Madecassoside composition with skin repair effect and use thereof

Abstract

Provided are a madecassoside composition with a skin repair effect and use thereof. A scratch experiment demonstrated that madecassoside derivatives I and II have good effects in promoting the closure of the intercellular gaps between human keratinocytes. A subsequent transdermal experiment and an assay of skin repair-related factor expression found that the efficacy can be potentially enhanced by compounding the madecassoside derivatives I and II. Formulations were then screened for optimal acne-removing effects in an efficacy study in humans. Finally, the safety and applicability of the derivatives as cosmetic starting materials were verified in a safety study. The studies on madecassoside derivatives facilitate the application and development of Centella asiatica in acne-removing cosmetics.

Description

Madecassoside composition with skin repairing effect and application thereof Technical Field

[0001] The present invention belongs to the fields of medicine and cosmetics, and in particular relates to a madecassoside composition having skin repairing effect and application thereof. Background Art

[0002] The skin is a highly adaptable, multifunctional organ that serves as the first barrier to protect the human body from external microbial invasion. However, external environmental factors such as chemical, physical, and UV radiation often lead to skin wounds. The stages of wound healing mainly include coagulation, inflammation, proliferation, and remodeling. Disorders at any stage of the healing process may delay wound healing, worsen the wound, and even turn it into a chronic wound. In order to treat chronic wounds, it is crucial to understand the pathophysiology of wounds. Despite different causes, chronic wounds share certain common characteristics, including excessive levels of proinflammatory cytokines, persistent infection, the formation of drug-resistant microbial biofilms, and senescent cells that do not respond to reparative stimuli. Chronic wounds (mainly venous ulcers, pressure ulcers, and diabetic foot ulcers) are a major area of ​​unmet clinical need. Therefore, it is crucial to find and develop drugs that promote skin repair.

[0003] Acne vulgaris is one of the most common skin diseases worldwide and is considered a chronic inflammatory disease. Key mechanisms involved in the development of acne include excessive sebum secretion, follicular hyperkeratinization, inflammation, and infection with Propionibacterium acnes, leading to a persistent and recurrent inflammatory process. However, conventional anti-inflammatory treatments often have drawbacks, including antibiotic resistance and skin irritation associated with the use of benzoyl peroxide or salicylic acid. Therefore, safe and effective alternative therapies have become a research hotspot.

[0004] Centella asiatica (L.) Urb. is a perennial herbaceous plant of the genus Centella in the Apiaceae family. Its entire plant has been used as a medicinal herb for over 3,000 years. It is found in many provinces of China, as well as in countries such as India, Sri Lanka, Malaysia, and Indonesia. It is known as the "Oriental longevity grass." Its main components include pentacyclic triterpenoid saponins such as asiaticoside and madecassoside, as well as flavonoids such as quercetin and kaempferol. Its main functions are to clear heat and dampness, detoxify, and reduce inflammation. It is commonly used to treat damp-heat jaundice, heatstroke-induced diarrhea, carbuncles, sores, and traumatic injuries. Centella asiatica also possesses antioxidant, anti-inflammatory, wound-healing, and anti-allergic properties. However, the content of madecassoside derivatives I and II in Centella asiatica is extremely low, and their applications in skin wound healing are rarely studied.

[0005] CN202110775063.4 introduces a preparation method of Centella asiatica fermentation filtrate and its anti-inflammatory effect. However, the content of hydroxy Centella asiatica disoside and hydroxy Centella asiatica monoside is only 0.5-4 g / L of water-soluble liquid, but the skin repair effect is not achieved, and it is difficult to meet the application requirements of various dosage forms of cosmetics. Summary of the Invention

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a madecassoside composition having skin repairing efficacy.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: a madecassoside composition having skin repairing effect, comprising madecassoside bioside and madecassoside monoside;

[0010] Wherein, the mass ratio of the hydroxy Centella asiatica bioside to the hydroxy Centella asiatica monoside is 1 to 3:1, and the structural formula of the hydroxy Centella asiatica bioside is as follows (I):

[0011] The structural formula of the hydroxy Centella asiatica monoglycoside is as follows (II):

[0012] As a preferred embodiment of the composition of the present invention, the mass ratio of the madecassoside to the madecassoside monoside is 3:1.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a madecassoside composition having skin repair efficacy for use in the preparation of products for improving skin beauty.

[0014] As a preferred embodiment of the application of the present invention, the improvement of skin beauty includes treating mild to moderate acne, allergic redness, promoting wound healing and alleviating scar formation.

[0015] As a preferred embodiment of the application of the present invention, the product is a health product, medicine or cosmetic.

[0016] As a preferred embodiment of the application of the present invention, the dosage form of the medicine or cosmetic includes ointment, paste, patch, toner, essence water, lotion or cream.

[0017] As a preferred embodiment of the application of the present invention, the cosmetic comprises an anti-acne cream, which comprises, by weight percentage, 0.5-2% of a skin conditioner, 2-5% of an emulsifier, 0.5-1.5% of a thickener, 9-15% of a moisturizer, 8-14% of an emollient, 0.1-0.6% of a preservative, 0.05-0.2% of a colorant, 0.01-0.1% of a fragrance, 0.5-5% of a madecassoside composition, and the remainder being water.

[0018] As a preferred embodiment of the application of the present invention, wherein: the skin conditioning agent is a carbohydrate isomer;

[0019] The emulsifier is one or more of sucrose polystearate, glyceryl stearate, sodium stearoyl lactylate, and myristic acid;

[0020] The thickener includes one or more of ammonium acryloyldimethyltaurate / VP copolymer, sodium polyacryloyldimethyltaurate, sodium hyaluronate, xanthan gum, glycerol polypropionate, acrylic acid (ester) copolymer, sodium chloride, and potassium chloride;

[0021] The moisturizing agent includes one or more of glycerol polyether-26, glycerin, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, sorbitol, panthenol, hyaluronic acid, ceramide, niacinamide, allantoin, and tremella polysaccharide;

[0022] The emollient includes one or more of squalane, hydrogenated ethylhexyl olive oil, hydrogenated olive oil unsaponifiables, Murumuru palm fat, babassu seed oil, cetearyl alcohol, and C10-18 fatty acid triglycerides;

[0023] The preservative includes one or more of phenoxyethanol, ethylhexylglycerin, and p-hydroxyacetophenone;

[0024] The colorant is guaiazulene.

[0025] As a preferred embodiment of the application of the present invention, the preparation method of the acne-removing cream comprises:

[0026] Weigh the formulated amount of emulsifier, emollient, colorant and fragrance, stir evenly to prepare phase A, which is the oil phase, and heat for later use;

[0027] Weigh the thickener, skin conditioner, moisturizer, preservative and appropriate amount of water in the formula, stir and mix to prepare phase B, which is the aqueous phase, and heat for later use;

[0028] Weigh the formulated amount of the madecassoside composition and the balance of water, stir and mix thoroughly to prepare phase C, and set aside;

[0029] Slowly pour the dissolved phase B into phase A, stir evenly while hot, add phase C after cooling, and stir evenly;

[0030] Stop stirring to prepare the anti-acne cream.

[0031] Beneficial effects of the present invention:

[0032] (1) The present invention provides a hydroxy-madecassoside composition, and experiments have verified that hydroxy-madecassoside derivatives I and II have a good effect on promoting the migration of human keratinocytes without affecting cell proliferation and morphology, which greatly enhances the application and development value of Centella asiatica in treating acne, promoting tissue repair, and improving skin beauty.

[0033] (2) The madecassoside derivatives I and II used in the present invention have been verified to have good transdermal properties through experiments, and can effectively improve the absorption of madecassoside derivatives as external skin preparations at target skin sites.

[0034] (3) The hydroxymadecassoside derivative composition of the present invention is a compound preparation made from I and II as raw materials in a suitable weight ratio. The determination experiment of skin repair related factors proves that its synergistic effect is much higher than that of a single component.

[0035] (4) The present invention also provides an anti-acne facial cream containing a madecassoside derivative composition. The addition of the madecassoside derivative composition to the facial cream enables it to have good moisturizing properties and regulate skin oiliness, which is of great significance for further expanding the application of Centella asiatica in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0037] FIG1 is a high performance liquid chromatogram of asiaticoside derivatives I and II of the present invention, wherein the standard substances include asiaticoside and asiatic acid;

[0038] FIG2 is a graph showing the experimental results of the effects of different types of madecassoside derivatives on HaCaT cell migration in Example 2 of the present invention;

[0039] FIG3 is a graph showing a comparison of the transdermal performance of madecassoside derivatives with different ratios in Example 3 of the present invention;

[0040] FIG4 is a graph showing the experimental results of the effects of madecassoside derivatives in different ratios on TLR4 factor expression in Example 3 of the present invention;

[0041] FIG5 is a graph showing the experimental results of the effects of madecassoside derivatives in different ratios on STAT3 expression in Example 3 of the present invention;

[0042] FIG6 is a graph showing the efficacy evaluation results of the anti-acne cream containing the composition of madecassoside derivatives I and II in Example 5 of the present invention (change rate of moisture content);

[0043] FIG7 is a graph showing the efficacy evaluation results of the anti-acne cream containing the composition of madecassoside derivatives I and II in Example 5 of the present invention (transepidermal water loss change rate);

[0044] FIG8 is a graph showing the efficacy evaluation results of the anti-acne cream containing the composition of madecassoside derivatives I and II in Example 5 of the present invention (change rate of oil content). DETAILED DESCRIPTION

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0046] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0048] The madecassoside used in the experiments of the present invention was purchased from Xi'an Shouhe Biotechnology Co., Ltd.

[0049] Centella asiatica derivatives I and II (purity >95%) were prepared by ethanol gradient recrystallization method; the specific method is as follows:

[0050] To 5 g / L madecassoside dissolved in 80% ethanol solution, a final concentration of 2% H2O2 solution and 0.05 mol / L glacial acetic acid solution were added, and the mixture was reacted at 55-60°C for 5 hours to obtain a suspension solution;

[0051] Filter, add water to the filtrate until the ethanol concentration reaches 50%, precipitate at 4°C overnight, collect the precipitate, filter, recrystallize twice with 50% ethanol, and freeze-dry the collected solid to obtain Centella asiatica glycoside derivative I.

[0052] The filtrate was collected and concentrated, and water was added to the ethanol concentration to reach 20%. The mixture was precipitated at 4°C overnight, filtered, and recrystallized twice with 20% ethanol solution. The collected solid was freeze-dried to obtain Centella asiatica glycoside derivative II.

[0053] The HPLC determination is shown in Figure 1. Chromatographic detection conditions: mobile phase: acetonitrile (phase A), water (phase B); C-18 column (4.6 mm × 250 mm × 5.0 μm); column temperature: 25°C; injection volume: 10 μL; peak detection at 205 nm. Mobile phase elution conditions: gradient elution (25-65% phase A); flow rate: 1 mL / min; elution time: 90 min. It can be seen that the elution times of asiaticoside derivatives I and II are between madecassoside and madecassic acid.

[0054] Example 1

[0055] 1) Recovery, culture, passaging, and cryopreservation of HaCaT cells:

[0056] (1) Cell recovery: Take the cryovial out of the liquid nitrogen tank and accelerate thawing in a 37°C water bath. After disinfection with 75% alcohol, aspirate the liquid in the cryovial into a centrifuge tube containing approximately 6 mL of complete culture medium. Centrifuge at 800 rpm for 5 min. Discard the upper layer of liquid, resuspend with 1 mL of new culture medium, and transfer to a culture dish containing 6 mL of culture medium.

[0057] (2) Cell culture: HaCaT cells were cultured in DMEM complete medium (10% FBS + 1% double antibody) in a constant temperature incubator at 37°C and 5% CO2.

[0058] (3) Cell passaging: When the cell density reaches 80-90%, discard the supernatant, wash twice with PBS, digest with 0.25% trypsin + 0.03% EDTA, keep incubator for 3-5 minutes, centrifuge at 1200 rpm for 5 minutes, and pass the cells at a ratio of 1:2.

[0059] (4) Cell cryopreservation: Collect cells, add serum-free non-programmed cell cryopreservation solution, transfer to cryopreservation tubes, mark the name, number, time, and name, and store the cryopreservation tubes in a -80℃ refrigerator. The next day, transfer the cryopreservation tubes to a liquid nitrogen tank for long-term storage.

[0060] The present invention detects the gap closure rate of human immortalized keratinocytes HaCaT cells (purchased from Beina Chuanglian Biotechnology Research Institute), evaluates and compares the effects of various Centella asiatica glycoside derivatives on cell migration, and verifies the excellent effect of hydroxy Centella asiatica bioside and hydroxy Centella asiatica monoside in promoting wound healing.

[0061] A Centella asiatica glycoside derivative with a mass concentration of 0.02 mg / mL was selected for the scratch test.

[0062] 2) Cell scratch assay:

[0063] (1) Insert fixation: Use sterile tweezers to carefully pick up the ibidi culture-inserts (2 well) and fix them in the middle of each well of a 24-well plate.

[0064] (2) Cell inoculation: HaCaT cells in the logarithmic growth phase were taken and plated at 5.0×10 5 The cells were inoculated into the inserts at a density of 100 μg / mL (cultured in DMEM complete medium), with 70 μL per well. The cells were divided into blank group, model group and experimental group, with three replicates per group.

[0065] (3) Grouped drug addition: After each group of cells is incubated, carefully remove the plug with sterile tweezers to create a blank gap of uniform width of 500 μm similar to a scratch. After discarding the supernatant, carefully wash the cells 2-3 times with PBS buffer to remove the remaining cells or floating dead cells in the scratch.

[0066] Blank group: fresh DMEM medium was added and cultured for 12 h and 24 h;

[0067] Control group: add fresh DMEM complete medium (containing 10% FBS, 1% double antibody) and continue culturing for 12 h and 24 h;

[0068] Experimental group: fresh DMEM medium (containing test samples No. 1-6, namely madecassoside, madecassobioside, madecassoside monoside, madecassoic acid, asiaticoside, and asiatic acid) was added and cultured for 12 h and 24 h.

[0069] (4) Observation under microscope: Immediately observe and record the scratch area at 0 h using an optical microscope, and take photos at 12 h and 24 h and save the scratch images at each time point.

[0070] (5) Data processing: ImageJ software was used to calculate the scratch area of ​​each group at each time point, and the cell migration rate of each group was calculated; T-test analysis was performed using GraphPad for statistical analysis, **p < 0.01 compared with the blank group.

[0071] The experimental results are shown in FIG2 . Madecassoside and monoglycoside have the best effect in promoting the migration of human keratinocytes, demonstrating that they have a better effect in promoting wound healing and repairing skin damage.

[0072] Example 2

[0073] To explore the effects of different ratios of asiaticoside derivatives on transdermal properties and the expression of skin repair-related factors:

[0074] Samples: Added with the same mass concentration but different ratios of the Centella asiatica derivative composition, divided into samples 1-6 as follows;

[0075] No. 1 (the ratio of madecassoside:madecassobioside:madecassoside monoside is 3:1:1);

[0076] No. 2 (the ratio of madecassoside:madecassobioside:madecassoside monoside is 2:1:1);

[0077] No. 3 (the ratio of madecassoside:madecassobioside:madecassoside monoside is 1:1:1);

[0078] No. 4 (the ratio of madecassoside:madecassobioside:madecassoside monoside is 0:1:0);

[0079] No. 5 (the ratio of madecassoside:madecassobioside:madecassoside monoside is 0:0:1);

[0080] No. 6 (the ratio of madecassoside:madecassobioside:madecassoside monoside is 0:1:1);

[0081] 1. In vitro percutaneous absorption test

[0082] Strat-M TM The artificial membrane was directly fixed between the sample reservoir and the receiving reservoir. The sample reservoir contained a 1 mg / mL solution of sample Nos. 1-6 (solvent: 20% ethanol-water). The receiving reservoir had a volume of 7.5 mL and the receiving solution was also ultrasonically degassed in 20% ethanol-water. The instrument was placed in a transdermal diffusion apparatus and maintained at a constant temperature and constant stirring (37°C, 500 rpm). The instrument was allowed to equilibrate for 1 hour and remove bubbles. At 0, 2, 4, 6, 8, 10, and 12 hours, 0.2 mL of receiving solution was aspirated and supplemented with blank receiving solution. The receiving solution at each time point was centrifuged at 12,000 rpm for 10 minutes.

[0083] The mass concentration of the supernatant was determined by HPLC, and the cumulative transdermal amount of the asiaticoside derivatives at each time point (Q n , μg):

[0084] Where, V: receiving chamber volume (mL); Vs: sampling volume (mL); C n : drug concentration in the receiving solution measured at the nth sampling point (μg / mL); C i : Drug concentration (μg / mL) at the i-th (i≤n-1) sampling point. The permeability is Q at each time point. n Ratio to the initial mass of the sample.

[0085] The experimental results are shown in Figure 3. The skin permeation rates of samples 4-6 are significantly better than those of samples 1-3, and hydroxy Centella asiatica diglycoside has the best transdermal absorption properties, and can achieve better transdermal effects when combined with hydroxy Centella asiatica monoglycoside.

[0086] 2. ELISA determination of TLR4 and STAT3 expression in HaCaT cells

[0087] The TLR4 / NF-κB pathway is key to promoting intracellular inflammation induced by Chlamydia acnes and also plays a crucial role in diseases such as atopic dermatitis, psoriasis, and skin cancer. In acute trauma, the recognition of various ligands by TLRs and NF-κB can induce the expression of inflammatory factors and delay the skin's repair process. Therefore, inhibiting the expression of TLR4 and NF-κB can treat diseases such as acne and atopic dermatitis.

[0088] (1) Cell grouping and processing

[0089] HaCaT cells in logarithmic growth phase were taken and 2*10 5 Cells were seeded at a density of 1.5 mL / well in a 6-well plate. Three replicates were set up for each of the blank, model, and experimental groups. Cultured in complete DMEM until 70–90% confluence was achieved, the medium was removed, and the cells were carefully rinsed twice with PBS.

[0090] Blank group: remove the culture medium, carefully rinse twice with PBS, add fresh DMEM medium, and continue to culture for 24 hours;

[0091] Model group: Remove the culture medium, carefully rinse twice with PBS, add DMEM medium containing 500 μM H2O2, and continue to culture for 24 h;

[0092] Experimental group: Remove the culture medium, carefully rinse twice with PBS, add DMEM medium containing 500 μM H2O2 and 0.02 mg / mL samples 1-6, and continue to culture for 24 hours.

[0093] (2) Collect cell lysis supernatant

[0094] After removing the culture medium, rinse twice with PBS. Add 1 mL of PBS and gently scrape the cells with a cell scraper into a sterile EP tube. Centrifuge in a high-speed benchtop refrigerated centrifuge at 4°C, 5000 rpm for 5 min. Discard the supernatant. Add 100 μL of cell lysis buffer (containing PMSF) to each well to resuspend the cells. Disrupt the cell suspension in an ice bath using an ultrasonic cell disruptor for a total of 30 min at 90% power. Collect the cell lysate, centrifuge at 4°C, 12000 rpm for 15 min, and remove the supernatant for analysis.

[0095] (3) The levels of TLR4 and STAT3 in the cell supernatant were detected using ELISA kits according to the instructions, and the protein content in the cell lysate supernatant was detected using a BCA protein detection kit for protein correction.

[0096] (4) Statistical analysis

[0097] Graphpad was used to perform significant difference analysis (One-way ANOVA Tukey's Method)

[0098] The experimental results are shown in Figures 4-5. Samples 4-6 can significantly inhibit the expression of TLR4 and STAT3, among which the combination of hydroxy Centella asiatica bioside and hydroxy Centella asiatica monoside has the best inhibitory effect.

[0099] In summary, the combination of hydroxy Centella asiatica disoside and hydroxy Centella asiatica monosaccharide has excellent potential for treating skin diseases such as acne and has good transdermal absorption properties.

[0100] Example 3

[0101] Preparation of anti-acne cream containing madecassoside derivatives I and II:

[0102] The steps include:

[0103] (1) Solution preparation: Weigh 4 g of glyceryl stearate, 4 g of sodium stearoyl lactylate, 6 g of squalane, 10 g of babassu seed oil, 3 g of cetearyl alcohol, 4 g of hydrogenated ethylhexyl olive oil, 4 g of hydrogenated olive oil unsaponifiables, 0.2 g of guaiazulene, and 0.06 g of essence, stir well, and prepare phase A, which is the oil phase. Heat and set aside.

[0104] Weigh 1.4 g sodium polyacryloyldimethyl taurate, 2 g saccharide isomers, 4 g glycereth-26, 10 g butylene glycol, 6 g niacinamide, and 130 g water, stir and mix to prepare phase B, which is the aqueous phase, and heat for later use;

[0105] Weigh 0.25 g of the combination of madecassoside derivatives I and II and 10 g of water, stir and mix to prepare phase C, and set aside;

[0106] (2) Slowly pour the dissolved phase B into phase A, stir evenly while hot, add phase C after cooling, and stir evenly;

[0107] (3) Stop stirring to prepare a facial cream containing hydroxy-madecassoside derivatives I and II.

[0108] Example 4

[0109] Experimental study on the efficacy of anti-acne cream containing madecassoside derivatives I and II:

[0110] Referring to the "Diagnostic Criteria and Treatment Principles of Cosmetic Contact Dermatitis", a total of 60 volunteers aged 20-25 years were recruited and randomly divided into 6 groups, with 10 people in each group.

[0111] Sample: The facial cream prepared in Example 4 of the present invention was used for the experiment, wherein:

[0112] Sample No. 1: Madecassoside derivatives I and II in a ratio of 1:3;

[0113] Sample No. 2: Madecassoside derivatives I and II in a ratio of 1:2;

[0114] Sample No. 3: Madecassoside derivatives I and II in a ratio of 1:1;

[0115] Sample No. 4: Madecassoside derivatives I and II in a ratio of 3:1;

[0116] Sample No. 5: Madecassoside derivatives I and II in a ratio of 2:1;

[0117] Reference substance No. 6 - madecassoside triglycoside;

[0118] Test indicators: skin moisture content MMV value; skin transepidermal water loss TEWL value; skin oil content.

[0119] Test environment requirements: constant temperature and humidity environment, set temperature: 20℃, set humidity: 50%.

[0120] Test method: The test site is the face, and the product must be applied evenly. Volunteers use the product on the acne-affected areas according to the instructions, once in the morning and once in the evening every day, with a dosage of 0.5g each time (the size of two peanuts). The use period is 4 weeks. During the test period, similar cosmetics are discontinued; Test instrument: Multi Probe Adapter (Germany CK Company), connecting probes include Sebumeter SM815 (oil test probe), Corneometer CM825 (moisture test probe), Tewameter TM300 (moisture loss test probe); After the experimenter put the tape on the volunteer's left cheek, another experimenter used the oil test probe to test the oil content next to the tape on the inside of the cheek, and then tested the water loss (moisture loss test probe) and moisture content (moisture test probe) in turn.

[0121] Data collection: A total of three data collections were conducted, divided into initial value collection and data collection after one, two, and four weeks of using acne-removing products. Volunteers cleaned the test area and sat quietly for 20 minutes before testing.

[0122] Data processing: The final result is expressed as a rate of change. A greater rate of change in moisture content indicates better moisturizing performance. The rates of change in transepidermal water loss and oil content are negative, and larger absolute values ​​represent reduced water loss and reduced oil secretion, respectively.

[0123] Experimental Results: As shown in Figures 6-8, compared to control sample No. 6, all samples demonstrated superior effects in increasing skin moisture, reducing transepidermal water loss, and reducing oil secretion. This indicates that the combination of madecassoside bioside and madecassoside monoside exhibits a more pronounced acne-removing effect than pure madecassoside. Over 90% of the volunteers experienced reduced or eliminated facial acne / acne scar symptoms after using the product, with no new acne developing. This demonstrates that the test samples are effective in repairing the skin barrier, regulating oil content, and removing acne / acne scars. Sample No. 4 (madecassoside derivatives I and II in a 3:1 ratio) demonstrated the most significant acne-removing effect.

[0124] Example 5

[0125] Safety evaluation of the combination of madecassoside derivatives I and II (ratio 3:1):

[0126] 1. Sensitization test

[0127] A direct peptide binding assay (DPRA) was used, replacing animal testing, to determine the sensitization effect of the samples based on the consumption of cysteine ​​and lysine peptides. The experiment was designed according to the OECD standards and relevant group standards, with certain modifications. Cysteine ​​peptides were dissolved in 0.1M PBS buffer (pH 7.5), and lysine peptides were dissolved in 0.1M ammonium acetate buffer (pH 10.2), both at a concentration of 0.667 mM. These were then serially diluted to a series of standard solutions of 0.2670, 0.1335, 0.0667, 0.0334, and 0.0167 mM.

[0128] Madecassoside derivatives I and II composition samples and the positive control substance cinnamaldehyde were diluted with acetonitrile to a concentration of 0.1M.

[0129] During testing, add the reagents according to Table 1 to the injection bottle. Mix thoroughly and incubate at 25±2.5℃ in the dark for 24±2h.

[0130] Table 1 DPRA test sample preparation table

[0131] The detection conditions of the liquid chromatography system were as follows: mobile phase: 0.1% trifluoroacetic acid in water, phase A; 0.1% trifluoroacetic acid in acetonitrile, phase B; C-18 column (4.6 mm × 250 mm × 5.0 μm); column temperature 25°C; injection volume 10 μL; peak detection at 220 nm;

[0132] The mobile phase elution conditions were as follows: cysteine ​​peptides: 78% phase A, flow rate 1 mL / min, elution time 15 min; lysine peptides: 82% phase A, flow rate 1 mL / min, elution time 15 min.

[0133] The test results are expressed as polypeptide consumption rates, which are calculated according to formula (IV), with one decimal place retained, and the sensitization status is predicted based on the consumption rate results.

[0134] The results were considered to be within the confidence interval if the following conditions were met: the average peptide consumption rate of the positive control was between 60.8% and 100% for cysteine ​​peptides, with a SD of <14.9%, and between 40.2% and 69.4% for lysine peptides, with a SD of <11.6%; the SD of the sample cysteine ​​peptide consumption rate was <14.9%, and the SD of the lysine peptide consumption rate was <11.6%. The sample results were determined according to Table 2.

[0135] Table 2 DPRA result judgment table

[0136] The experimental results are shown in Table 3.

[0137] Table 3 Results of sensitization evaluation experiment

[0138] Therefore, it can be determined that the combination of madecassoside derivatives I and II will not cause allergic reactions.

[0139] 2. Mutagenicity test

[0140] The Ames test was used to preliminarily evaluate the mutagenicity of the combination of madecassoside derivatives I and II. This method primarily involves culturing auxotrophic mutant strains and observing whether the test substance can correct or compensate for the mutations carried by the mutants, returning them to a normal wild-type strain, thereby determining their mutagenicity.

[0141] The experiment used five strains of histidine-deficient Salmonella typhimurium, TA97, TA98, TA100, TA102, and TA1535, for testing. After the strains were revived, their biological characteristics were first identified to confirm that the strains used were correct.

[0142] The strain was cultured in nutrient broth (5.0 g beef extract, 10.0 g tryptone, 5.0 g sodium chloride, 2.6 g potassium phosphate dibasic, 1 L deionized water, pH = 7.4) at 37°C. Four doses of 5000, 2500, 1250, and 625 μg / dish were used, diluted with sterile water, and filtered through a 0.1 μm sterile filter. In addition to the sample groups, an untreated control group, a solvent control group, and a positive control group (TA97, TA98, and TA102 using dichlorodiphenylmethane, and TA100 and TA1535 using sodium azide) were set up. The experiment was conducted using the plate incorporation method. To 2 mL of top culture medium insulated at 45°C, add 0.1 mL of the test substance or control and 0.1 mL of fresh enrichment solution of the test strain, mix thoroughly, and quickly pour onto the bottom culture medium. Rotate the culture dish to evenly distribute the culture medium on the bottom layer. After solidification, invert the dish in a 37°C incubator. Incubate for 48 hours and observe the results. Count the number of revertant colonies. Prepare three replicate dishes for each dose.

[0143] The experimental results are shown in Table 4.

[0144] Table 4 Mutagenicity evaluation test results

[0145] Therefore, it can be preliminarily determined that the combination of hydroxy-madecassoside derivatives I and II will not produce mutagenicity.

[0146] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A centella asiatica total glycosides composition with skin repair efficacy, characterized in that: It includes asiaticoside disaccharide and asiaticoside monosaccharide; Among them, the mass ratio of the asiaticoside diglycoside to the asiaticoside monoglycoside is 1 to 3:1, and the structural formula of the asiaticoside diglycoside is as follows formula (I): The structural formula of the asiaticoside monoglycoside is as follows in formula (II):

2. The composition according to claim 1, wherein: The mass ratio of the asiaticoside disaccharide to the asiaticoside monosaccharide is 3:

1.

3. Use of the composition according to claim 1 or 2 in the preparation of a product for improving skin beauty.

4. The application according to claim 3, characterized in that: The improvement of skin beauty includes treating mild to moderate acne, allergic flushing, promoting wound healing and reducing scar formation.

5. The application according to claim 3, characterized in that: The product is a health care product, a drug or a cosmetic.

6. The application according to claim 3, characterized in that: The dosage form of the drug or cosmetic includes ointment, paste, patch, toner, essence water, lotion or cream.

7. The application according to claim 5 or 6, characterized in that: The cosmetic includes an anti-acne cream, which, calculated by weight percentage, includes 0.5-2% of a skin conditioner, 2-5% of an emulsifier, 0.5-1.5% of a thickener, 9%-15% of a humectant, 8%-14% of an emollient, 0.1-0.6% of a preservative, 0.05-0.2% of a colorant, 0.01-0.1% of a fragrance, 0.5-5% of an asiaticoside composition and the balance of water.

8. The application according to claim 7, characterized in that: The skin conditioner is a carbohydrate isomer; The emulsifier is one or more of sucrose poly stearate, glyceryl stearate, sodium stearoyl lactylate, myristic acid; The thickener includes one or more of acryloyldimethyltauramide / VP copolymer, sodium polyacryloyldimethyltaurate, sodium hyaluronate, xanthan gum, glyceryl polyacrylate, acrylate copolymer, sodium chloride, potassium chloride; The humectant includes one or more of glycerol polyether-26, glycerol, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, sorbitol, panthenol, hyaluronic acid, ceramide, niacinamide, allantoin, tremella polysaccharide; The emollient includes one or more of squalane, ethylhexyl hydroxystearate, unsaponifiable matter of hydrogenated olive oil, murumuru butter, babassu oil, cetearyl alcohol, C10-18 triglycerides; The preservative includes one or more of phenoxyethanol, ethylhexylglycerin, p-hydroxyacetophenone; The colorant is azulene; 9. The application according to claim 7, wherein: The preparation method of the anti-acne cream includes, Weigh the formulated amount of emulsifier, emollient, colorant and fragrance, stir evenly to prepare Phase A, and Phase A is an oil phase, and heat it for standby; Weigh the formulated amount of thickener, skin conditioner, humectant, preservative and appropriate amount of water, stir and mix evenly to prepare Phase B, and Phase B is an aqueous phase, and heat it for standby; Weigh the formulated amount of asiaticoside composition and the balance of water, stir and mix evenly to prepare Phase C, and keep it for standby; Slowly pour the dissolved Phase B into Phase A, stir evenly while it is hot, add Phase C after cooling, and stir evenly; Stop stirring to obtain the anti-acne cream.

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