Nymphaea and myrtle composite make-up gel for repairing uvb damage and preparation method thereof

Abstract

The application discloses a lotus and myrtle compound make-up gel for repairing UVB damage and a preparation method thereof. The application provides application of myrtle extract or myrtle extract and lotus polysaccharide extract in preparation of cosmetics or medicines for whitening, sunscreen and skin repair. The prepared make-up gel can significantly improve the survival rate of HaCaT cells after UVB damage, effectively inhibit cell apoptosis, repair UVB-induced HaCaT cell damage, and has excellent moisturizing and skin moistening effects. The lotus polysaccharide and myrtle fruit extract used in the lotus and myrtle compound make-up gel for repairing UVB damage are derived from natural plant resources with the same medicinal and edible functions, have small stimulation to skin damaged parts, low toxicity and few side effects, and have a very good development and application prospect in preparation of make-up products.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics or biopharmaceuticals, specifically involving the extraction or preparation method of water lily polysaccharide, myrtle fruit extract, and the synergistic preparation of a myrtle-based cosmetic gel. The cosmetic gel prepared by this method has a repairing effect on UVB-induced HaCaT cell damage and also has excellent moisturizing and skin-nourishing effects. Background Technology

[0002] As the largest biological organ in the human body, the skin primarily performs functions such as protection, regulation, buffering, and resistance. As an organ in direct contact with the external environment, the skin is subject to aging due to external factors and changes in the body itself, which can be categorized into natural aging and photodamage. Ultraviolet (UV) radiation belongs to electromagnetic radiation. Based on wavelength, it is divided into UVA, UVB, and UVC. Studies have shown that at the same dose, the biological effect ratio of UVA to UVB is 1:1000. The main effect of UV radiation on the skin is oxidative damage. UV radiation stimulates the body to produce oxidative stress, inducing a large production of reactive oxygen species (ROS), leading to cell or tissue damage. UVB can act on cells by inducing ROS production or causing DNA damage, promoting cell apoptosis, accelerating cell proliferation, and inducing skin cancer. Furthermore, skin exposure to UV radiation can cause excessive secretion of micronutrients (MMPs), leading to collagen breakdown, pigment accumulation, and problems such as skin leatheriness and aging. Therefore, how to effectively protect against and repair skin damage caused by UVB is a crucial research direction in the fields of skin care and medical aesthetics.

[0003] The search for and development of functional substances that protect against and repair skin damage caused by UVB has been a hot research topic for scientists in the pharmaceutical and cosmetic fields. Currently, inorganic UVB-protective ingredients such as ethylene compounds like TiO2, ZnO, PbO, CaCO3, and talc have been discovered. These components shield against UVB by reflecting and scattering ultraviolet light, thus achieving the effect of protecting against UV damage. However, these inorganic components are typically used only as UV shielding agents in cosmetics and do not possess the function of repairing UV damage. Researchers have also found that some organic compounds with conjugated double bonds can also play a role in UV shielding and repairing UV damage, such as benzophenones and benzotriazoles. However, most of these organic chemical components have strong toxic side effects on the human body, easily causing skin allergies or chapping, and are often expensive. Currently, most commonly used functional ingredients for repairing UV damage on the market are artificially synthesized products, and as functional raw materials for repairing UV damage, they all have the aforementioned natural drawbacks. Therefore, developing natural, green, and highly efficient functional raw materials for repairing UV damage derived from plants has become an inevitable trend for the future of the cosmetic and pharmaceutical industries.

[0004] Rhodomyrtus tomentosa, also known as mountain myrtle, is mainly found in the tropical and subtropical regions of southeastern and southwestern my country. It is endemic to my country and a valuable strategic wild plant resource. The plant has high medicinal value; its roots, leaves, and fruits are all used medicinally, possessing significant astringent, antidiarrheal, blood-activating, antibacterial, and qi-regulating / fetal-stabilizing pharmacological effects. Furthermore, the fruit is often used as a nourishing, edible, and medicinal wild fruit to enhance immunity and exhibits strong antioxidant activity. Studies have shown that the main chemical components of rhodomyrtus tomentosa include phloroglucinols, triterpenoids, flavonoids, tannins, and polysaccharides, which possess good anti-tumor, anti-inflammatory, antibacterial, antioxidant, and hepatoprotective properties. However, to date, there are no research reports on rhodomyrtus tomentosa's ability to repair UV damage, indicating its significant potential application in the development of cosmetic raw materials for repairing UV damage.

[0005] Water lilies are perennial aquatic herbaceous plants belonging to the genus *Nymphaea* in the family Nymphaeaceae, widely distributed in temperate, subtropical, and tropical regions. These plants not only possess rich ornamental and medicinal value, but some are also edible, either directly or processed into various delicacies, or used in herbal teas. Water lilies have been important medicinal plants used in traditional medicine, but current research on their functional activities is limited. Our previous findings revealed that water lilies (fragrant lotus) contain abundant polysaccharides with moisturizing, firming, antibacterial, and whitening properties. Recent studies have shown that the viscosity of these polysaccharide molecules gradually increases with increasing concentration, forming a uniform, smooth gel. This natural gel exhibits excellent breathability, achieving moisturizing and water-locking properties, maintaining long-lasting skin-friendliness. Therefore, water lily polysaccharides also possess significant potential economic value and a broad market space in the preparation of medical aesthetic gel products. Summary of the Invention

[0006] The purpose of this invention is to provide a composite beauty gel of water lily and myrtle that repairs UVB damage and its preparation method, in order to solve key industry problems in the beauty market such as the shortage of natural functional cosmetics for whitening, sun protection and skin repair, the tendency of common UVB products to cause allergic reactions, skin dehydration and cracking caused by long-term use, and high raw material costs. At the same time, it meets the urgent needs of the modern medical beauty industry for natural, green and safe products with the function of repairing UVB damage.

[0007] This invention provides the use of myrtle extract, or myrtle extract and water lily polysaccharide extract, in the preparation of cosmetics or pharmaceuticals for anti-ultraviolet damage and skin repair.

[0008] Preferably, the skin repair described is the repair after ultraviolet damage.

[0009] Preferably, the preparation method of the myrtle extract is as follows: crush fresh myrtle fruit, extract with anhydrous ethanol, concentrate the extract by rotary evaporator to obtain crude extract; disperse the crude extract in ultrapure water, and extract with petroleum ether, ethyl acetate and n-butanol in sequence; concentrate the extract under reduced pressure to obtain four fraction extracts, freeze-dry to obtain four fraction crude extracts, namely petroleum ether extract, ethyl acetate extract, n-butanol extract and aqueous extract, which are all myrtle extracts.

[0010] Further preferably, the myrtle extract is an ethyl acetate extract.

[0011] Preferably, the water lily polysaccharide is prepared by the following method: fresh water lily flowers are weighed, crushed, and firstly extracted with ethanol. Then, sterile distilled water is added to the treated water lily flowers, and extraction is carried out at room temperature. The mixture is then filtered using a 500-mesh filter membrane. The filtered extract is concentrated under reduced pressure. Ethanol is then added to the concentrated water lily flower extract, stirred evenly, and allowed to precipitate. The precipitate is filtered through gauze and then vacuum dried to obtain a crude polysaccharide extract. The crude polysaccharide extract is dissolved in water and dialyzed using a dialysis membrane with a molecular weight cutoff of 20 kDa. After dialysis, ethanol is added to the aqueous solution of the macromolecular water lily polysaccharide, and the mixture is stirred until the precipitate is completely precipitated and then filtered. The filtered macromolecular polysaccharide is further freeze-dried to obtain water lily polysaccharide.

[0012] Preferably, the myrtle extract and water lily polysaccharide extract are formulated into a water lily and myrtle composite cosmetic gel.

[0013] Preferably, the specific preparation method is to dissolve the myrtle extract in ethanol, then add it to the water lily polysaccharide solution, and stir evenly to obtain the water lily and myrtle composite beauty gel.

[0014] Preferably, the myrtle extract content in the water lily and myrtle composite beauty gel is 0.5%-4%, and the water lily polysaccharide content is 5%.

[0015] This invention also provides a composite beauty gel of water lily and myrtle.

[0016] This invention patent is the first to discover that myrtle extract has significant pharmacological effects in repairing UV damage. The UV damage repair capabilities of PE, EA, and nBU extracts from myrtle fruit are far superior to the positive control drug ATRA. In particular, the EA extract from myrtle fruit at 3.125 µg / mL can effectively repair human epidermal HaCaT cells damaged by UVB, significantly increasing the survival rate of human epidermal HaCaT cells by inhibiting cell apoptosis, with effects significantly better than the positive control drug ATRA. Simultaneously, this project reports for the first time a method for preparing a moisturizing and firming medical aesthetic gel using water lily polysaccharides. Furthermore, based on the synergistic effect of water lily polysaccharides and myrtle fruit extract, this patent innovatively develops a method for preparing a water lily and myrtle composite cosmetic gel with UVB damage repair efficacy. The water lily and myrtle composite cosmetic gel prepared by this invention can be widely used in natural anti-UV damage, moisturizing and firming cosmetics and medical aesthetic products, possessing significant economic potential and application value.

[0017] Technological innovation

[0018] This patent is the first to discover that myrtle fruit extract has a significant effect on repairing UVB-damaged cells, and the first to report a method for preparing a moisturizing cosmetic gel using water lily polysaccharide; based on the above findings, this patent is the first to prepare a composite cosmetic gel of water lily and myrtle, which has a repairing effect on HaCaT cell damage induced by medium-wave ultraviolet (UVB) and has excellent moisturizing and skin-nourishing effects. Attached Figure Description

[0019] Figure 1 It is the repairing effect of myrtle extract on UVB damage;

[0020] Figure 2 This is an evaluation of the activity of myrtle extract in reducing UVB damage;

[0021] Figure 3 It is the repair activity of the water lily and myrtle compound beauty gel against UVB cell damage;

[0022] Figure 4 This is an evaluation of the activity of the water lily and myrtle composite cosmetic gel in reducing UVB damage. Detailed Implementation

[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0024] Example 1:

[0025] I. Preparation of Myrtle Extract

[0026] The myrtle fruit of this invention was harvested from the South China Botanical Garden in July 2023. The specific method for preparing the crude extract is as follows: 700 g of fresh fruit was crushed and extracted with three times its volume of anhydrous ethanol for 24 h, repeated three times. The extract was concentrated using a rotary evaporator to obtain a crude extract. The crude extract was dispersed in a small amount of ultrapure water and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol, with each extraction repeated three times. After concentration under reduced pressure, four fractions of extract were obtained. After freeze-drying, four crude extracts were obtained: petroleum ether (PE) extract (2.0 g), ethyl acetate (EA) extract (2.9 g), n-butanol (nBU) extract (3.2 g), and aqueous extract (2.3 g). The four myrtle fruit extracts were prepared into a 20 mg / mL stock solution using cell-grade dimethyl sulfoxide (DMSO) and stored at -20 ˚C for experimental use.

[0027] II. Extraction of Water Lily Polysaccharides

[0028] Weigh 1.0 kg of fresh water lily flowers and pulverize them into 100-mesh powder. First, add 10 L of ethanol and extract three times, 24 hours each time, to remove pigments and small molecule phenolic acid compounds from the water lily flowers. Then, add 5 L of sterile distilled water to the treated water lily flowers and extract at room temperature for 24 hours, followed by filtration using a 500-mesh filter membrane. Concentrate the filtered extract to 500 mL under reduced pressure. Then, add 800 mL of 100% ethanol to the concentrated water lily extract, stir well, and allow it to precipitate. Filter the precipitate through gauze and vacuum dry to obtain a crude polysaccharide extract (127 g). Dissolve the crude polysaccharide extract in 1.0 L of sterile distilled water and dialyze using a 20 kDa molecular weight cutoff dialysis membrane for 2 days. Place the dialyzed macromolecular water lily polysaccharide aqueous solution in a 3.0 L beaker, add 1.5 L of 100% ethanol, stir until the precipitate is completely formed, and then filter. The filtered macromolecular polysaccharide was further freeze-dried and then ground to a fineness of less than 300 mesh at low temperature to obtain the target macromolecular water lily polysaccharide (77 g).

[0029] III. Repairing effect of myrtle extract on UVB-induced HaCaT cell damage

[0030] Inoculate 0.8 × 10⁶ cells into 96-well plates. 4HaCaT cell suspension at a cell / well density was added to each well with 90 µL of cell suspension. After culturing for 24 h, the plate was irradiated with UVB for 30 min. After irradiation, 10 µL of different concentrations of drugs were added to each well of a 96-well plate (positive control: all-trans retinoic acid (ATRA); negative control: PBS; drug groups: different concentrations of myrtle fruit extract). The plates were then incubated at 37 ˚C in a 5% CO2 incubator for another 24 h. After incubation, 10 µL of MTT solution (5 mg / mL) was added, and the plates were incubated for another 4 h. After incubation, the supernatant was carefully aspirated, and 100 µL of DMSO was added to dissolve the crystals. The OD value of each well at 490 nm was then measured using a microplate reader. Cell viability was calculated as: Cell viability = OD 490样品组 / OD 490 CK组 ×100%

[0031] like Figure 1 As shown, compared with the positive control drug ATRA (a retinoic acid receptor agonist commonly used clinically to treat UV-induced skin problems), the PE, EA, and nBU extracts of myrtle fruit exhibited better UV damage repair capabilities, with significantly higher cell survival rates. The crude EA extract, at 3.125 µg / mL, effectively repaired cells after UVB damage, increasing cell survival. Therefore, myrtle EA extract has a significant effect on repairing UVB damage.

[0032] IV. Evaluation of the apoptosis-reducing activity of myrtle extract in reducing UVB-damaged HaCaT cells

[0033] HaCaT cells in logarithmic growth phase were digested with trypsin and adjusted to a size of 1×10⁻⁶. 6 Cells were seeded at a density of [number] cells / mL in 6-well plates, with 2 mL of culture medium added to each well. When cell confluence reached 80%, the plate lid was removed to create a UVB damage model. UV light irradiation lasted 30 min, with the blank control group wrapped in aluminum foil. After irradiation, different concentrations of the crude extract to be tested were added to each well, and the plates were incubated for another 24 h. After incubation, the culture medium from each well was transferred to centrifuge tubes. After washing twice with PBS, 200 µL of trypsin was added to each well to digest the adherent cells. After digestion, the cells were mixed with the remaining culture medium to stop digestion, then centrifuged to remove the culture medium, and washed three times with PBS. After washing, the cells were resuspended in PBS and the concentration adjusted to 1 × 10⁻⁶. 6 cells / mL; FITC and PI were added to the cell suspension collected in the above steps, and the cells were stained in the dark for 20 min, and then analyzed by the instrument. The data obtained were processed using Flowjo.

[0034] Flow cytometry was used to detect apoptosis in HaCaT cells after UVB irradiation, and the results are as follows: Figure 2 As shown, after UVB irradiation, the survival rate of HaCaT cells was only 51.8%, but the addition of myrtle EA extract could repair the cell damage caused by UVB irradiation. Compared with the positive control drug ATRA, the myrtle EA extract could effectively reduce the apoptosis rate of HaCaT cells. When the concentration was 12.50 µg / mL, the apoptosis rate was 29.06%, which was much lower than that of the UVB damage model (apoptosis rate 48.28%). Moreover, with the increase of EA extract concentration, the apoptosis rate of HaCaT cells gradually decreased. When the concentration was 25 µg / mL, the apoptosis rate of HaCaT cells was 23.62%, which was significantly better than that of the positive control drug.

[0035] V. Preparation of Water Lily Makeup Gel

[0036] Example 1: Weigh 0.05 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 1.

[0037] Example 2: Weigh 0.1 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 2.

[0038] Example 3: Weigh 0.2 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 3.

[0039] Example 4: Weigh 0.4 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 4.

[0040] Example 5: Weigh 0.6 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 5.

[0041] Example 6: Weigh 0.8 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 6.

[0042] Example 7: Weigh 1.0 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 7.

[0043] Example 8: Weigh 1.2 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 8.

[0044] Example 9: Weigh 1.4 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 9.

[0045] Example 10: Weigh 1.6 g of water lily polysaccharide and place it in a 100 mL mortar. Add 20 mL of sterile deionized water and grind evenly to obtain water lily beauty gel 10.

[0046] Table 1. Morphology of water lily polysaccharide gel

[0047]

[0048] By configuring different concentrations of water lily polysaccharides, it was found that when the amount of water lily polysaccharides added is less than 2%, a fluid hydrogel can be successfully prepared; when the concentration of water lily polysaccharides added is between 3% and 4%, a non-fluid gel is obtained, and the gel can exhibit different morphologies to a certain extent; when the concentration of water lily polysaccharides added is 5% or higher, the resulting water lily gel is semi-solid and has high viscosity. The low-concentration hydrogel prepared by this patent can be used in cosmetic serums and lotions, the medium-concentration water lily polysaccharide gel is suitable for cosmetic creams, and the high-concentration water lily polysaccharide gel can be used in cream-based cosmetic products.

[0049] VI. Preparation of Water Lily and Myrtle Composite Makeup Gel

[0050] Example 1: Weigh 0.05 g of myrtle EA extract into a 50 mL beaker, then add 0.45 mL of anhydrous ethanol and stir to ensure complete dissolution. Simultaneously, weigh 1.0 g of water lily polysaccharide into a 100 mL mortar, add 18.5 mL of sterile deionized water, and grind until homogeneous. Then add the aforementioned myrtle EA extract ethanol solution and stir until homogeneous to obtain a thick water lily and myrtle composite cosmetic gel 1 (Poly-EA-1).

[0051] Example 2: Weigh 0.10 g of myrtle EA extract into a 50 mL beaker, then add 0.40 mL of anhydrous ethanol and stir to ensure complete dissolution. Simultaneously, weigh 1.0 g of water lily polysaccharide into a 100 mL mortar, add 18.5 mL of sterile deionized water, and grind until homogeneous. Then add the aforementioned myrtle EA extract ethanol solution and stir until homogeneous to obtain a thick water lily and myrtle composite cosmetic gel 2 (Poly-EA-2).

[0052] Example 3: Weigh 0.20 g of myrtle EA extract into a 50 mL beaker, then add 0.80 mL of anhydrous ethanol and stir to ensure complete dissolution. Simultaneously, weigh 1.0 g of water lily polysaccharide into a 100 mL mortar, add 18.0 mL of sterile deionized water, and grind until homogeneous. Then add the aforementioned myrtle EA extract ethanol solution and stir until homogeneous to obtain a thick water lily and myrtle composite cosmetic gel 3 (Poly-EA-3).

[0053] Example 4: Weigh 0.40 g of myrtle EA extract into a 50 mL beaker, then add 1.60 mL of anhydrous ethanol and stir to ensure complete dissolution. Simultaneously, weigh 1.0 g of water lily polysaccharide into a 100 mL mortar, add 17.0 mL of sterile deionized water, and grind until homogeneous. Then add the aforementioned myrtle EA extract ethanol solution and stir until homogeneous to obtain a thick water lily and myrtle composite cosmetic gel 4 (Poly-EA-4).

[0054] Example 5: Weigh 0.80 g of myrtle EA extract into a 50 mL beaker, then add 1.20 mL of anhydrous ethanol and stir to ensure complete dissolution. Simultaneously, weigh 1.0 g of water lily polysaccharide into a 100 mL mortar, add 17.0 mL of sterile deionized water, and grind until homogeneous. Then add the aforementioned myrtle EA extract ethanol solution and stir until homogeneous to obtain a thick water lily and myrtle composite cosmetic gel 5 (Poly-EA-5).

[0055] VII. Moisturizing and skin-nourishing activities of the water lily and myrtle complex beauty gel

[0056] Testing methods for moisturizing and skin-nourishing activities: The degree of skin dryness or smoothness is directly related to skin moisture. Loss of skin moisture directly leads to skin laxity, wrinkles, dryness, and even cracking. Therefore, by measuring the inhibition rate of elastase by water lily polysaccharides, the firming and anti-wrinkle effects can be detected.

[0057] The previously prepared water lily and myrtle composite beauty gels (samples 1-5) were freeze-dried and ground into a fine powder below 200 mesh at low temperature to obtain water lily and myrtle composite beauty gel freeze-dried powder (samples 1-5). A clean, dry 10 mL flat weighing bottle with a 25 mm mouth was selected, and 500 mg of the previously prepared water lily and myrtle composite beauty gel freeze-dried powder was weighed into it. Then, 200 mg of sterile deionized water was slowly added, and the weighing bottle was gently shaken to allow the water lily and myrtle composite beauty gel freeze-dried powder to fully absorb the sterile deionized water. Next, the water-absorbed water lily and myrtle composite beauty gel freeze-dried powder was placed in a glass desiccator with 200 g of anhydrous color-changing silica gel at the bottom and placed at 25°C. oThe hydration rate of the freeze-dried powder of water lily and myrtle composite cosmetic gel was then tested in a C biochemical incubator. Hyaluronic acid was used as a positive control, and 700 mg of deionized sterile water was used as a blank control. The flat weighing bottles were removed and weighed at 2, 4, 6, 8, 10, 12, and 24 h, and the hydration rate was calculated using the following formula: Hydration rate (%) = (mo – m) / mo × 100%; where mo is the initial water content of the sample, and m is the water content of the sample after water loss.

[0058] The results showed that the freeze-dried powder of the water lily and myrtle composite cosmetic gel possessed excellent moisturizing activity. Compared with the sterile deionized water (the blank control), the freeze-dried powder of the water lily and myrtle composite cosmetic gel exhibited a very significant moisturizing effect for more than 4 hours. Furthermore, the freeze-dried powder of the water lily and myrtle composite cosmetic gel showed even better moisturizing activity when the proportion of water lily polysaccharides was higher; in particular, composite gel freeze-dried powder 1 had a moisturizing effect similar to hyaluronic acid. These research results indicate that the water lily and myrtle composite cosmetic gel has outstanding moisturizing and skin-nourishing effects.

[0059] Table 2. Moisturizing effect of freeze-dried powder of water lily and myrtle composite beauty gel.

[0060]

[0061] 8. Repair ability of water lily and myrtle composite beauty gel on UVB damage model

[0062] Inoculate 0.8 × 10⁶ cells into 96-well plates. 4 HaCaT cell suspension at a cell / well density was added to each well with 80 µL of cell suspension. After culturing for 24 h, the plate was irradiated with UVB for 30 min. After irradiation, 20 µL of a composite beauty gel of different concentrations of water lily and myrtle was added to each well of a 96-well plate (positive control: all-trans retinoic acid (ATRA); negative control: PBS). The plates were then incubated at 37 ˚C in a 5% CO2 incubator for another 24 h. After incubation, 10 µL of MTT solution (5 mg / mL) was added, and the plates were incubated for another 4 h. After incubation, the supernatant was carefully aspirated, and 100 µL of DMSO was added to dissolve the crystals. The OD value of each well at 490 nm was then measured using a microplate reader. Cell viability was calculated as: Cell viability = OD 490 Sample group / OD 490 CK group × 100%.

[0063] As above Figure 3As shown, after 20 minutes of UVB irradiation, the cell viability in the PBS-treated group significantly decreased to 28.74%, indicating that UVB caused severe damage to HaCaT cells. Under treatment with different concentrations of Poly-EA gel, cell viability showed a trend of gradual improvement with increasing concentration. Specifically, the 20 mg / mL (Water Lily and Myrtle Complex Cosmetic Gel 4, Poly-EA-4) and 40 mg / mL (Water Lily and Myrtle Complex Cosmetic Gel 5, Poly-EA-5) groups showed significant repair effects, with viability recovering to 98.03% and 218.89%, respectively, significantly higher than other low-concentration groups. The results indicate that high-concentration Poly-EA has a very prominent protective and repairing effect on UVB-damaged cells.

[0064] IX. Evaluation of the apoptosis-reducing activity of myrtle extract in HaCaT cells damaged by UVB.

[0065] HaCaT cells in logarithmic growth phase were digested with trypsin and adjusted to a size of 1×10⁻⁶. 6 Cells were seeded at a density of [number] cells / mL in 6-well plates, with 2 mL of culture medium added to each well. When cell confluence reached 80%, the plate lid was removed to create a UVB damage model. UV light irradiation lasted 30 min, while a blank control group was wrapped in aluminum foil. After irradiation, the previously prepared water lily and myrtle composite beauty gel was diluted 100-fold, and 20 µL was added to each well. The plates were then incubated for 24 h. After incubation, the culture medium from each well was transferred to centrifuge tubes. After washing twice with PBS, 200 µL of trypsin was added to each well to digest the adherent cells. After digestion, the cells were mixed with the remaining culture medium to stop digestion, then centrifuged to remove the culture medium, and washed three times with PBS. After washing, the cells were resuspended in PBS and the concentration adjusted to 1 × 10⁻⁶. 6 cells / mL; FITC and PI were added to the cell suspension collected in the above steps, and the cells were stained in the dark for 20 min, and then analyzed by the instrument. The data obtained were processed using Flowjo.

[0066] The effect of Poly-EA gel on the apoptosis rate of UVB-damaged HaCaT cells was detected by flow cytometry. The results are as follows: Figure 4As shown, after UVB irradiation, the apoptosis rate of HaCaT cells was 64.95%, but the addition of Poly-EA gel could repair the cell damage caused by UVB irradiation and reduce the apoptosis rate. When the concentration was 10 mg / mL (water lily and myrtle composite beauty gel 3, Poly-EA-3), the apoptosis rate of cells was 62.64%, showing a weak repair ability compared with the UVB model group. When the concentration was 20 mg / mL (water lily and myrtle composite beauty gel 4, Poly-EA-4), the damaged HaCaT cells were repaired, and the apoptosis rate decreased to 38.94%. Moreover, with the increase of the concentration, the apoptosis rate of HaCaT cells gradually decreased. When the concentration was 40 mg / mL (water lily and myrtle composite beauty gel 5, Poly-EA-5), the apoptosis rate of HaCaT cells was 23.62%, which significantly reduced the cell apoptosis caused by UVB damage and effectively restored cell growth.

[0067] In summary, this patent is the first to discover that myrtle fruit extract has a significant effect on repairing UVB-damaged cells, and the first to report a method for preparing a moisturizing cosmetic gel using water lily polysaccharide. Based on the above findings, this patent is the first to prepare a composite cosmetic gel of water lily and myrtle, which has a repairing effect on UVB-induced HaCaT cell damage and also has excellent moisturizing and skin-nourishing effects.

Claims

1. The application of myrtle extract, or myrtle extract and water lily polysaccharide extract, in the preparation of cosmetics or drugs for anti-ultraviolet damage and skin repair, wherein the skin repair refers to repair after ultraviolet damage, and the preparation method of myrtle extract is as follows: crush fresh myrtle fruit, extract with anhydrous ethanol, concentrate the extract by rotary evaporator to obtain crude extract; disperse the crude extract in ultrapure water, and extract successively with petroleum ether, ethyl acetate and n-butanol; concentrate the extract under reduced pressure to obtain four fraction extracts, freeze-dry to obtain four fraction crude extracts, namely petroleum ether extract, ethyl acetate extract, n-butanol extract and aqueous extract, which are all myrtle extracts.

2. The application according to claim 1, characterized in that, The myrtle extract is an ethyl acetate extract.

3. The application according to claim 1, characterized in that, The water lily polysaccharide is prepared by the following method: Fresh water lily flowers are weighed, crushed, and firstly extracted with ethanol. Then, sterile distilled water is added to the treated water lily flowers, and extraction is carried out at room temperature. The mixture is then filtered using a 500-mesh filter membrane. The filtered extract is concentrated under reduced pressure. Ethanol is then added to the concentrated water lily flower extract, stirred evenly, and allowed to precipitate. The precipitate is filtered through gauze and then vacuum dried to obtain a crude polysaccharide extract. The crude polysaccharide extract is dissolved in water and dialyzed using a dialysis membrane with a molecular weight cutoff of 20 kDa. After dialysis, ethanol is added to the aqueous solution of the macromolecular water lily polysaccharide, and the mixture is stirred until the precipitate is completely precipitated and then filtered. The filtered macromolecular polysaccharide is further freeze-dried to obtain the water lily polysaccharide.

4. The application according to claim 1, characterized in that, The myrtle extract and water lily polysaccharide extract are used to make a water lily and myrtle composite cosmetic gel.

5. The application according to claim 4, characterized in that, The specific preparation method involves dissolving myrtle extract in ethanol, then adding it to a water lily polysaccharide solution and stirring until a water lily and myrtle composite beauty gel is obtained.

6. The application according to claim 1, characterized in that, The myrtle extract content in the water lily and myrtle complex beauty gel is 0.5%-4%, and the water lily polysaccharide content is 5%.

7. A composite beauty gel of water lily and myrtle, characterized in that, The product is prepared by the following method: Myrtle extract is dissolved in ethanol and then added to a water lily polysaccharide solution. The mixture is stirred until homogeneous to obtain a composite beauty gel of water lily and myrtle. The preparation method of the myrtle extract is as follows: Fresh myrtle fruit is crushed and extracted with anhydrous ethanol. The extract is concentrated using a rotary evaporator to obtain a crude extract. The crude extract is dispersed in ultrapure water and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol. The extract is concentrated under reduced pressure to obtain a four-fragment extract paste. After freeze-drying, four crude extracts are obtained, namely, petroleum ether extract, ethyl acetate extract... The extracts, including n-butanol extract and aqueous extract, are all myrtle extracts. The water lily polysaccharide is prepared by the following method: fresh water lily flowers are weighed, crushed, and firstly, ethanol is added for extraction. Then, sterile distilled water is added to the treated water lily flowers, and extraction is carried out at room temperature. The mixture is then filtered using a 500-mesh filter membrane. The filtered extract is concentrated under reduced pressure, and then ethanol is added to the concentrated water lily flower extract. After stirring evenly, the extract is allowed to precipitate. The precipitate is filtered through gauze and then vacuum dried to obtain a crude polysaccharide extract. The crude polysaccharide extract is dissolved in water and dialyzed using a dialysis membrane with a molecular weight cutoff of 20 kDa. After dialysis, ethanol is added to the aqueous solution of the macromolecular water lily polysaccharide, and the mixture is stirred until the precipitate is completely precipitated and then filtered. The filtered macromolecular polysaccharide is further freeze-dried to obtain water lily polysaccharide.

8. The water lily and myrtle composite beauty gel according to claim 7, characterized in that, The myrtle extract content in the water lily and myrtle complex beauty gel is 0.5%-4%, and the water lily polysaccharide content is 5%.

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