Preparation method of a dewberry extract and application thereof

Abstract

The application provides a preparation method of Rubus corchorifolius extract and application thereof, and belongs to the technical field of traditional Chinese medicine pharmacy. The preparation method is simple in steps and does not produce toxic and harmful substances. The Rubus corchorifolius extract prepared by the application can inhibit skin tissue structure damage, collagen fiber reduction, elastic fiber damage and reduction of mast cell infiltration, and is effectively used for skin photodamage. Compared with a UVB model group, after intervention of a TFDI low-dose group and a TFDI high-dose group, skin photodamage scores are reduced by 1.7306 and 2.3184 respectively; epidermal thickness is reduced by 0.8572 microns and 1.3627 microns respectively; dermal thickness is reduced by 0.4767 microns and 0.8370 microns respectively; and collagen content is increased by 64.95% and 126.65% respectively. The Rubus corchorifolius cream prepared by the application can treat and / or prevent skin photodamage, and has no obvious toxicity in an in-vitro experiment, and exhibits good safety.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine pharmaceutical technology, specifically relating to a method for preparing a snakeberry extract and its application. Background Technology

[0002] As the body's first line of defense, the skin is susceptible to damage from ultraviolet (UV) radiation. Among these, long-wave ultraviolet (UVA) and medium-wave ultraviolet (UVB) are the main pathogenic factors. UVB single photons have high energy and, although they only reach the epidermal layer, they can cause skin erythema, DNA damage, and even skin cancer, making them the main cause of photodamage to the skin.

[0003] Acute photodamage can cause skin laxity and wrinkles, the core of which is the alteration of the extracellular matrix (ECM) in the dermis: the function of elastic fibers is affected, and collagen is degraded due to the upregulation of matrix metalloproteinases (MMPs); at the same time, UVB will generate excessive reactive oxygen species (ROS), disrupting the oxidative balance, reducing the activity of endogenous antioxidant enzymes, and inducing inflammatory responses. Through NF-κB and AP-1, a vicious cycle of "free radical-oxidative stress-inflammation" is formed, which in turn activates the MAPK signaling pathway (including ERK, JNK, and p38), mediates keratinocyte apoptosis, and aggravates skin damage.

[0004] Current methods for preventing and treating photodamage have limitations. Physical and chemical sunscreens can be too heavy on sensitive skin, antioxidants such as vitamin C and E have poor stability, and medical aesthetic treatments are expensive. The 0.05% all-trans retinoic acid cream approved by the US FDA is prone to causing retinoid dermatitis and has a slow effect. Therefore, the development of highly effective and stable anti-photodamage products has become a research hotspot.

[0005] Traditional Chinese medicine believes that acute photodamage to the skin is related to "phototoxicity" and requires nourishing yin and blood, clearing heat and detoxifying. (Snakeberry) Duchesnea indica Andr. Focke Duchesnea indicum (also known as Three-Clawed Phoenix, Dragon Spitting Pearl, Oriental Strawberry, etc.) is the dried whole herb of a plant in the Rosaceae family. It is widely distributed in Liaoning, Yunnan, and Guizhou provinces of China and possesses properties such as clearing heat and detoxifying, dispersing blood stasis and reducing swelling, and cooling the blood and stopping bleeding. However, there are currently no systematic reports on whether duchesnea indicum extract has a protective effect against photodamage to the skin. Summary of the Invention

[0006] To address the aforementioned technical problems, the primary objective of this invention is to provide a method for preparing snakeberry extract. The preparation method of this invention features simple extraction steps, produces no toxic or harmful substances, is low-cost, and is suitable for large-scale industrial production. The snakeberry extract obtained by this invention can inhibit skin tissue structure damage, collagen fiber reduction, elastic fiber damage, and reduce mast cell infiltration, thereby effectively addressing photodamage to the skin.

[0007] A second objective of the present invention is to provide a snakeberry cream that can treat and / or prevent photodamage to the skin.

[0008] A third objective of this invention is to provide the use of the above-mentioned snakeberry extract and snakeberry cream in the preparation of products for the prevention and / or treatment of photodamage to the skin.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing snakeberry extract, comprising the following steps: The crude powder of wild strawberry was mixed with an ethanol solution, the pH was adjusted to 8.0-10.0 with an alkaline solution, and the mixture was refluxed for extraction. The mixture was then filtered, the filtrate was collected, and the solution was concentrated to obtain a concentrate. The concentrate was then adjusted to pH 3.0-5.0 with an acid solution, allowed to stand, filtered, and the precipitate was collected. The precipitate was washed with water and ether, filtered, and dried to obtain the wild strawberry extract.

[0010] Preferably, the wild strawberry coarse powder is prepared by pulverizing the dried whole wild strawberry plant to 55-65 mesh using a pulverizer; the material-to-liquid ratio of the wild strawberry coarse powder to the ethanol solution is 1g:10-20mL; and the volume percentage of the ethanol solution is 65%-75%.

[0011] Preferably, the reflux extraction time is 1-2 hours; the water washing is 2-3 times with distilled water; and the ether washing is 1-2 times with petroleum ether or diethyl ether.

[0012] This invention provides a snakeberry cream comprising an oil phase, an aqueous phase, and a snakeberry extract prepared by the above-described preparation method.

[0013] Preferably, the oil phase comprises one or more of stearic acid, white petrolatum, and liquid paraffin; the aqueous phase comprises one or more of sodium lauryl sulfate, ethylparaben, glycerin, and distilled water; the oil phase is stearic acid, white petrolatum, and liquid paraffin, and the mass-to-volume ratio of stearic acid, white petrolatum, and liquid paraffin is 1.5~3.0g:1.5~2.5g:1.0~2.0mL; the aqueous phase is sodium lauryl sulfate, ethylparaben, glycerin, and distilled water, and the mass-to-volume ratio of sodium lauryl sulfate, ethylparaben, glycerin, and distilled water is 0.1~0.7g:0.01~0.03g:0.5~2.5mL:8.0~16.0mL; the snakeberry extract accounts for 3%~15% of the total weight of the snakeberry cream.

[0014] This invention provides a method for preparing the above-mentioned snakeberry cream, comprising the following steps: The stearic acid, white petrolatum and liquid paraffin were heated in a water bath at 70-90°C and stirred to dissolve, thus obtaining the oil phase. The sodium lauryl sulfate, ethylparaben, glycerol, and water were heated in a water bath at 70-90°C and stirred to dissolve, thus obtaining an aqueous phase. The aqueous phase is kept at 70~90℃. The aqueous phase is added to the oil phase, and then the snakeberry extract is added and stirred to obtain the snakeberry cream.

[0015] This invention provides the application of the snakeberry extract or the snakeberry cream prepared by the above preparation method in the preparation of products for preventing and / or treating photodamage to the skin.

[0016] Preferably, the skin photodamage is acute skin photodamage.

[0017] Preferably, the pharmaceutical dosage form includes a topical preparation, which includes creams, ointments, gels, liniments, lotions, sprays, patches, or films.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing snakeberry extract. This method involves simple extraction steps, produces no toxic or harmful substances, is low-cost, and is suitable for large-scale industrial production. The snakeberry extract obtained by this invention can inhibit skin tissue structure damage, collagen fiber reduction, elastic fiber damage, and reduce mast cell infiltration, and can be effectively applied to the repair and improvement of photodamage to the skin. Compared with the UVB model group, after intervention with the low-dose TFDI group and the high-dose TFDI group: the skin photodamage score decreased by 1.7306 and 2.3184, respectively; the epidermal thickness decreased by 0.8572 μm and 1.3627 μm, respectively; the dermal thickness decreased by 0.4767 μm and 0.8370 μm, respectively; and the collagen content increased by 64.95% and 126.65%, respectively. The snakeberry cream obtained by this invention can treat and / or prevent photodamage to the skin, and shows no significant toxicity in in vitro experiments, demonstrating good safety. Attached Figure Description

[0019] Figure 1 Images of the skin on the back of mice under different treatments at 0, 4, 7, 10, and 15 days of UVB irradiation.

[0020] Figure 2 The scoring criteria for acute photodamage in mice after UVB irradiation under different treatments are shown, with skin images of mice scoring 0-6 from left to right.

[0021] Figure 3 Statistical graph of appearance scores of mice with different treatments after 15 days of UVB irradiation (n=4). # indicates that compared with the control group, P<0.01; ## indicates that compared with the UVB group, P<0.01.

[0022] Figure 4 HE staining results of mice with different treatments on day 15 after UVB irradiation; A represents HE staining results of KM mice after different pretreatments; B shows the statistical results of HE epidermal thickness; C shows the statistical results of HE dermal thickness; (n=4). # indicates that compared with the control group, P<0.01; ## indicates that compared with the UVB group, P<0.01.

[0023] Figure 5 Gomori staining results of mouse skin tissue from mice under different treatments on day 15 of UVB irradiation.

[0024] Figure 6 Representative images of Masson staining results and collagen fiber content statistics for mice under different UVB irradiation on day 15; A is a representative image of Masson staining results; B is the statistical result of collagen fiber content; (n=4). The value of P<0.01 indicates that the result is significantly different from the control group; ## indicates that the result is significantly different from the UVB group; and ### indicates that the result is significantly different from the UVB group.

[0025] Figure 7 The results of mast cell staining in mouse skin tissue on day 15 after UVB irradiation in mice under different treatments are shown. Black arrows indicate mast cells. Detailed Implementation

[0026] This invention provides a method for preparing snakeberry extract, comprising the following steps: The crude powder of wild strawberry was mixed with an ethanol solution, the pH was adjusted to 8.0-10.0 with an alkaline solution, and the mixture was refluxed for extraction. The mixture was then filtered, the filtrate was collected, and the solution was concentrated to obtain a concentrate. The concentrate was then adjusted to pH 3.0-5.0 with an acid solution, allowed to stand, filtered, and the precipitate was collected. The precipitate was washed with water and ether, filtered, and dried to obtain the wild strawberry extract.

[0027] In this invention, the wild strawberry is sourced from commercially available sources; the wild strawberry can be from Guizhou Province; the medicinal part of the wild strawberry is preferably the whole plant; the coarse powder of the wild strawberry has a mesh size of 55-65 mesh, preferably 58-62 mesh, and more preferably 60 mesh; in one feasible embodiment, the pulverization is completed by a pulverizer, ball mill, crusher, or air jet mill. This invention increases the extraction efficiency by pulverization, thereby increasing the contact area, and avoids affecting the subsequent hot reflux extraction process due to excessive pulverization. The ethanol solution is an aqueous ethanol solution; the volume concentration of the ethanol solution is 65%-75%, preferably 67%-73%, and more preferably 70%, where the volume percentage refers to the percentage of ethanol volume in the total volume of the ethanol solution; the ratio of wild strawberry coarse powder to ethanol solution is 1g:10-20mL, preferably 1g:13-17mL, and more preferably 1g:14mL, 1g:15mL, or 1g:16mL; the wild strawberry coarse powder is soaked at 25℃ for 15 minutes to ensure complete ethanol saturation. The alkaline solution is preferably a 0.1 mol / L NaOH solution; the pH of the alkaline solution is adjusted to 8.0~10.0, preferably 8.5~9.5, and more preferably 9.0. The reflux extraction temperature is 70~80℃, preferably 72~78℃, and more preferably 75℃. Stable heating extraction at this temperature prevents excessively high temperatures from causing charring of the raw materials or decomposition of the active ingredients. The reflux extraction time is preferably 1h~2h, and the number of reflux extractions is 2~3 times, preferably 2 or 3 times. At the extraction time and number of extractions described in this invention, the active ingredients can be extracted to the maximum extent, avoiding excessively long extraction times that lead to increased impurities or component degradation. In this invention, the above extracts are combined, filtered, and concentrated by heating to remove ethanol to obtain a concentrated solution. The heating and concentration temperature is 50~70℃.

[0028] In this invention, the acid solution is preferably 1-2 mol / L hydrochloric acid, such as 1 mol / L hydrochloric acid, 1.5 mol / L hydrochloric acid, or 2 mol / L hydrochloric acid; the pH of the acid solution is adjusted to 3.0-5.0, preferably pH 3.5-4.5, and more preferably pH 4.0; the acid solution is slowly added dropwise to the concentrate while stirring; the settling is preferably done at room temperature for 4 hours or at 4°C overnight. The settling temperature and time specified in this invention ensure more complete precipitation, and room temperature refers to 20-25°C.

[0029] In this invention, each water wash involves adding 10-15 mL of distilled water; the number of water washes is 2-3 times, preferably 2 or 3 times, which maximizes the removal of water-soluble impurities. The ether is preferably petroleum ether or diethyl ether, with 10 mL of petroleum ether or diethyl ether added each time; the number of ether washes is 1-2 times, preferably 1 or 2 times, which maximizes the removal of fat-soluble impurities. The filtration time is preferably until dry; the drying is preferably done in a drying oven, with the temperature preferably 35-40°C, more preferably 37°C.

[0030] This invention provides a snakeberry cream comprising an oil phase, an aqueous phase, and a snakeberry extract prepared by the above-described preparation method.

[0031] In this invention, the oil phase comprises one or more of stearic acid, white petrolatum, and liquid paraffin; the aqueous phase comprises one or more of sodium lauryl sulfate, ethylparaben, glycerin, and distilled water; the mass-to-volume ratio of stearic acid, white petrolatum, and liquid paraffin is 1.5~3.0g:1.5~2.5g:1.0~2.0mL; the aqueous phase comprises sodium lauryl sulfate, ethylparaben, glycerin, and distilled water, and the mass-to-volume ratio of sodium lauryl sulfate, ethylparaben, glycerin, and distilled water is 0.1~0.7g:0.01~0.03g:0.5~2.5mL:8.0~16.0mL. In a preferred embodiment, the oil phase comprises stearic acid, white petrolatum, and liquid paraffin, wherein the mass-to-volume ratio of stearic acid, white petrolatum, and liquid paraffin is 1.8-2.5 g : 1.5-2.5 g : 1.3-1.5 mL; the aqueous phase comprises sodium lauryl sulfate, ethylparaben, glycerin, and distilled water, wherein the mass-to-volume ratio of sodium lauryl sulfate, ethylparaben, glycerin, and distilled water is 0.2-0.5 g : 0.01-0.03 g : 1-2 mL : 10-14 mL; and the snakeberry extract comprises 3%-15% of the total weight of the snakeberry cream, such as 5% or 10%.

[0032] This invention provides a method for preparing a snakeberry cream, comprising the following steps: heating stearic acid, white petrolatum, and liquid paraffin in a water bath at 70-90°C and stirring to dissolve them, thereby obtaining an oil phase; heating sodium lauryl sulfate, ethylparaben, glycerin, and water in a water bath at 70-90°C and stirring to dissolve them, thereby obtaining an aqueous phase; maintaining the aqueous phase at 70-90°C, adding the aqueous phase to the oil phase, then adding snakeberry extract, and stirring to obtain the snakeberry cream.

[0033] In this invention, the water bath heating temperature is 70~90℃, such as 70℃, 80℃, or 90℃. As a preferred embodiment, the preparation method of the snakeberry cream includes the following steps: heating 2.5g stearic acid, 2.0g white petrolatum, and 1.5mL liquid paraffin in a water bath at 80℃, stirring to dissolve, and obtaining an oil phase; heating 0.5g sodium lauryl sulfate, 0.02g ethylparaben, 2.0g glycerin, and 14mL water in a water bath at 80℃, stirring to dissolve, and obtaining an aqueous phase; maintaining the aqueous phase at 80℃, adding the aqueous phase to the oil phase, then adding 2g snakeberry extract, stirring, and obtaining the snakeberry cream. As another preferred embodiment, the preparation method of the snakeberry cream includes the following steps: heating 1.8g stearic acid, 2.0g white petrolatum, and 1.3mL liquid paraffin in a water bath at 80℃, stirring to dissolve, and obtaining an oil phase. 0.2 g sodium lauryl sulfate, 0.02 g ethylparaben, 1.0 g glycerin, and 10 mL of water were heated in a water bath at 80 °C and stirred until dissolved to obtain an aqueous phase. The aqueous phase was kept at 70 °C and added to the oil phase, followed by the addition of 2 g of wild strawberry extract. The mixture was stirred to obtain the wild strawberry cream. The active ingredient in the wild strawberry cream is wild strawberry extract.

[0034] This invention provides the use of the snakeberry extract or the snakeberry cream prepared by the above-described method in the preparation of products for the prevention and / or treatment of photodamage to the skin.

[0035] This invention utilizes wild strawberry extract or wild strawberry cream to inhibit skin tissue structure damage, collagen fiber reduction, elastic fiber damage, and reduce mast cell infiltration, thereby reducing skin photodamage score, epidermal thickness, dermal thickness, and collagen content, and can be effectively applied to the repair and improvement of skin photodamage.

[0036] In this invention, the skin photodamage is acute skin photodamage; the product includes pharmaceuticals or cosmetics; the pharmaceutical dosage form includes topical preparations, which include creams, ointments, gels, liniments, lotions, sprays, patches, or films.

[0037] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] In the following examples, SPF-grade female Kunming (KM) mice, 6 weeks old and weighing 20-28g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (License No.: SCXK (Xiang) 2021-0002). All animals were housed in the SPF-grade animal facility of the Key Laboratory of Basic Pharmacology, Ministry of Education, Zunyi Medical University, under the following conditions: ambient temperature 18-25℃, humidity 40%-70%, 12-hour light-dark cycle, and free access to food and water.

[0040] The UVB lamp (TL20W / 01) was purchased from Philips GmbH, Germany; the digital camera was purchased from Sony GmbH, Japan; the TP1020 tissue dehydrator was purchased from Leica GmbH, Germany; the EG1150 paraffin embedding machine, the RM2245 rotary microtome, and the TKY-TKA slide spreader / batter were purchased from Hubei Taikang Company, China; the Milli QA pure water processor was purchased from Millipore, France; and the ultrasonic cleaner was purchased from Shanghai Yijing Ultrasonic Instrument Co., Ltd., China.

[0041] Hematoxylin was purchased from Wuhan Sewell Biotechnology Co., Ltd., catalog number G1004; the elastic fiber staining kit (modified Gomori aldehyde fuchsin method) was purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number G1593; the modified Masson trichrome staining kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number G1346; and the G1346 mast cell staining solution (toluidine blue method) was purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number G3670.

[0042] Statistical analysis of data: Experimental data were analyzed using SPSS 21.0 statistical software. All data are expressed as (Mean ± SEM). One-way ANOVA was used to compare the differences in means between groups. The LSD test was used to test the homogeneity of variance. Dunnett's T3 was used to compare the differences in means between groups. P < 0.05 was considered statistically significant.

[0043] Example 1 A method for preparing snakeberry extract (TFDI) includes the following steps: (1) Raw material pretreatment: 300g of dried whole snakeberry plant was crushed by a pulverizer and passed through a 60-mesh sieve to obtain snakeberry coarse powder for later use.

[0044] (2) Ethanol-alkali extraction: Weigh out the crude snakeberry powder and add 70% ethanol aqueous solution at a ratio of 1g:15mL. Soak at 25℃ for 15min to ensure complete saturation of the powder. Adjust the pH to 9.0 with 0.1 mol / L NaOH solution and then reflux at 75℃ for 1h. Filter while hot to obtain the first filtrate and the first residue. Then, add 70% ethanol aqueous solution at a ratio of 1g:15mL to the first residue and soak at 25℃ for 15min to ensure complete saturation of the powder. Adjust the pH to 9.0 with 0.1 mol / L NaOH solution and then reflux at 75℃ for 1h. Filter to obtain the second filtrate and the second residue. Combine all filtrates.

[0045] Then, to the second filter residue, a 70% ethanol aqueous solution was added at a material-to-liquid ratio of 1:15 (g:mL). The pH was then adjusted to 9.0 with 0.1 mol / L NaOH solution, and the mixture was heated to reflux at 75°C for 1 hour. After filtration, the third filtrate was obtained. The three filtrates were combined, heated, and concentrated to remove the ethanol, yielding a concentrated solution.

[0046] (3) Acid precipitation: Slowly add 1.5 mol / L hydrochloric acid to the above concentrated solution while stirring. After adjusting the pH to 4.0, let it stand at room temperature for 4 hours to allow complete precipitation. Filter the solution and collect the precipitate to obtain the first precipitate.

[0047] (4) Washing and purification: The first precipitate was washed twice with distilled water, adding 10 mL of distilled water each time, to remove water-soluble impurities. Then it was washed twice with petroleum ether, adding 10 mL of petroleum ether each time, to remove fat-soluble impurities. The precipitate was then filtered to obtain the second precipitate.

[0048] (5) Drying: The second precipitate was placed in an oven at 37°C and dried to constant weight to obtain 6.8g of snakeberry extract.

[0049] Example 2 A method for preparing a wild strawberry extract, comprising the following steps: (1) Raw material pretreatment: 300g of dried whole snakeberry plant was crushed by a pulverizer and passed through a 55-mesh sieve to obtain snakeberry coarse powder for later use.

[0050] (2) Ethanol-alkali extraction: Weigh out the crude snakeberry powder and add 65% ethanol aqueous solution at a material-to-liquid ratio of 1g:10mL. Soak at 25℃ for 15min to ensure complete saturation of the powder. Adjust the pH to 8.5 with 0.1 mol / L NaOH solution and then reflux at 70℃ for 1h. Filter while hot to obtain the first filtrate and the first filter residue. Then, add 65% ethanol aqueous solution at a material-to-liquid ratio of 1g:10mL to the first filter residue and soak at 25℃ for 15min to ensure complete saturation of the powder. Adjust the pH to 8.5 with 0.1 mol / L NaOH solution and then reflux at 70℃ for 1h. Filter to obtain the second filtrate and the second filter residue. Combine all filtrates.

[0051] Then, in the second filter residue, a 65% ethanol aqueous solution was added at a material-to-liquid ratio of 1:10 (g:mL), and the mixture was soaked at 25℃ for 35 minutes to ensure complete saturation of the wild strawberry powder. The pH was then adjusted to 8.5 with 0.1 mol / L NaOH solution, and the mixture was heated to reflux at 70℃ for 1 hour. The mixture was then filtered to obtain the third filtrate. The three filtrates were combined, heated, and concentrated to remove the ethanol, yielding a concentrated solution.

[0052] (3) Acid precipitation: Slowly add 1.5 mol / L hydrochloric acid to the above concentrated solution while stirring. After adjusting the pH to 3.0, let it stand at room temperature for 4 hours to allow complete precipitation. Filter the solution and collect the precipitate to obtain the first precipitate.

[0053] (4) Washing and purification: The first precipitate was washed twice with distilled water, adding 10 mL of distilled water each time, to remove water-soluble impurities. Then it was washed once with petroleum ether, adding 10 mL of petroleum ether each time, to remove fat-soluble impurities. The precipitate was then filtered to obtain the second precipitate.

[0054] (5) Drying: The second precipitate was placed in an oven at 35°C and dried to constant weight to obtain snakeberry extract.

[0055] Example 3 A method for preparing a wild strawberry extract, comprising the following steps: (1) Raw material pretreatment: 300g of dried whole snakeberry plant was crushed by a pulverizer and passed through a 65-mesh sieve to obtain snakeberry coarse powder for later use.

[0056] (2) Ethanol-alkali extraction: Weigh out the crude snakeberry powder and add 75% ethanol aqueous solution at a ratio of 1g:20mL. Soak at 25℃ for 15min to ensure complete saturation of the powder. Adjust the pH to 10.0 with 0.1 mol / L NaOH solution and reflux at 80℃ for 2h. Filter while hot to obtain the first filtrate and the first residue. Then, add 80% ethanol aqueous solution at a ratio of 1g:20mL to the first residue and soak at 25℃ for 15min to ensure complete saturation of the powder. Adjust the pH to 10.0 with 0.1 mol / L NaOH solution and reflux at 80℃ for 2h. Filter to obtain the second filtrate and the second residue. Combine the two filtrates and concentrate by heating to remove the ethanol, obtaining the concentrated solution.

[0057] (3) Acid precipitation: Slowly add 1.5 mol / L hydrochloric acid to the above concentrated solution while stirring. After adjusting the pH to 4.0, let it stand at room temperature for 4 hours to allow complete precipitation. Filter the solution and collect the precipitate to obtain the first precipitate.

[0058] (4) Washing and purification: The first precipitate was washed three times with distilled water, adding 10 mL of distilled water each time, to remove water-soluble impurities. Then it was washed twice with diethyl ether, adding 10 mL of diethyl ether each time, to remove fat-soluble impurities. The precipitate was then filtered to obtain the second precipitate.

[0059] (5) Drying: The second precipitate was placed in an oven at 40°C and dried to constant weight to obtain the snakeberry extract.

[0060] Example 4 A method for preparing a wild strawberry cream, comprising the following steps: (1) Oil phase: 1.8g stearic acid, 2.0g white petrolatum and 1.3mL liquid paraffin were heated in a water bath at 80℃ and stirred to dissolve, thus obtaining the oil phase.

[0061] (2) Aqueous phase: 0.2g sodium lauryl sulfate, 0.02g ethylparaben, 1.0g glycerol and 10mL water were heated in a water bath at 80℃ and stirred to dissolve, thus obtaining the aqueous phase.

[0062] (3) Emulsification: The aqueous phase is kept at 70°C. The aqueous phase is added to the oil phase, and 1g of the snakeberry extract prepared in Example 1 is added and stirred continuously. The mixture is condensed and ground at room temperature to form an O / W type emulsion, resulting in a 5% snakeberry cream (low-dose snakeberry cream).

[0063] Example 5 A method for preparing a wild strawberry cream, comprising the following steps: (1) Oil phase: 2.5g stearic acid, 2.0g white petrolatum and 1.5mL liquid paraffin were heated in a water bath at 80℃ and stirred to dissolve.

[0064] (2) Aqueous phase: 0.5g sodium lauryl sulfate, 0.02g ethylparaben, 2.0g glycerol and 14mL water were heated in a water bath at 80℃ and stirred to dissolve.

[0065] (3) Emulsification: At 80°C, the aqueous phase was added to the oil phase in a thin stream, and 2g of the snakeberry extract prepared in Example 1 was added and stirred continuously. The mixture was then cooled and homogenized at room temperature to form an O / W type emulsion, resulting in a 10% snakeberry cream (high-dose snakeberry cream).

[0066] Example 6 Efficacy evaluation of snakeberry extract against acute photodamage to mouse skin (1) Grouping: Six-week-old female KM mice were selected and acclimatized under normal feeding conditions for one week. A hairless area of ​​approximately 3cm × 5cm was created on the back of the mice, and the hair was shaved every 3 days. After hair removal, the mice were randomly divided into five groups: control group (Control), model group (UVB), UVB + vitamin E group (UVB + VE), UVB + low-dose duchesne extract group (UVB + TFDI-L), and UVB + high-dose duchesne extract group (UVB + TFDI-H).

[0067] (2) Modeling Irradiation chamber preparation: Stainless steel front-opening box (67cm×54.5cm×54cm), with 4 312nm UVB lamps installed at 3cm intervals on the top of the box; a corrugated cardboard box is placed in the center of the bottom of the box and the position of the fixation device is marked. The distance between the fixation device and the lamp is 28cm. The mouse is placed perpendicular to the lamp. During irradiation, a green cloth is used to shield the light and prevent leakage.

[0068] Preparation before irradiation: Preheat the UVB lamp for 15 minutes, measure the intensity using an ultraviolet irradiator (retest periodically), and determine the irradiation dose (mJ / cm²). 2 ) Maintain consistent dosage during adjustment; fix mice in a self-made restraint device, cover their heads with a black cloth and expose their backs.

[0069] MED (the minimum dose that still produces erythema after 24 hours) was determined as follows: 80 mJ / cm² was selected for preliminary experiments. 2 This is the minimum erythema dose.

[0070] Modeling protocol: Acute photodamage to the skin of mice was induced by UVB, with continuous irradiation for 14 days (once a day). The irradiation started at 2 MED, increased by 2 MED every 3 days, and maintained at 8 MED for the last 5 days; the control group did not receive irradiation.

[0071] Mice in the UVB group developed skin wrinkles starting from day 4 of irradiation. As the irradiation time increased, the degree of erythema and wrinkles continued to deepen. On day 15 after irradiation, the skin thickness increased significantly, with deep wrinkles and a leathery appearance, and even local ulceration. This indicates that the acute photodamage model of the skin was successfully established.

[0072] (3) Administration Control group (Control) was treated with petroleum jelly: 0.2g of petroleum jelly was applied to each mouse from the first day of modeling, once a day for 14 consecutive days; The model group (UVB) was treated with petroleum jelly: 0.2g of petroleum jelly was applied to each mouse from the first day of modeling, once a day for 14 consecutive days. UVB+Vitamin E group (UVB+VE) applied Vitamin E (170mg / kg): starting from the first day of modeling, apply Vitamin E at a rate of 170mg / kg based on the mouse's body weight, once daily for 14 consecutive days; The UVB + low-dose snakeberry extract group (UVB + TFDI-L) was applied to the 5% snakeberry cream prepared in Example 4: that is, from the first day of modeling, 0.2g of 5% snakeberry cream was applied to each mouse once a day for 14 consecutive days. UVB + high-dose snakeberry extract group (UVB+TFDI-H) was applied to the 10% snakeberry cream prepared in Example 5: that is, from the first day of modeling, 0.2g of 10% snakeberry cream was applied to each mouse once a day for 14 consecutive days.

[0073] The medication is applied to the back and administered daily, followed by ultraviolet irradiation 30 minutes later.

[0074] (4) Sampling and specimen preparation On day 15 post-irradiation, mice were intraperitoneally injected with 100 mg / kg sodium pentobarbital. Immediately after anesthesia, images of the mouse's back skin were recorded using a digital camera. The back skin was wiped with 75% alcohol, and the skin was harvested parallel to the spine at the injection site. A portion of the skin was trimmed into 1 cm wide strips and fixed in 4% neutral paraformaldehyde solution for 24–36 h. The remaining skin was cut into pieces weighing approximately 20 mg each and placed in EP tubes for storage at -80°C for later use.

[0075] Figure 1The results showed that the skin condition of mice on their backs differed at 0, 4, 7, 10, and 15 days after UVB irradiation. The control group, which had not undergone UVB irradiation, exhibited normal skin texture, smoothness, and no wrinkles or erythema. Mice in the UVB group developed skin wrinkles starting from day 4 of irradiation. The severity of erythema and wrinkles increased with irradiation time. By day 15, skin thickness had significantly increased, with deep wrinkles and a leathery appearance, even accompanied by local ulceration, indicating a successful establishment of an acute photodamage model. The aforementioned photodamage symptoms were significantly improved in the UVB+TFDI-L, UVB+TFDI-H, and UVB+VE groups. The UVB+TFDI-H and UVB+VE groups showed similar effects, with lower skin roughness and a significantly reduced erythema area. These results demonstrate that *Duchesnea indica* extract can effectively improve photodamage symptoms.

[0076] (5) Results Analysis A macroscopic skin scoring system was used to quantitatively assess the degree of photodamage to the skin. The scoring criteria for acute photodamage in mice followed the evaluation criteria of Bissett et al. Three evaluators, other than the experimenters, assessed the skin appearance using the rating scale shown in Table 1, calculating appearance scores ranging from 0 to 6 (0 indicating normal skin, 6 indicating severely damaged skin). The animal grouping process was single-blind to the evaluators. Figure 2 As shown, the statistical results are as follows: Figure 3 As shown.

[0077] Table 1 Evaluation criteria for acute photodamage to the skin

[0078] Figure 2 The results showed that, from left to right, the images were of mouse skin with scores ranging from 0 to 6, and the higher the score, the more severe the photodamage.

[0079] The results of the mouse skin photodamage scoring showed that UVB radiation significantly aggravated skin photodamage, with the damage score increasing by 5.5306 compared to the control group. Compared to the UVB model group, the skin photodamage scores of the UVB+VE group, UVB+TFDI-L group, and UVB+TFDI-H group decreased by 1.6327, 1.7306, and 2.3184 respectively after intervention. All three groups could effectively alleviate UVB-induced skin photodamage, and the UVB+TFDI-H group showed the best improvement.

[0080] Figure 3The results showed that the macroscopic skin score of mice in the UVB group was significantly higher than that in the control group; while the macroscopic scores of the UVB+TFDI-L group, UVB+TFDI-H group and UVB+VE group were significantly lower than those in the UVB group. This quantitative result is consistent with the qualitative observation conclusion of skin appearance, further verifying that the snakeberry extract has a protective effect against UVB-induced acute photodamage to the skin in mice.

[0081] (6) Protective effect of Duchesnea inflicted on skin tissue structure in UVB-induced photodamage Preparation method of eosin dye: Dissolve 1g of eosin powder directly in 100mL of distilled water and stir until completely dissolved. Add 2 drops of glacial acetic acid, mix well, and filter before use. No special storage is required.

[0082] The specific steps for H&E staining are as follows: Xylene I and II, 10 min each; followed by staining with anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol, 2 min each, then washing with distilled water; hematoxylin staining for 5 min, followed by rinsing with running water for 60 s; differentiation with 1% hydrochloric acid ethanol for 3-5 s, then rinsing with running water for 5 min; counterstaining with eosin for 5 min, then rinsing off excess stain with running water for 30 s; followed by staining with 70% ethanol for 2 s, 80% ethanol for 2 s, 95% ethanol for 10 s, and anhydrous ethanol for 15 s; after the sections have air-dried, they are mounted with neutral resin containing xylene. The results are as follows: Figure 4 As shown.

[0083] HE staining analysis of epidermal thickness showed that UVB irradiation induced significant epidermal thickening, with an increase of 1.5512 μm (relative value) compared to the control group. Compared to the UVB model group, the epidermal thickness decreased by 1.0408 μm, 0.8572 μm, and 1.3627 μm (relative value) after intervention in the UVB+VE group, UVB+TFDI-L group, and UVB+TFDI-H group, respectively. All three groups effectively alleviated UVB-induced epidermal hyperplasia and thickening, with the UVB+TFDI-H group showing the best improvement.

[0084] HE staining analysis of dermal thickness showed that UVB irradiation caused dermal thickening, with a relative increase of 0.9436 μm compared to the control group. Compared to the UVB model group, the UVB+VE group, UVB+TFDI-L group, and UVB+TFDI-H group showed a reduction in dermal thickness of 0.4710 μm, 0.4767 μm, and 0.8370 μm (relative values) after intervention. All three groups effectively alleviated UVB-induced dermal thickening damage, with the UVB+TFDI-H group showing the best improvement.

[0085] Figure 4The results showed that the control group had intact skin tissue structure, distinct layers, and no obvious structural abnormalities or hyperkeratosis; the epidermis was of uniform thickness, the epidermal-dermal junction was wavy, and abundant epidermal ridges and dermal papillae were visible, with tight epidermal-dermal connections; the hair follicles in the dermis were neatly arranged and structurally normal. The UVB model group exhibited typical photodamage pathological features, with significant changes in skin tissue structure, characteristic sunburn cells in the epidermis, flattening of the dermal-epidermal junction, disappearance of epidermal ridges and dermal papillae, epidermal thickening, and abnormal hair follicle morphology; in addition, significant thickening of the dermis was observed. After intervention in the UVB+TFDI-L group, UVB+TFDI-H group, and UVB+VE group, the overall skin structural damage was improved. Among them, in the UVB+TFDI-H group and UVB+VE group, the stratum corneum thickness returned to normal, the epidermal layer thickness was significantly reduced, the dermal papillae and epidermal ridge structures reappeared, and the overall skin structural damage was significantly improved. The above results indicate that snakeberry extract can maintain the normal tissue structure of the skin and has a protective effect against tissue damage caused by photodamage.

[0086] (7) Protection of elastic fibers in UVB-induced skin photodamage by wild strawberry extract The procedure was performed according to the instructions for the elastic fiber staining kit (modified Gomori aldehyde fuchsin method), and the results are as follows. Figure 5 As shown.

[0087] Figure 5 The results showed that the elastic fibers in the control group exhibited a regular network structure, with long, thin fibers arranged in an orderly manner, without any curling or twisting. In the UVB model group, the elastic fiber network showed a flocculent distribution, exhibiting typical photodamage characteristics, including fiber thickening, curling, disordered arrangement, accompanied by breakage, twisting, and abnormal proliferation. In some areas, interwoven and fused fibers were observed, and the number of elastic fibers was significantly reduced. The elastic fiber damage in the mouse skin of the UVB+TFDI-L, UVB+TFDI-H, and UVB+VE groups all showed varying degrees of repair. In the UVB+TFDI-H and UVB+VE groups, the fiber morphology recovered to a long, interwoven shape, with a more orderly arrangement, and the network structure was reconstructed in some areas, with a significant increase in the number of elastic fibers. These results indicate that *Duchesnea indica* extract can maintain the normal physiological structure of elastic fibers and has a protective effect against photodamage-induced elastic fiber damage.

[0088] (8) Protective effect of wild strawberry extract on collagen fibers in UVB-induced skin photodamage. The procedure was performed according to the instructions for the modified Masson trichrome staining kit. Masson staining results represent statistical results related to collagen fiber content, such as... Figure 6 As shown.

[0089] Masson staining results showed that UVB radiation significantly reduced skin collagen content, with the model group showing a 61.56% decrease in collagen content compared to the control group (relative value). Compared to the UVB model group, the UVB+VE group, UVB+TFDI-L group, and UVB+TFDI-H group showed increases in collagen content of 135.52%, 64.95%, and 126.65% (relative value) respectively after intervention, indicating that all three groups effectively alleviated UVB-induced collagen loss.

[0090] Figure 6 The results showed that the collagen fibers in the skin of the control group mice exhibited a typical wavy structure, with uniform and orderly arrangement and dense distribution. The UVB model group, however, showed significant collagen fiber damage, specifically a marked decrease in collagen content (P<0.05), disordered and irregular fiber arrangement in the dermis, and photodamage characteristics such as fiber breakage and thinning in some areas. The collagen fiber structure in the UVB+TFDI-L, UVB+TFDI-H, and UVB+VE groups was significantly improved. Among these, the overall morphology of the collagen fiber structure in the UVB+TFDI-H and UVB+VE groups was closer to that of the control group, with significantly better fiber arrangement and distribution density than the UVB model group. These results indicate that *Duchesnea indica* extract can effectively inhibit UVB-induced reduction of collagen fibers in skin tissue and has a protective effect against photodamage-induced damage to the extracellular matrix of dermal cells.

[0091] (9) Protection of mast cells by wild strawberry extract in UVB-induced skin photodamage The staining was performed according to the instructions for G1346 mast cell staining solution (toluidine blue method), and the results are as follows. Figure 7 As shown.

[0092] Figure 7 The results showed that the black arrows represent mast cells. The number of mast cells in the skin tissue of the UVB model group was significantly increased (P<0.05), indicating that UVB irradiation can activate the skin inflammatory cascade by promoting mast cell proliferation. After treatment with the UVB+TFDI-L group, UVB+TFDI-H group, and UVB+VE group, the number of mast cells was significantly reduced compared to the UVB model group (P<0.05), with the UVB+TFDI-H group and UVB+VE group showing comparable inhibition. These results indicate that *Duchesnea indica* extract can effectively reduce mast cell infiltration and has a protective effect against photodamage-induced mast cell infiltration.

[0093] In summary, snakeberry extract can effectively improve the symptoms of photodamage and has a protective effect against photodamage.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a snakeberry extract, characterized in that, Includes the following steps: The crude powder of wild strawberry was mixed with an ethanol solution, the pH was adjusted to 8.0-10.0 with an alkaline solution, and the mixture was refluxed for extraction. The mixture was then filtered, the filtrate was collected, and the solution was concentrated to obtain a concentrate. The concentrate was then adjusted to pH 3.0-5.0 with an acid solution, allowed to stand, filtered, and the precipitate was collected. The precipitate was washed with water and ether, filtered, and dried to obtain the wild strawberry extract.

2. The preparation method according to claim 1, characterized in that, The crude powder of wild strawberry is prepared by pulverizing the dried whole wild strawberry herb to 55-65 mesh using a pulverizer; the ratio of the crude powder of wild strawberry to ethanol solution is 1g:10-20mL; the volume percentage of the ethanol solution is 65%-75%.

3. The preparation method according to claim 1, characterized in that, The reflux extraction time is 1-2 hours; the water washing is 2-3 times with distilled water; the ether washing is 1-2 times with petroleum ether or diethyl ether.

4. A snakeberry cream, characterized in that, Includes oil phase, aqueous phase, and snakeberry extract prepared by the preparation method according to any one of claims 1 to 3.

5. The snakeberry cream according to claim 4, characterized in that, The oil phase comprises one or more of stearic acid, white petrolatum, and liquid paraffin; the aqueous phase comprises one or more of sodium lauryl sulfate, ethylparaben, glycerin, and distilled water; the oil phase is stearic acid, white petrolatum, and liquid paraffin, with a mass-to-volume ratio of 1.5~3.0g:1.5~2.5g:1.0~2.0mL; the aqueous phase is sodium lauryl sulfate, ethylparaben, glycerin, and distilled water, with a mass-to-volume ratio of 0.1~0.7g:0.01~0.03g:0.5~2.5mL:8.0~16.0mL; the wild strawberry extract accounts for 3%~15% of the total weight of the wild strawberry cream.

6. The method for preparing the snakeberry cream according to claim 4 or 5, characterized in that, Includes the following steps: The stearic acid, white petrolatum and liquid paraffin were heated in a water bath at 70-90°C and stirred to dissolve, thus obtaining the oil phase. The sodium lauryl sulfate, ethylparaben, glycerol, and water were heated in a water bath at 70-90°C and stirred to dissolve, thus obtaining an aqueous phase. The aqueous phase is kept at 70~90℃. The aqueous phase is added to the oil phase, and then the snakeberry extract is added and stirred to obtain the snakeberry cream.

7. The use of the snakeberry extract prepared by the preparation method according to any one of claims 1 to 3, or the snakeberry cream according to claim 4 or 5, or the snakeberry cream prepared by the preparation method according to claim 6, in the preparation of products for the prevention and / or treatment of photodamage to the skin.

8. The application according to claim 7, characterized in that, The skin photodamage mentioned refers to acute skin photodamage.

9. The application according to claim 7, characterized in that, The products include pharmaceuticals or cosmetics.

10. The application according to claim 9, characterized in that, The dosage form of the medicine includes topical preparations, which include creams, ointments, gels, liniments, lotions, sprays, patches, or films.

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