CN113876614A - Glycyrrhizic anhydride and tremella polysaccharide degradation product compound composition and application thereof - Google Patents
Glycyrrhizic anhydride and tremella polysaccharide degradation product compound composition and application thereof Download PDFInfo
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- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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Abstract
The invention discloses a glycyrrhizic acid anhydride (GAMS) and tremella polysaccharide degradation product (TAP) compound composition, wherein the active ingredients of the compound composition consist of the glycyrrhizic acid anhydride and the tremella polysaccharide degradation product; the weight ratio of the glycyrrhetic anhydride to the tremella polysaccharide degradation product is 1:1-1: 10. It was found that administration of a combination of gam and TAP, particularly when the weight ratio is 1:1-1: when 10, the moisturizing synergistic effect is good; the inventor determines the protection effect of a sample on a dry damaged HaCaT cell and the influence of the sample on the content of AQP3 in the dry damaged HaCaT cell by in vitro moisturizing activity, adds the moisturizing composition into a cosmetic variety, and determines the per-epidermal water loss rate at different times, which shows that the moisturizing composition has the capability of improving the self-drying resistance of the cell on the molecular level and the cosmetic has stronger moisturizing effect.
Description
Technical Field
The invention relates to the technical field of cosmetics, in particular to a compound composition of glycyrrhizic anhydride and tremella polysaccharide degradation products and application thereof.
Background
Skin moisturization is the most essential requirement for skin care. The stratum corneum of human skin is mainly composed of anucleate keratinocytes, keratin filaments, amino acid compounds and natural moisturizing factors, and has the functions of retaining moisture, a barrier and the like. The water source in the stratum corneum of the skin is mainly sweat secreted by sweat glands, and 20% of water is tightly bound to natural moisturizing factors and skin lipids, so that the formed sebum membrane emulsified by sweat and water and the natural moisturizing factors become two most important factors for skin moisturizing. The moisturizing effect of cosmetics and raw materials can be evaluated by indexes such as Natural Moisturizing Factors (NMF), Aquaporins (AQPs), tight junction proteins (Claudins), Hyaluronic Acid (HA) and the like in skin keratinocytes cultured in vitro.
Aquaporins (AQPs) are proteins located on cell membranes that significantly increase the permeability of the cell membrane to water molecules. Since there are no blood vessels in the epidermis and the water in the epidermis diffuses from the dermis, AQPs between epidermal cells constitute the major pathway for intercellular water transport. To date, 13 AQPs have been found, of which AQP3 is the most abundant aquaporin subtype expressed in human skin, and AQP3 is permeable to glycerol and the like in addition to water molecules. At present, the change of AQP3 content is quantitatively detected mainly by Western Blot hybridization, immunohistochemical staining method semi-quantitative detection and commercial kit ELISA method, and can be used as one of evaluation indexes of effective components of cosmetic moisturizing effect.
At present, a great deal of research shows that glycyrrhizin (Glycyrrhiza Acid Monoamonium Salt n-Hydrate, GAMS) has the effects of resisting virus, protecting liver, reducing transaminase, regulating immunity, protecting cell membrane, relieving inflammatory reaction and the like (the clinical observation that Chaihei A-2b interferon is combined with compound glycyrrhizin to treat chronic hepatitis [ J ] Shandong medicine, 2011, 33 (1): 108-109.). The glycyrrhizic acid preparation has similar pharmacological effects to glucocorticoid medicine in anti-inflammatory effect and no obvious side effect, and may be used clinically in treating bronchitis, acute and chronic hepatitis caused by different reasons and AIDS virus resisting effect. Glycyrrhizic acid drugs are getting more and more attention, and some other effects thereof are slowly recognized. Guo et al found that AQP 3expression was increased in HaCat cells treated with appropriate concentrations of Compound Glycyrrhizin (CG), and speculated that CG may have a role in repairing skin barrier function in addition to anti-inflammatory effects (Guo Bo, Song Weimin, Hu Lingling. Effect of compound glycyrrhizin on the aqua porrin 3expression in HaCaT cell cultured in vitro [ J ]. Chinese Journal of Dermatoveneology of Integrated genetic & Western Medicine 2011, 10(4): 219-.
Golden fungus (Tremella aurantiaba, TA) is a rare and precious edible fungus, is recorded in Chinese medicinal fungi, has warm and cold properties and sweet taste, can reduce phlegm and relieve cough, is mainly used for treating diseases such as lung heat, excessive phlegm, asthma and the like, and has the effects of lubricating skin and the like due to fine colloid, white and smooth taste and fragrant smell of the golden fungus. Tremella aurantialba polysaccharides have relatively large molecular weight and are difficult to absorb, and the absorption rate of TAP by human body is improved after the molecular weight of Tremella Aurantialba Polysaccharides (TAP) is reduced, and the research on the moisturizing effect of Polysaccharide acid degradation products after the aqueous acid degradation of Tremella aurantialba has also been reported (Zhicai Zhang, Yin Li; Kechang Zhang, Application of Statistical Analysis for the Optimization of Mycelia and Polysaccharide Production by Tremella aurantia, Food Technology and Biotechnology, Volume 45, Issue 1.2007.PP 45-50).
However, no one has found the synergistic effect of the glycyrrhizic anhydride and the tremella aurantialba polysaccharide degradation product in the research process.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention mainly aims to provide a compound composition of glycyrrhiza uralensis anhydride and tremella aurantialba polysaccharide degradation products; the active ingredients of the compound composition consist of oxalic anhydride and tremella polysaccharide degradation products.
The invention also aims to provide the application of the compound composition in preparing moisturizing cosmetics; pharmacological tests prove that the compound composition has the function of cooperative moisture retention.
A compound composition of glycyrrhizic acid anhydride and tremella polysaccharide degradation product, the active ingredient of the compound composition is made up of glycyrrhizic acid anhydride and tremella polysaccharide degradation product; the weight ratio of the glycyrrhetic anhydride to the tremella polysaccharide degradation product is 1:1-1: 10.
Preferably, the tremella aurantialba polysaccharide degradation product is an acid degradation product of tremella aurantialba extracted by an aqueous enzymatic method.
More preferably, the degradation product of the tremella polysaccharide has a total sugar content of 68.58% as measured by phenol-sulfuric acid method.
More preferably, the enzyme in the aqueous enzymatic extraction is pectinase, and the weight ratio of the pectinase to the tremella aurantialba powder is 1: 10.
the compound composition also contains one or more of emulsifier, thickener, antiseptic, bacteriostatic agent, humectant, penetrant and whitening agent which can be added during cosmetic preparation.
Preferably, the weight ratio of the glycyrrhetic anhydride to the aureobasidin degradation product is 1:5-1: 8.
The compound composition of the degradation products of the glycyrrhiza uralensis anhydride and the tremella aurantialba polysaccharide is applied to preparing moisturizing cosmetics.
Preferably, the dosage of the compound composition of the glycyrrhiza uralensis anhydride and the tremella polysaccharide degradation product is 0.1% -3%. The compound composition is added into cosmetics, and the moisturizing cosmetics are prepared according to a conventional preparation process.
Compared with the prior art, the invention has the beneficial effects that: the inventor finds that the molecular weight of polysaccharide is reduced after tremella aurantialba is subjected to aqueous enzymatic extraction and acid degradation, the polysaccharide is favorable for absorption, and the GAMS and the TAP are combined for administration, particularly when the weight ratio of the liquiritin (GAMS) to the tremella aurantialba polysaccharide degradation product (TAP) is 1:1-1:10 has good moisture-keeping synergistic effect when being compatible; the inventor shows that the moisturizing composition has the effects of improving the self-drying resistance of cells and enhancing the moisturizing effect of skin on the molecular level and has no toxic or side effect on HaCaT cells through in-vitro moisturizing activity determination, namely the protective effect of a sample on the HaCaT cells with dry damage and the influence of the sample on the content of AQP3 in the HaCaT cells with dry damage. The moisturizing cream is proved to have good moisturizing effect by adding the moisturizing composition into cosmetic varieties and measuring the skin hydration degree of healthy people at different times and the per-epidermal water loss rate at different times.
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FIG. 1 skin hydration levels at different times
FIG. 2 trans-epidermal water loss at different times
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Experiment 1: preparation of tremella aurantialba polysaccharide and degradation product thereof
Cleaning and drying Auricularia auricula (provided by Xishuangbanna tropical garden of Chinese academy of sciences), pulverizing, weighing above dry powder 30g, adding 10 times of water, adding pectase (purchased from Fluka company) 3g, performing enzymolysis at 35 deg.C for 2h, adding 20 times of water, soaking in boiling water for 2h, centrifuging (7000r/min,10min), collecting supernatant, adding anhydrous ethanol to reach 80%, precipitating with ethanol at 4 deg.C for 24h, centrifuging (3000r/min,10min), collecting precipitate, and drying to obtain Auricularia auricula polysaccharide. The Tremella aurantialba polysaccharides are formulated as a solution and analyzed by HPLC to have a molecular weight of about 12000 kD.
Taking 0.6g of tremella aurantialba polysaccharide, and mixing the raw materials according to the weight ratio of 1: adding 0.5mol/L HCl into the mixture according to the proportion of 30, stirring the mixture evenly, performing magnetic stirring reaction at 80 ℃ for degradation for 4 hours, centrifuging the mixture at 6000r/min for 10 minutes, drying and grinding the mixture in a 65 ℃ oven for later use. The tremella aurantialba polysaccharide degradation product is prepared into a solution, and the molecular weight of the solution is about 67kD by HPLC analysis.
Experiment 2: determination of polysaccharide content
Adopts a phenol-sulfuric acid method and takes glucose as a standard substance.
And (3) preparing a standard curve: drying the glucose standard product to constant weight at 105 ℃, taking 20mg, preparing a solution by using ultrapure water, and fixing the volume to 100mL in a volumetric flask to obtain the glucose standard product solution. The standard solution was diluted with ultrapure water to obtain a set of solutions having concentrations of 30, 50, 100, 150, and 200. mu.g/mL. Sucking 1.0mL of glucose solution, placing the glucose solution in 5 test tubes in sequence, adding 0.5mL of 5% phenol and 2.5mL of concentrated sulfuric acid respectively, shaking gently, mixing, placing at 25 ℃ for 20min, and measuring the absorbance value at 490nm of a spectrophotometer. And drawing a standard curve by taking the concentration of the glucose solution as an abscissa and the absorbance value as an ordinate. The method for measuring the sugar content in the tremella aurantialba polysaccharide before and after degradation is the same as that of a standard product, the polysaccharide after being dried and constant in weight is prepared into solution, the absorbance value is measured at 490nm according to the method, and the polysaccharide content is calculated according to a standard curve. The total sugar content of the tremella aurantialba polysaccharide is about 54.38% measured by a phenol-sulfuric acid method, and the total sugar content of the degradation product of the tremella aurantialba polysaccharide is 68.58%.
Experiment 3: in vitro moisturizing Activity
Detecting in vitro moisturizing activity by weighing method, and collecting saturated K2CO3Solution and saturated (NH)4)SO4Placing the solution in a sealed desiccator, placing the desiccator in a climatic chamber at 20 deg.C to make the Relative Humidity (RH) of the desiccator 43%, removing fat layer of fresh Corii Sus Domestica, unhairing with forceps, cleaning, and refrigerating at-20 deg.C for use. In application, the refrigerated pigskin is thawed in Phosphate Buffered Saline (PBS) with pH 7.4 for 30 min. Taking the pigskin with a smooth and undamaged surface, cutting the pigskin into a square with the side length of about 1.3cm, sucking 10 mu L of prepared solution to be detected on the surface of the pigskin, immediately placing the pigskin in a drier, and weighing the mass of the pigskin and the sample after 24 hours. In the experiment, pigskin without sample is used as a blank control group, and only liquiritin (GAMS) is coated to be used as a comparative example 1, only aurora degrading polysaccharide (TAP) is coated to be used as a comparative example 2, and only glycerin is coated to be used as a comparative example 3. Each sample was measured 3 times in parallel, and the average value was taken, and the moisturizing rate was calculated by the following formula.
In the formula: hn-the mass of the pigskin and the sample after a period of time, g;
H0-mass of pigskin and sample when sample is not applied, g.
Table 1: in vitro moisture retention of samples with different concentration ratios
The moisturizing composition of the present invention is used in an amount of 0.1% to 3% in various applications. As can be seen from table 1, the moisturizing composition has a moisturizing effect in a weight ratio of 1:10, wherein the effect is the best at 1:8, compared to the moisturizing rate of the pigskin of the comparative example without the sample; compared with the conventional glycerin humectant, the moisturizing composition has a moisturizing effect basically equivalent to that of glycerin when the weight ratio is 1:3 and the dosage is 0.5%.
Experiment 4: effect of sample samples of different concentrations on HaCaT cytotoxicity
Cell Counting Kit-8(CCK-8) is a colorimetric method with high sensitivity, no radioactivity and low cytotoxicity for measuring the number of living cells. Dehydrogenase is continuously generated in living cells, WST-8 in the CCK-8 reagent is oxidized and reduced by dehydrogenase to generate orange-yellow formazan in the presence of a Methoxy PMS electron carrier, the formazan dye can be dissolved in a culture medium, and the amount of the formazan is in direct proportion to the number of the living cells. I.e., the greater the number of viable cells, the higher the o.d. value. Because the samples need to be contacted with the skin to exert moisturizing effect, in order to ensure the safety of the samples, the CCK-8 method is adopted to evaluate the viability of HaCaT cells (cell bank of the type culture Collection of the Chinese academy of sciences) treated by samples with different mass concentrations. The determination method comprises the following steps:
taking 10-30 generations of HaCaT cells in logarithmic growth phase, and diluting the HaCaT cells into 1 × 10 by using a DMEM medium5Cell suspension at a concentration of 100. mu.L/well was seeded into 96-well plates and cultured in 5% CO2Culturing at 37 deg.C in incubator with saturated humidity for 24 hr, and removing culture solution from the well when cell fusion degree reaches 80%. Adding 100 μ L sample solution with different mass concentrations prepared with DMEM medium into each well, culturing for 24 hr, removing culture solution from each well, washing with PBS for 2 times, adding 10 per wellmu.L of 10% CCK-8 solution, and incubating in an incubator for 1h in the absence of light. Immediately after the incubation, OD at 450nm was measured with a microplate reader. The average value of 3 multiple holes arranged in each group is obtained, and the experiment is repeated for 3 times.
In the experiment, wells containing DMEM medium and CCK-8 solution are used as a control group, and wells containing CCK-8 solution only are used as a blank group.
Table 2: effect of sample samples of different concentrations on HaCaT cytotoxicity
As can be seen from table 2, the moisturizing composition has no toxic or side effect on HaCaT cells in the range of 0.1% to 3%, the cell survival rate is greater than 100%, and when the moisturizing composition is used in an amount of 2.0%, GAMS/TAP ═ 1: HaCaT cells survived the greatest at 8 hours, 120.21%.
Experiment 5: protection of samples against desiccation-damaged HaCaT cells
A cell drying damage model is established, and the moisturizing activity of the substance is explored at the cellular level. The CCK-8 method was used to assess the viability of the desiccated, damaged HaCaT cells treated with samples of varying mass concentrations. The method for establishing the cell drying damage model comprises the following steps: taking 10-30 generations of HaCaT cells in logarithmic growth phase, and diluting the HaCaT cells into 1 × 10 by using a DMEM medium5Cell suspension at a concentration of 100. mu.L/well was seeded into 96-well plates and cultured in 5% CO2Culturing at 37 deg.C for 24 hr in incubator with saturated humidity, and drying when cell fusion degree reaches 80%. The drying method comprises the following steps: placing the 96-hole plate in a clean workbench, keeping the room temperature, and setting the drying air speed to be 0.4 m/s; all culture solution in the holes is removed in sequence and placed in a clean workbench for 20min, and HaCaT cell drying damage experiments show that the longer the cell drying time is, the smaller the cell volume is, and the lower the activity is. Drying the cells for 20min, and the survival rate of the cells is 5045%, approaching half-cell lethality. Then 100 mul of sample to be tested prepared by DMEM medium is added to culture for 24h, and the sample concentration is a series of concentrations which are non-toxic to cells or have proliferation effect on cells. Wells containing DMEM medium alone were used as negative controls in the experiment, and the positive controls were commercial grade ceramide CER-3 and tremella aurantialba polysaccharide degradation product (TAP). The measurement method was the same as in experiment 4.
Table 3: protection of Dry damaged HaCaT cells by samples of different concentrations
As can be seen from table 3, the moisturizing composition was applied to HaCaT cells in an amount of 0.1% to 3% and was applied after drying, and the moisturizing composition had a wound repairing effect in the concentration range of the experiment, and compared to experiment 4, experiment 5 was also applied in an amount of 2.0% and GAMS/TAP ═ 1: the survival rate of the HaCaT cells is the largest at 8 hours and is 102.01 percent, so the damage repairing effect on the cells is closely related to the cell proliferation effect, the more obvious the cell proliferation effect is, the stronger the capacity of repairing damaged cells is, and the protection effect of the HaCaT cells on dry damage is obviously stronger than that of commercial grade ceramide CER-3.
Experiment 6: effect of samples on AQP3 content in Dry damaged HaCaT cells
Taking 10-30 generations of HaCaT cells in logarithmic growth phase, and diluting the HaCaT cells into 1 × 10 by using a DMEM medium5The cell suspension with the concentration of each hole is inoculated in a 6-hole plate, the cell is dried and damaged after being cultured for 24 hours according to the method of experiment 5, DMEM culture medium is used for preparing serial sample concentrations without cytotoxicity or with cell proliferation effect, samples with different concentrations are added into a sample group, DMEM culture medium and commercial grade ceramide CER-3 are added into a control group, and each hole is 2 mL. Culturing for 24h, and removing the culture medium from the holeAnd (3) culturing the cell, adding 2mL PBS buffer solution into each hole for washing, putting the 6-hole plate on ice, adding 1mL PBS and 200 mu L of medium-speed RIPA lysate into each hole, fully lysing, and transferring the cell lysate into a 1.5mL centrifuge tube. The cells were further disrupted with an ultrasonic cell disrupter for 1min, centrifuged at 1000g for 3min, and the supernatant was stored in a 2mL centrifuge tube for further use.
An enzyme-linked immunosorbent assay (ELISA) kit is used for determining the content of AQP3 in a sample by using a double-antibody sandwich method, the selected antibody is human aquaporin3, the stability and the inter-plate accuracy are good, a standard product in the human aquaporin 3(AQP3) ELISA kit (Beinanchuanglian biotechnology limited) is diluted into 2000, 1000, 500, 250 and 125ng/L standard solution, and the light absorption value of a cell lysate at 450nm is determined according to the operation of the instruction steps.
Table 4: effect of samples on AQP3 content in Dry damaged HaCaT cells
As shown in table 4, the experiment of the ELISA kit to measure the content of AQP3 in the cells shows that examples 1-7 can up-regulate the level of AQP3 in the HaCaT cells with dry injury, and when the amount of the moisturizing composition is 2.0%, GAMS/TAP is 1: compared with the commercial grade ceramide CER-3 of the comparative example 1, the content of 8 hours is also up-regulated by 27.48 percent, so that the experiments can prove that the moisturizing composition has the effects of improving the self-drying resistance of cells and enhancing the moisturizing effect of skin on a molecular level.
Experiment 7: the composition is prepared into moisturizing cream according to the following formula:
the preparation process comprises the following steps: adding the water phase except the moisturizing composition into a water phase pot, heating to 85 ℃ for dissolving, standing by, adding the oil phase into an oil phase pot, heating to 80 ℃ for dissolving, standing by, preheating an emulsifying pot to 85 ℃, adding the water phase, adding the oil phase, homogenizing and emulsifying (3200R/min) for 5min, stirring (25R/min), vacuumizing to 0.4 atmospheric pressure, slowly cooling with cooling water to 45 ℃ for standing by, adding the moisturizing composition, stirring (15R/min) for 15 min, homogenizing, discharging at 38 ℃, and storing and standing.
Experiment 8 and experiment 7 determination of moisturizing performance of moisturizing cream
30 volunteers aged 20-55 years, with healthy skin and no cosmetic allergy history were selected. Before testing, the left and right forearms are cleaned and dried, the test areas with the length of 3cm multiplied by 3cm are respectively marked on the left and right forearms after the skin temperature and humidity are balanced with the environment in a constant temperature and humidity room with the temperature of 20-22 ℃ and the relative humidity of 55 percent for at least 30min, the left arm is a control sample smearing area, and the right arm is a sample smearing area to be tested.
The TEWL change of the subject's inner forearm mark points before and after 10 and 120min of application of the sample was determined using a Tewameter TM300 through the epidermal water loss test probe and averaged for 30s for each mark point.
The water content of stratum corneum at the test site was measured using a CM825 skin water content test probe, and the result was expressed as MMV, taking the average of 5 measurements as the test value. Then taking the moisturizing cream according to the ratio of (2.0 +/-0.1) mg sample/cm2The dose of (2) was applied to the test site and after 10 and 120min, the MMV of the stratum corneum of the skin was measured in the same way. The MMV change rate is calculated as follows.
The high-efficacy moisturizing cream prepared in experiment 7 was subjected to measurement of skin hydration degree (MMV) and trans-epidermal water loss rate (TEWL), and the results are shown in fig. 1 and 2. As can be seen from figures 1 and 2, the high-efficiency moisturizing cream is a product with good instant moisturizing capability and can better restore the barrier function of the skin. After the skin care product is used, the testee considers that the skin becomes soft and smooth, and the condition of dry skin can be improved.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (8)
1. A compound composition of glycyrrhizic anhydride and tremella polysaccharide degradation products is characterized in that: the active ingredients of the compound composition consist of oxalic anhydride and tremella polysaccharide degradation products; the weight ratio of the glycyrrhetic anhydride to the tremella polysaccharide degradation product is 1:1-1: 10.
2. The compound composition of glycyrrhizic anhydride and tremella polysaccharide degradation product according to claim 1, wherein: the tremella aurantialba polysaccharide degradation product is an acid degradation product of tremella aurantialba extracted by an aqueous enzymatic method.
3. The compound composition of glycyrrhizic anhydride and tremella polysaccharide degradation product according to claim 2, wherein: the total sugar content of the tremella aurantialba polysaccharide degradation product is 68.58% measured by a phenol-sulfuric acid method.
4. The compound composition of claim 3, wherein the degradation product of the glycyrrhiza uralensis anhydride and the tremella polysaccharide is: the enzyme in the aqueous enzymatic extraction is pectinase, and the weight ratio of the pectinase to the tremella aurantialba powder is 1: 10.
5. the compound composition of glycyrrhizic anhydride and tremella polysaccharide degradation product according to claim 1, wherein: the weight ratio of the glycyrrhetic anhydride to the tremella polysaccharide degradation product is 1:5-1: 8.
6. The compound composition of glycyrrhizic anhydride and tremella polysaccharide degradation product according to claim 1, wherein: the compound composition also contains one or more of emulsifier, thickener, antiseptic, bacteriostatic agent, humectant, penetrant and whitening agent which can be added during cosmetic preparation.
7. Use of the composite composition of glycyrrhizic anhydride and tremella polysaccharide degradation product according to any one of claims 1 to 6 for preparing a moisturizing cosmetic.
8. The use of the compound composition of glycyrrhizic anhydride and tremella polysaccharide degradation product according to claim 7 in the preparation of moisturizing cosmetics, wherein: the dosage of the compound composition of the oxalic anhydride and the tremella polysaccharide degradation product is 0.1 to 3 percent by weight.
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| CN115058482A (en) * | 2022-05-06 | 2022-09-16 | 仙乐健康科技股份有限公司 | Method for evaluating moisturizing and/or dry repairing performance of material |
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| CN113876614B (en) | 2023-05-02 |
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