schizophyllum commune fermentation comprises the following components: 2% of glucose, 0.3% of beef extract and NH4Cl 0.1％、KH2PO40.05、MgSO40.05% and the rest of distilled water.
4.2 irritation test
The irritancy of actives a-D was compared according to the 3.2 skin irritation test and the 14 day integrated mean per animal and the daily integrated mean per animal were calculated and the results are shown in table 7 below.
TABLE 7 comparison of the results of the reduction in irritation for each active
Test article Mean of daily score for each animal Intensity of stimulus
Herba gynostemmatis segeti 80% alcoholic extract 3.88 Middle irritation
Active A 0.36 Has no irritation
Active substance B 0.48 Has no irritation
Active C 0.27 Has no irritation
Active substance D 2.54 Middle irritation
As can be seen from Table 7, the addition of two microorganisms for fermentation can reduce the integral average value of each animal per day, and has the effect of reducing skin irritation; the addition of Schizophyllum commune for fermentation reduces the average value of each animal per day compared with that before fermentation (3.88 is reduced to 2.54), but the stimulation intensity is still moderate; the addition of Xylaria nigripes for fermentation obviously has a more direct effect on reducing skin irritation, and the integral mean value of each animal per day is 0.36-0.48.
4.3 whitening and moisturizing Performance test
The whitening activity of actives a-D was tested according to test 3.1, with the results shown in table 8 below (only data at a concentration of 100 μ g/mL are shown); the moisture retention effect of the active substances A to D was measured by using a Corneometer CM 825 from Courage + Khazaka electronic GmbH, and a positive control group was set, and the increase rate of the skin moisture content was calculated according to the following formula, and the results were as shown in Table 9 below, and it was examined whether the types of microorganisms have potential influences on the whitening and moisture retention properties of the active substances.
Figure BDA0002385904230000081
In the formula, MMV0MMV on skin before application; MMVtMMV of the skin t time after application.
TABLE 8 results of the Effect of different microorganisms on the whitening Activity of actives after fermentation
Figure BDA0002385904230000082
Figure BDA0002385904230000091
Note: compared with the gynura divaricata 80% alcohol extract group,*P≤0.05，**P≤0.01。
TABLE 9 Effect of different microorganisms on the moisturizing Properties of actives after fermentation
Figure BDA0002385904230000092
Note: compared with the 80 percent alcohol extract group of gynura divaricata in the same time period,*P≤0.05，**P≤0.01。
as can be seen from the above table 7, the Xylaria nigripes fermented gynura divaricata alcohol extract has very obvious advantage in the aspect of reducing the irritation, the irritation strength of the active matter to the skin is changed from moderate irritation to non-irritation, and compared with Schizophyllum commune, the Xylaria nigripes is more beneficial to reducing the irritation of the gynura nigripes alcohol extract to the skin.
In addition, as can be seen from tables 8 to 9, the whitening activity was increased to a very small extent after fermentation with Xylaria nigripes, but the effect was not significant, and it was seen that the fermentation was performedThe whitening effect on the active substances is not greatly contributed, and the whitening effect plays a great role in reducing stimulation; meanwhile, the active substance A obtained after the fermentation of the Xylaria nigripes is found to have obviously improved moisturizing performance compared with the 80 percent alcohol extract group of the gynura divaricata, and has very obvious difference (**P is less than or equal to 0.01), which shows that the active matter is endowed with better moisturizing performance after the Xylaria nigripes is fermented. In addition, the moisturizing performance of the active substances after the schizophyllum commune fermentation is also obviously improved compared with that before the schizophyllum commune fermentation, which is probably because the schizophyllum commune fermentation can generate schizophyllan to play a certain role in moisturizing.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (9)

1. A preparation method of a nonirritating gynura divaricata active substance is characterized by comprising the following steps:
s1, preparing the gynura divaricata alcohol extract: taking roots or stems of gynura divaricata as a raw material, cleaning, crushing, taking 65-85% ethanol solution as an extraction solvent, wherein the material-liquid ratio is 1: 5-10, performing heating reflux, removing ethanol, and concentrating to prepare an ethanol extract;
s2, fermentation: inoculating the Xylaria nigripes subjected to secondary culture into a 30L fermentation tank with the inoculation amount of 10%, adding the gynura divaricata alcohol extract into the fermentation tank, and fermenting at 25-30 ℃ for 40-80 h to obtain fermentation filtrate;
s3, filtering the fermentation filtrate by an ultrafiltration membrane to obtain the gynura divaricata active matter.
2. The method according to claim 1, wherein in step S1, the ethanol solution has a concentration of 80%; the material-liquid ratio is 1: 8; the extraction temperature is 60-85 ℃; the crushing refers to crushing the roots or stems of the gynura divaricata into powder with the granularity of 30-60 meshes.
3. The method according to claim 2, wherein the extraction temperature is 70 ℃ in step S1; pulverizing root or stem of Gynura divaricata into powder with particle size of 40 mesh.
4. The method of claim 1, wherein the fermentation medium used in the fermentation of step S2 comprises: 60-80 g/L of glucose, 5-10 g/L of peptone, 1-3 g/L of citric acid, 1-3 mg/L of magnesium sulfate and 1-2 mg/L of zinc sulfate, and the pH value is 5.0-6.0.
5. The preparation method of claim 1, wherein the amount of the gynura divaricata alcohol extract added in the step S2 is 1-15%.
6. The method according to claim 1, wherein the fermentation temperature in step S2 is 25 ℃ and the fermentation time is 60 hours.
7. The method according to claim 1, wherein in the fermentation process of step S2, the stirring speed is 100-200 rpm, the aeration rate is 1-4 vvm, and the tank pressure is 0.03-0.05 MPa.
8. Use of the gynura divaricata active substance as defined in any one of claims 1 to 7 in preparation of non-irritating skin care products with whitening and moisturizing effects.
9. A skin care product comprising the gynura divaricata active substance according to any one of claims 1 to 7.
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