Epimedium essence as well as preparation method and application thereof

Abstract

The invention relates to herba epimedii essence as well as a preparation method and application thereof. The preparation method of the herba epimedii essence disclosed by the invention comprises the following steps: S1, taking fresh herba epimedii stems and leaves, cleaning and draining; s2, putting the clean herba epimedii stems and leaves into a closed microwave vacuum tank, performing microwave heating to 30-60 DEG C under a vacuum decompression condition, and performing decompression extraction for 1-4 hours to obtain steam; s3, the steam is led out and condensed, condensate is collected, and the herba epimedii essence is obtained. According to the preparation method, a microwave vacuum reduced-pressure drying technology is adopted, an organic solvent is completely abandoned, the risk of solvent residue is avoided, meanwhile, a reduced-pressure environment can accelerate diffusion of intracellular components to the surface of a material, and the extraction efficiency is improved; the prepared herba epimedii essence has the effects of inhibiting melanin, resisting wrinkles, tightening skin, preserving moisture, controlling oil, relieving, relieving itching, removing acnes and the like, and can be effectively applied to medicines or skin care products for preventing or treating skin problems.

Description

Technical Field

[0001] This invention relates to the field of epimedium extraction technology, and in particular to an epimedium extract, its preparation method, and its application. Background Technology

[0002] Epimedium extract is derived from Epimedium, a plant belonging to the genus Epimedium in the family Berberidaceae. Epimedium brevicomu Maxim., Epimedium sarcodactylis Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Epimedium pubescens Maxim. or Korean Epimedium Epimedium koreanum The stems and leaves of *Epimedium nakai* are a plant extract rich in flavonoid active ingredients. This type of plant is widely distributed in my country, mainly produced in Gansu, Sichuan, and Shaanxi provinces. Its medicinal and health-promoting value has long been recorded in traditional texts. According to the 2020 edition of the Chinese Pharmacopoeia, its properties are pungent, sweet, and warm, and it enters the liver and kidney meridians. It has the effects of tonifying kidney yang, strengthening tendons and bones, and dispelling wind and dampness. It is often used for impotence and seminal emission caused by kidney yang deficiency, weakness of tendons and bones, rheumatic pain, numbness, and contractures. It is a commonly used traditional Chinese medicine in clinical practice. However, to date, there are no reports of its application in the prevention or treatment of skin problems.

[0003] Currently, the industrial preparation of Epimedium extract is still based on solvent extraction. This method relies on the difference in solubility of active ingredients in solvents to achieve separation and extraction. It has advantages such as low equipment requirements, mature technology, and simple operation, and is the mainstream technology in the industry. However, in practical applications, solvent extraction has inherent technical shortcomings in solvent selection, use, and subsequent processing. The physicochemical properties of solvents can easily cause degradation of active ingredients, reducing the efficacy of the extract, which contradicts the core needs of various application fields. Flavonoids in Epimedium (especially icariin) are sensitive to temperature and solvent environment. Traditional solvent extraction methods often require reflux heating (60~80℃). Under prolonged high temperature, the polarity of the solvent accelerates the glycosidic bond breakage of flavonoid glycosides, leading to the degradation and transformation of target components. This not only reduces the content of active ingredients in the extract, weakening its anti-inflammatory, analgesic, kidney-tonifying, and bone-strengthening effects in the pharmaceutical field, but also fails to effectively exert its antioxidant and barrier-repairing effects in cosmetics. It may also generate unknown degradation products, affecting the product's efficacy stability and safety.

[0004] Secondly, solvent residue directly threatens the safety and applicability of extracts, thus limiting their application in core scenarios. In industrial production, 30%–70% ethanol aqueous solutions are often used as extraction solvents to improve the yield of flavonoids; in some cases, organic solvents such as methanol and ethyl acetate are also used. While these solvents can improve the dissolution efficiency of active ingredients, they are difficult to completely remove during subsequent concentration and dealcoholization processes, easily leaving residues in the extract. For end products such as cosmetics and oral health supplements, organic solvent residues may cause skin irritation, allergic reactions, and even potential endocrine disruption risks.

[0005] Furthermore, solvent use brings dual pressures of environmental protection and cost control. In industrial production, solvent extraction consumes large amounts of solvents. The recovery of organic solvents such as ethanol and methanol relies on distillation and rectification equipment, which is extremely energy-intensive. Moreover, the recovery process still results in solvent loss, increasing raw material costs. Simultaneously, improper handling of residual solvents and impurities in the extraction wastewater can cause water and soil pollution, requiring additional investment in environmental protection equipment and funds for wastewater treatment to meet national emission standards, further exacerbating the production burden on enterprises. While using water as a solvent avoids the residue and environmental problems of organic solvents, the low yield and excessive impurities necessitate increasing the number of extraction cycles to compensate for the insufficient yield, leading to low production efficiency and difficulty in meeting the needs of large-scale production. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a method for preparing Epimedium extract, which employs microwave vacuum drying technology to directly process fresh Epimedium stems and leaves, efficiently extracting flavonoids such as icariin at low temperatures, combining the advantages of speed, energy saving, and good preservation of heat-sensitive components. This preparation method abandons traditional solvent extraction, using water as the sole medium, simplifying subsequent separation and purification steps, reducing environmental costs and product safety risks. The Epimedium extract prepared by this microwave vacuum drying method has the effects of inhibiting melanin synthesis, anti-wrinkle and firming skin, moisturizing, oil control, soothing, anti-itching and acne-reducing, and can be used to prepare pharmaceutical preparations or skin care products for the prevention or treatment of skin problems.

[0007] This invention provides a method for preparing Epimedium extract, comprising the following steps: S1. Take fresh Epimedium stems and leaves, wash and drain them; S2, place the clean Epimedium stems and leaves in a sealed microwave vacuum container, microwave heat to 30~60℃ under vacuum and depressurization conditions and extract under depressurization for 1~4 hours to obtain steam; S3, the steam is extracted and condensed, and the condensate is collected to obtain Epimedium extract.

[0008] In this invention, the water content of fresh stems and leaves of Epimedium is used as the extraction medium. Cell wall disruption is achieved through microwave directional heating. Microwave energy preferentially acts on water molecules in the material, causing them to vibrate at high speed and generate internal frictional heat. This instantly causes a surge in intracellular pressure and cell wall rupture. At the same time, by reducing the ambient vacuum, the boiling point of water can be lowered, avoiding the destruction of heat-sensitive flavonoids by high temperatures. Furthermore, the reduced pressure environment can accelerate the diffusion of intracellular components to the surface of the material, thereby improving extraction efficiency.

[0009] The technical solution of this invention completely eliminates organic solvents, avoids the risk of solvent residue, and at the same time retains flavonoid active ingredients such as icariin.

[0010] Preferably, the internal heating temperature of the microwave vacuum chamber is 45~55℃, and the extraction time is 2-3 hours to prevent the oxidative degradation of icariin in the extract, which would cause it to lose some of its activity. At the same time, within this range, the cell walls of fresh epimedium stems and leaves can be fully broken down, while avoiding local scorching.

[0011] Furthermore, in this step, the vacuum degree of the microwave vacuum tank during the extraction process is -0.03 to -0.095 MPa to lower the boiling point of water. Within this pressure range, the active substances in the cells evaporate along with the water by microwave heating to a certain temperature.

[0012] Preferably, the vacuum degree of the microwave vacuum tank during the extraction process is -0.06 to -0.75 MPa.

[0013] In this invention, the stems and leaves of the Epimedium genus plant are selected from Epimedium, a plant belonging to the Berberidaceae family. Epimedium brevicomu Maxim., Epimedium sarcodactylis Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Epimedium pubescens Maxim. or Korean Epimedium Epimedium koreanum One or more of the Nakai plants.

[0014] The epimedium extract prepared by this invention comprises one or more of the following: epimedin, epimedin A, epimedin B, epimedin C, phenylethanol, leaf alcohol, and 2,4-di-tert-butylphenol.

[0015] The Epimedium extract prepared by the method of this invention can be widely used in the preparation of drugs and / or skin care products for the prevention or treatment of skin problems.

[0016] Furthermore, the amount of Epimedium extract added to the pharmaceutical preparation or the skin care product is 0.01~100%.

[0017] Specifically, the Epimedium extract prepared by this invention has the effect of inhibiting melanin synthesis and can be used as an active ingredient in pharmaceutical preparations or skin care products that inhibit melanin synthesis.

[0018] Specifically, the Epimedium extract prepared by this invention has a certain inhibitory effect on elastase degradation, can tighten the skin, and also has anti-wrinkle effects. It can effectively improve or treat problems such as loose skin and enlarged pores. It can be used to prepare anti-wrinkle and skin-tightening pharmaceutical preparations and / or cosmetics, and is widely used in daily basic care and medical aesthetics.

[0019] Specifically, the Epimedium extract prepared by this invention has moisturizing effects and can maintain a high moisturizing rate within a certain time range. It can be used to prepare pharmaceutical preparations or skin care products for skin moisturizing.

[0020] Specifically, the Epimedium extract prepared by this invention contains soothing or anti-allergic ingredients, has a certain inhibition rate on hyaluronidase, and can soothe the skin; it also has oil-controlling, anti-itch, and acne-removing effects, can effectively inhibit harmful microorganisms on the skin surface, eliminate itching caused by microbial imbalance, effectively eliminate skin inflammation, inhibit intracellular lipid content, improve acne inflammation, and reduce the proportion of red area in acne. It has the effects of anti-acne, acne removal, and repairing acne-prone skin, and can be used to prepare pharmaceutical preparations or skin care products for improving or treating oily skin, acne, and seborrheic dermatitis.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Microwave vacuum decompression drying technology is used to directly process the fresh stems and leaves of Epimedium, and extract flavonoids such as epimedin at low temperature. It has the advantages of being fast, energy-saving and retaining heat-sensitive components well. (2) The preparation method of the present invention abandons the traditional solvent extraction and uses water as the only medium, which simplifies the subsequent separation and purification steps, reduces environmental protection costs and product safety risks, and is more conducive to the end application of the extract. (3) The Epimedium extract prepared by the present invention has the effect of inhibiting melanin synthesis and can be used to prepare pharmaceutical preparations or skin care products that inhibit melanin synthesis and whiten skin. (4) The Epimedium extract prepared by the present invention has skin-tightening and anti-wrinkle effects, and can be used to prepare anti-wrinkle and skin-tightening pharmaceutical preparations or cosmetics; (5) The Epimedium extract prepared by this invention has moisturizing effect and can be used to prepare pharmaceutical preparations or skin care products that soothe, moisturize and repair the skin; (6) The Epimedium extract prepared by the present invention has the effects of oil control, soothing and antipruritic and acne removal. It can effectively inhibit harmful microorganisms on the skin surface, eliminate itching caused by microbial imbalance, effectively eliminate skin inflammation and inhibit intracellular lipid content, and reduce the proportion of red area of ​​acne. It can be used to prepare drug preparations or skin care products to improve or treat oily skin, acne and seborrheic dermatitis.

[0022] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings. Attached Figure Description

[0023] Figure 1 The diagram shows the inhibitory activity of melanin production in zebrafish embryos in test example 2; Figure 2 The image shows the firming effect test result for Test Example 3; Figure 3 The image shows the anti-wrinkle test results for Test Example 4; Figure 4 The figure shows the test results of the soothing efficacy of test example 5; Figure 5 The result of the moisturizing test for Example 6 is shown in the figure. Figure 6 The results of a satisfaction survey of the participants in the antipruritic test in Case 7 after using the product; Figure 7 To test the effect of different concentrations of the test substance on cell activity in the oil-controlling efficacy test of Example 8; Figure 8 A comparison of the relative intracellular lipid content between the sample group and the blank control group in the test of the oil-controlling effect in Example 8; Figure 9 This is a comparison chart showing the percentage of red acne area before and after product use in Test Example 9; Figure 10 This is a comparison chart of the number of facial acne marks on the subjects before and after product use in Test Example 9; Figure 11 For comparison of standard facial images and facial red area of ​​test subject 9. Figure 1 ; Figure 12 For comparison of standard facial images and facial red area of ​​test subject 9. Figure 2 . Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 Take fresh Epimedium leaves Epimedium sagittatum (Sieb. et Zucc.) Maxim. Wash and drain the fresh stems and leaves; then place the clean Epimedium stems and leaves in a sealed microwave vacuum chamber, set the gauge pressure of the microwave vacuum chamber to -0.075MPa, control the microwave heating temperature to 45℃ for 3 hours to extract steam, then export and condense the steam, collect the condensate to obtain Epimedium extract.

[0026] Example 2 Epimedium leaves Epimedium sagittatum (Sieb. et Zucc.) Maxim: Wash and drain fresh stems and leaves; then place the clean Epimedium stems and leaves in a sealed microwave vacuum chamber, set the gauge pressure of the microwave vacuum chamber to -0.03MPa, control the microwave heating temperature to 30℃ for 4 hours to extract steam, then export and condense the steam, collect the condensate to obtain Epimedium extract.

[0027] Example 3 Epimedium leaves Epimedium sagittatum (Sieb. et Zucc.) Maxim: Wash and drain fresh stems and leaves; then place the clean Epimedium stems and leaves in a sealed microwave vacuum chamber, set the gauge pressure of the microwave vacuum chamber to -0.05MPa, and control the microwave heating temperature to 55℃ for 2 hours to extract steam. Then, export and condense the steam, collect the condensate, and obtain Epimedium extract.

[0028] Example 4 Epimedium leaves Epimedium sagittatum (Sieb. et Zucc.) Maxim: Wash and drain fresh stems and leaves; then place the clean Epimedium stems and leaves in a sealed microwave vacuum chamber, set the gauge pressure of the microwave vacuum chamber to -0.095MPa, control the microwave heating temperature to 60℃ for 1 hour to extract steam, then export and condense the steam, collect the condensate to obtain Epimedium extract.

[0029] Test Example 1 The components of the Epimedium extracts prepared in Examples 1-4 were tested. 1 μL of each Epimedium extract prepared in Examples 1-4 was injected into GCMS for detection, and the results are shown in Table 1.

[0030] Table 1. Main components of the Epimedium extract prepared in Examples 1-4 Test Example 2 The effect of the Epimedium extract sample prepared in Example 1 on inhibiting melanin synthesis in a zebrafish embryo model was tested according to the method described in the group standard T / ZHCA012-2021 "Test Method for Melanin Inhibition Efficacy of Whitening Cosmetics in Zebrafish Embryos".

[0031] 1. Embryo preparation: The night before the experiment, sexually mature zebrafish older than 4 months were selected and placed in spawning boxes with baffles at a female-to-male ratio of 1:1 or 1:2, and kept in the dark. The next morning, the light source was turned on, and the baffles of the spawning boxes were removed to allow mating and spawning. Fertilized eggs were collected, observed under a biological microscope, and selected from those that met the experimental requirements.

[0032] 2. Formal Experiment: The formal experiments were grouped according to the following method: a) Blank control group: containing zebrafish embryos and embryo buffer water; b) Positive control group: containing positive control samples and zebrafish embryos; c) Test group: containing the test substance and zebrafish embryos, with multiple different concentration groups.

[0033] Ten normally developed zebrafish embryos at 6 hpf were placed into a six-well cell culture plate. Without harming the embryos, the standard dilution water was removed from the plate, and 3 mL of the corresponding concentration of the test substance dilution was quickly added to each well. After thorough mixing, the plate was covered and wrapped in aluminum foil. The plate was incubated in a (28.5±1.0)℃ biochemical incubator in the dark for (45±0.5) h until the incubation endpoint was reached. The fish were then transferred to a cellulose solution for fixation and photographed using a biological microscope with an electron eyepiece. After photographing, the zebrafish images were analyzed using image analysis software. The selected quantitative analysis target area was the edge of the zebrafish head (excluding the eyes) to the portion tangent to the yolk sac. The intensity of melanin signal in the zebrafish head was expressed as grayscale value, and 10 valid data points were collected from each group.

[0034] 3. Data processing: Calculate the mean (n = 10) standard deviation (SD) of the melanin inhibition rate for each group and the statistical significance of the differences between each experimental group and the blank / solvent control group. Using the blank / solvent control group as the standard, compare the melanin signal intensity of each experimental group. P < 0.05 indicates a statistically significant difference. The melanin inhibition rate is calculated using the following formula: In the formula: C - melanin inhibition rate of the test substance (%); The average melanin signal intensity of the S0-blank control group (n = 10); S1 - Average melanin signal intensity of the test group (n = 10).

[0035] 4. Test Results Table 2 Test Results of Test Example 2 *Note: P <0.050 indicates a significant difference between the sample group, the positive control group, and the blank control group.

[0036] Combine Table 2 and Figure 1 The test results showed that the melanin area of ​​zebrafish in the positive control group was significantly reduced compared with that in the blank group (P<0.05), indicating that the reaction system was effective. Compared with the blank control group, the melanin signal intensity of the test substance was significantly reduced (P<0.05), and the melanin inhibition rate was >20%, indicating that the test substance had the ability to inhibit melanin production at this concentration. When the sample concentration was 1%, the melanin inhibition rate in zebrafish was 32.12%, which was significantly reduced compared with the blank group (P<0.05), and the melanin inhibition rate was >20%, indicating that the Epimedium extract prepared in Example 1 had a whitening effect.

[0037] Test Example 3 The firming effect of the Epimedium extract sample prepared in Example 1 was tested.

[0038] 1. Testing Principle The biochemical method for assessing skin firming efficacy primarily characterizes the firming effect by evaluating the inhibition rate of elastase by the active ingredient. Elastase is the most important enzyme in the matrix metalloproteinase family, mainly synthesized and secreted by fibroblasts, and can degrade elastin in the skin, leading to skin aging.

[0039] 2. Instruments, Equipment and Reagents 2.1 Instruments and Equipment: Analytical balance; multi-wavelength microplate reader.

[0040] 2.2 Reagents and Consumables: Tris(hydroxymethyl)aminomethane; concentrated hydrochloric acid; elastase; EGCG; N-succinyl-alanine-alanine-p-nitroaniline (AAAPAN).

[0041] 2.3 Test substance Sample: Epimedium extract stock solution; Negative control: buffer solution; Positive control: 1% EGCG solution.

[0042] 3. Testing Method: Four groups (A, B, C, and D) were set up, with three replicates in each group. Buffer solution, sample solution, enzyme solution, and substrate were added sequentially to the 96-well plate. The 96-well plate was placed in a low-temperature shaking water bath at a specific temperature, and shaking was turned on. After the solutions had reacted completely, the absorbance values ​​of each group were measured at a wavelength of 410 nm.

[0043] elastase inhibition rate In the formula: A — is the absorbance of the reaction solution containing the sample and enzyme; B—is the absorbance of the reaction solution containing the sample and the reaction solution without the enzyme; C—is the absorbance of the reaction solution containing the enzyme but not the sample; D—The absorbance of the reaction solution without the sample and enzyme.

[0044] 4. Test Results Table 3 Test Results of Test Example 3 Combined with Table 3 and Figure 2 The test results showed that, under the premise of ensuring the effectiveness of the experimental reaction system (the elastase inhibition rate of the positive control was >50%), when the sample was the original solution, the elastase inhibition rate was 30.269%, which was significantly different from the negative control (P<0.05), indicating that the sample had a firming effect.

[0045] Test Example 4 The anti-wrinkle efficacy of the Epimedium extract sample from Example 1 was tested according to the group standard (T / SHRH 006-2018 Cosmetics - Experimental Method for Free Radical (DPPH) Scavenging).

[0046] 1. Testing Principle 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable, long-lived free radical. Its ethanol solution is deep purple and exhibits strong absorption around 517 nm. In the presence of free radical scavengers, the light absorption of the DPPH ethanol solution is reduced due to its pairing with unpaired electrons.

[0047] 2. Instruments, Equipment and Reagents 2.1 Instruments and Equipment: PR224ZH analytical balance; Agilent SYNERGY H1 full-wavelength multi-functional microplate reader; 2.2 Reagents and Consumables: 1,1-Diphenyl-2-trinitrophenylhydrazine; Vitamin E (purity ≥96%); 95% ethanol (AR); 2.3 Test substance Sample: Epimedium extract stock solution; Negative control: pure water; Positive control: 0.1% vitamin E solution.

[0048] 3. Testing Method: Set up sample tubes, blank tubes, and positive control tubes (T), and sample / blank and positive control background (T0); set up 3 replicates for each group, add ethanol solution, sample solution and DPPH solution to each, let stand at room temperature for 5 minutes, and after the solution in the test tube has reacted completely, measure the absorbance value of each group at a wavelength of 517 nm.

[0049] Free radical scavenging rate Where: T—absorbance value of the test sample tube, that is, absorbance value of the solution after the test sample reacts with DPPH; T0—Background absorbance of the test sample; The average of three absorbance values ​​of the C-DPPH tube, i.e. the absorbance of the DPPH solution without the addition of sample; C0 — Solvent background absorbance.

[0050] 4. Test Results Table 4 Test Results of Test Example 4 Combine Table 4 and Figure 3 The test results showed that, under the premise of ensuring the effectiveness of the experimental reaction system (positive control free radical scavenging rate > 50%), when the sample was the undiluted solution, the free radical scavenging rate was 20.735%, which was significantly different from the negative control. P <0.05) indicates that the sample has anti-wrinkle effects.

[0051] Test Example 5 The soothing efficacy of the Epimedium extract sample from Example 1 was tested using laboratory methods (hyaluronidase inhibition rate method).

[0052] 1. Testing Principle Hyaluronidase is involved in type I hypersensitivity reactions and is strongly correlated with inflammation and allergies. Referring to the Elson-Morgan hyaluronidase inhibition assay, soothing or anti-allergic components in the sample react with hyaluronidase in the presence of calcium chloride and sodium hyaluronate. The resulting reaction product is then developed with the chromogenic reagent P-DAB, and its absorbance is measured at 530 nm using a microplate reader. The result is then calculated and converted into a hyaluronidase inhibition rate.

[0053] 2. Instruments, Equipment and Reagents 2.1 Instruments and Equipment: PR224ZH electronic balance; SYNERGY-H1 full-wavelength multi-functional microplate reader; LC-HH-6 electric thermostatic water bath.

[0054] 2.2 Reagents and Consumables: Sodium hydroxide (AR); concentrated hydrochloric acid (GR); glacial acetic acid (AR); p-dimethylaminobenzaldehyde (AR); anhydrous sodium carbonate (AR); anhydrous sodium acetate (AR); anhydrous calcium chloride (AR); acetylacetone (AR); sodium hyaluronate (USP); hyaluronidase (BR); dipotassium glycyrrhizate, purity ≥98%.

[0055] 2.3 Test substance Sample: Epimedium extract stock solution; Negative / solvent control: buffer solution; Positive control: 1% dipotassium glycyrrhizate solution.

[0056] 3. Testing Method: Four groups (A, B, C, and D) were set up, with three replicates in each group. Calcium chloride, buffer solution, sample solution, sodium hydroxide, and acetylacetone were added sequentially to test tubes and mixed thoroughly. The tubes were then subjected to three different reaction environments: boiling water, ice water, and room temperature. P-DAB colorimetric reagent was then added to each of the four tubes. After the solutions had reacted completely, the absorbance values ​​of each group were measured at a wavelength of 530 nm.

[0057] Hyaluronidase inhibition rate In the formula: A — is the absorbance of the reaction solution containing the sample and enzyme; B—is the absorbance of the reaction solution containing the sample and the reaction solution without the enzyme; C—is the absorbance of the reaction solution containing the enzyme but not the sample; D—The absorbance of the reaction solution without the sample and enzyme.

[0058] 4. Test Results Table 5. Test Results of Test Example 5 Combined with Table 5 and Figure 4 The test results showed that, under the condition that the experimental system was effective (the positive control hyaluronidase inhibition rate was >50%), when the sample was the original solution, the hyaluronidase inhibition rate was 36.121%, which was significantly different from the negative control. P <0.05) indicates that the sample has a soothing effect.

[0059] Test Example 6 The moisturizing rate of the Epimedium extract sample from Example 1 was tested according to the laboratory method (Cosmetic Moisturizing Efficacy Test Method - Weighing Method).

[0060] 1. Testing Principle Depending on the differences in the hygroscopic and moisturizing properties of humectants, different humectant molecules have different forces acting on water molecules, resulting in varying abilities to absorb and retain moisture. Humectants with stronger forces have a greater binding affinity to water molecules and can absorb and retain a larger amount of water.

[0061] 2. Instruments, Equipment and Reagents 2.1 Instruments and Equipment: Electronic balance; constant temperature and humidity chamber.

[0062] 2.2 Reagents and Consumables: 7.5cm x 7.5cm glass plate; medical breathable tape; glycerin.

[0063] 2.3 Test substance: Sample group: Original sample of Epimedium extract for testing; Positive control group: 3% glycerol.

[0064] 3. Testing Methods The moisture retention rate of the sample and the positive control was calculated by measuring the ratio of the sample volume to the initial sample volume after 1 hour, 2 hours, and 4 hours of storage. The specific test steps are as follows: (1) Pretreatment of samples and positive controls; (2) Weigh a certain amount of sample and positive control and place them on the test glass plate; (3) Then place it in a constant temperature and humidity chamber (temperature: 20±1℃, relative humidity 50%±5%). (4) Calculate its moisture retention rate; Moisturizing rate ; Where: Mt: mass of the sample (positive control) and the plate after 1 h, 2 h, and 4 h; M0: Mass of the empty board; M: Mass of the sample (positive control) taken.

[0065] 4. Test Results Table 6. Results of Moisturizing Rate in Test Example 6 As shown in Table 6, the moisturizing rates of the test samples at 1h, 2h, and 4h were 76.45%, 54.06%, and 15.99%, respectively. These high moisturizing rates indicate that the test samples have a certain moisturizing effect, especially within the first 2 hours. Comparing the moisturizing rate of the test samples with that of the positive control, the moisturizing rate of the test samples was significantly higher. Combined with… Figure 5Similarly, it can be seen that the moisturizing rate of the test sample at 1h, 2h, and 4h was significantly higher than that of the positive control, indicating that the test sample has moisturizing effect.

[0066] Test Example 7 According to the "Evaluation Method for Consumer Use Testing of Cosmetics", the epimedium extract sample of Example 1 was tested for antipruritic effects.

[0067] 1. Purpose and Principle of the Test This experiment referenced the evaluation method of cosmetic efficacy evaluation—consumer use testing. It required 30 healthy male / female subjects to use the test product and then use a questionnaire to evaluate the antipruritic effect of the Epimedium extract sample of Example 1.

[0068] 2. Test Design Planned recruitment number: 32; Number of candidates screened: 30; Number of participants to be enrolled: 30; Test environment: The test was conducted in a constant environment with a temperature of 21±1℃ and a relative humidity of 50±10%RH.

[0069] 3. Data statistical analysis methods: A 5-point scale was used to statistically analyze the questionnaire options of the participants, and a statistical score of ≥4 was considered a valid score.

[0070] Rating method: 1 point indicates complete disagreement; 2 points indicates partial disagreement; 3 points indicates neutral / unfavorable; 4 points indicates partial agreement; 5 points indicates complete agreement.

[0071] The calculation formula is: Satisfaction = (Number of valid scores ≥ 4 / Total number of valid scores) × 100.

[0072] Results were determined based on the following criteria: more than 50% of the subjects agreed that the test sample had an antipruritic effect.

[0073] 4. Test Results Table 7 Results of the Self-Assessment Satisfaction Survey of Test Case 7 Note: 1 point indicates complete disagreement; 2 points indicates partial disagreement; 3 points indicates neutral / unfavorable; 4 points indicates partial agreement; 5 points indicates complete agreement.

[0074] After using the test product continuously for 7 days, the results of the questionnaire survey on the participants' satisfaction with the product showed that: Q1: 96.67% of the respondents agreed that the product had a pleasant scent; Q2: 100.00% of the test takers agreed that the product was gentle and non-irritating when used; Q3: 100.00% of the test takers agreed that the product felt refreshing and non-sticky on the skin when used; Q4: 96.67% of the respondents agreed that the product was quickly absorbed by the skin when used; Q5: 100.00% of the test takers agreed that the product made their skin feel comfortable and light after use; Q6: 100.00% of the test subjects agreed that the product effectively relieved itching and had an anti-itch effect; Q7: 100.00% of the test subjects agreed that the product effectively relieved dry, itchy, and red skin after use; Q8: 96.67% of the subjects were satisfied with the overall effect of the product.

[0075] Consumer questionnaire survey results: After using the product continuously for 7 days, no fewer than 29 of the 30 participants scored ≥4 on each satisfaction question. See Table 7 for details.

[0076] Combined with Table 7 and Figure 6 The subjects reported high satisfaction with the product. 96.67% of subjects agreed that the product had a pleasant scent; 100.00% agreed that the product was gentle and non-irritating; 100.00% agreed that the product felt refreshing and non-sticky on the skin; 96.67% agreed that the product was quickly absorbed by the skin; 100.00% agreed that the product left the skin feeling comfortable and light; 100.00% agreed that the product effectively relieved itching and had an anti-itch effect; 100.00% agreed that the product effectively relieved dry, itchy, and red skin; and 96.67% of subjects were satisfied with the overall effect of the product. The results indicate that after 7 days of continuous use, the subjects considered the product to have an anti-itch effect.

[0077] Test Example 8 The oil-controlling efficacy of the Epimedium extract sample from Example 1 was tested according to the laboratory method (determination of lipid content in human sebaceous gland cells).

[0078] 1. Testing Principle Human sebaceous gland cells (SZ95 cells) can be used as a cell model to study the oil-controlling ability of cosmetics. The lipid content in human sebaceous gland cells after administration of the test substance, the blank control and the test substance are measured by using Oil Red O (also known as Sudan Red 5B) staining solution to evaluate whether the test sample has the effect of oil control.

[0079] 2. Main Instruments, Equipment and Reagents 2.1 Main instruments and equipment: Yamato / HQIP610 CO2 incubator; BSC-1300IIA2 biosafety cabinet; HQ / HQICX inverted fluorescence microscope; DL-6000B low-speed refrigerated centrifuge; SYNERGY-H1 full-wavelength multi-functional microplate reader.

[0080] 2.2 Main reagents and consumables: Human sebaceous gland cells (SZ95 cells); DMEM high glucose medium; fetal bovine serum; penicillin-streptomycin mixture (100× penicillin-streptomycin); thiazolyl blue (MTT); Oil Red O kit.

[0081] 2.3 Test substance: Sample: Dilute the Epimedium extract to a certain concentration as the sample stock solution, then filter it through a 0.22 μm filter, and then dilute it to a series of concentrations after filtration; Blank control: basal culture medium.

[0082] 3. Testing Methods 3.1 Cell Culture: Take the frozen cell culture and inoculate it into the conventional culture medium at an appropriate density for the experiment.

[0083] 3.2 Cell viability assay: Cell viability was determined using the MTT assay at different test concentrations. After cell culture in different concentrations of test culture medium, cell status was observed and recorded. The culture medium was discarded, and cells were washed with PBS and then MTT working solution was added. Cells were then cultured in a CO2 incubator for 2 to 6 hours. The culture medium was discarded, and a certain amount of DMSO solution was added to each well and shaken. The absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated based on the results.

[0084] 3.3 Measurement of intracellular relative lipid content: Based on the cell viability assay results, the cells were cultured in a medium containing a certain amount of sample for a period of time. Oil Red O staining was then performed, and the treated solution was transferred to 96-well plates and the absorbance at 490 nm was measured using a microplate reader.

[0085] 4. Analytical Methods 4.1 Calculation formula: 4.1.1 Cell survival rate after intervention with different drug concentrations: Survival rate (%) = 4.1.2 Relative content of intracellular lipids: Relative lipid content (%) in human sebaceous gland cells = 4.2 Data Analysis: Statistical analysis using software showed that, at the selected concentrations of the test substance, a two-tailed test was used to compare the sample group and the blank control, with a significance level of α=0.05. P≥0.05 indicates no significant difference between the two groups; P<0.05 indicates a significant difference between the two groups.

[0086] 4.3 When the cell viability of the sample at the corresponding concentration is ≥90%, and there is no significant difference in morphology between the cells in the sample group and the blank group, it indicates that the sample has no significant cytotoxicity at that concentration, and this concentration should be selected for the experiment.

[0087] 4.4 Compared with the blank control, the relative content of intracellular lipids in the test group was reduced, and the difference was significant (P<0.05), indicating that the test substance has the ability to inhibit intracellular lipid content at the test concentration, and that the sample has oil-controlling effect.

[0088] 5. Test Results It should be noted that "*" indicates a significant difference between the sample and the blank control, and "ns" indicates no statistical difference. P ≥0.050; "*" indicates a significant difference, 0.010≤P<0.050 is marked with "*"; 0.001≤P<0.010 is marked with "**"; P<0.001 is marked with "***".

[0089] Combination Figure 7 and Figure 8 Based on the premise of ensuring the effectiveness of the experimental system, the cell activity was high when the concentration of the experimental sample was 0.016%, and the intracellular lipid content was about 87.30% of that of the blank control group. Compared with the blank control, the intracellular lipid content of the sample group was reduced, and the difference was significant (P<0.05). That is, the experimental sample at a concentration of 0.016% has the ability to inhibit the intracellular lipid content, indicating that the sample has the effect of controlling oil.

[0090] Test Example 9 Referring to the standard T / TDCA 004-2021 "Test Method for Acne-Removing Efficacy of Cosmetics", a human evaluation test was conducted on the acne-removing efficacy of the Epimedium extract in Example 1.

[0091] 1. Inspection basis and testing purpose Based on T / TDCA 004-2021 "Test Method for Acne-Clearing Efficacy of Cosmetics", the effectiveness and safety of the cosmetics used by the subjects in improving acne were evaluated.

[0092] 2. Test Design Planned number of recruits: 33; Number of candidates screened: 31; Number of candidates to be enrolled: 30.

[0093] The experiment was conducted under a constant temperature of 21±1℃ and a relative humidity of 50±10%RH.

[0094] Test area: face.

[0095] 3. Statistical Analysis Methods 3.1 Descriptive statistics: Statistical analysis software was used to perform descriptive statistics on the test values ​​of each test site, including quantity, mean, standard deviation, minimum, median and maximum values.

[0096] 3.2 Difference Analysis: The differences between the initial values ​​of the test sites and the values ​​measured at other time points were calculated. If the instrumental analysis data conformed to a normal distribution, a paired t-test was used for statistical analysis; if the test data did not conform to a normal distribution, a Wilcoxon signed-rank test was used for statistical analysis. All statistical methods used two-tailed tests, with a significance level of α = 0.05. Significance markers: "ns" indicates no statistical difference (p ≥ 0.050); p < 0.050 indicates a significant difference; 0.010 ≤ p < 0.050 is marked with "*"; 0.001 ≤ p < 0.010 is marked with "**"; p < 0.001 is marked with "***".

[0097] 3.3 Result determination: If the proportion of red area of ​​acne on the face and the number of blackheads on the subject's face are significantly different from their initial values ​​before and after product use during the test period (p<0.05), and the mean of the measured values ​​is better than the initial values, then the test product is considered to have acne-removing effect during the test period.

[0098] 4. Test Results: 4.1 Results of the percentage of red area on the face of the subjects with acne 4.1.1 Descriptive statistical results of the proportion of red area on acne Table 8. Descriptive statistics on the percentage of red area in acne lesions 4.1.2 Change rate of the proportion of red area of ​​acne Table 9. Change rate of the proportion of red area in acne Note: The data in the table are mean ± standard error.

[0099] 4.1.3 Results of the analysis on the difference in the proportion of red area of ​​acne Table 10 Results of the analysis of differences in the proportion of red area of ​​acne pimples The percentage of red area on pimples can be used to evaluate how well a product improves acne inflammation; the smaller the percentage of red area, the milder the inflammation.

[0100] Combining Table 8-10 and Figure 9 It can be clearly seen that after using the sample continuously for 28 days, the proportion of red area of ​​acne on the face of the subjects was significantly different from the initial value (p<0.05), with an improvement rate of 35.74%; the test results show that the test product has the effect of reducing the proportion of red area of ​​acne.

[0101] 4.2 Results of the number of facial acne in the subjects 4.2.1 Descriptive statistics on the number of acne pimples Table 11 Descriptive statistics of the number of facial acne in the subjects 4.2.2 Change rate of acne number Table 12. Rate of change in the number of facial acne in the subjects Note: The data in the table are mean ± standard error. 4.2.3 Results of the analysis of differences in the number of pimples Table 13 Results of the analysis of differences in the number of facial acne among subjects Combining Table 11-13 and Figure 10 It can be clearly seen that after using the sample continuously for 28 days, the number of facial acne in the subjects was significantly different from the initial value (p<0.05), with an improvement rate of 45.44%.

[0102] The number of facial pimples can be used to evaluate the product's effect on improving acne. The fewer the number of facial pimples, the less acne the subject experiences. The test results indicate that the product has the effect of reducing the number of facial pimples.

[0103] Combination Figure 11 and Figure 12 The comparison images of the subjects' standard facial features and facial redness areas show that after 28 days of continuous use of the sample, the subjects' acne improved after using the product. The results indicate that the test product has an acne-reducing effect on facial skin.

[0104] Therefore, the Epimedium extract prepared according to the method provided in this application has the effect of inhibiting melanin synthesis and can be used as an active ingredient in whitening pharmaceutical preparations or skin care products to inhibit melanin synthesis. Simultaneously, the Epimedium extract prepared by the method in this application has a certain inhibitory effect on elastase degradation, which can tighten the skin. It also has anti-wrinkle and moisturizing effects, and can be used to prepare moisturizing, anti-wrinkle, and skin-tightening pharmaceutical preparations and / or cosmetics. It can be effectively applied to skin care products or pharmaceutical preparations that improve or treat skin laxity, enlarged pores, and other problems, and is widely used in daily basic care and medical aesthetics.

[0105] Furthermore, the Epimedium extract obtained through the preparation method of this application contains soothing or anti-allergic components and has a certain inhibition rate on hyaluronidase, thus soothing the skin. Simultaneously, this Epimedium extract also has oil-controlling, anti-itch, and acne-controlling effects. It inhibits harmful microorganisms on the skin surface, eliminates itching caused by microbial imbalance, effectively eliminates skin inflammation, inhibits intracellular lipid content, improves acne inflammation, and reduces the proportion of red areas in acne. It has anti-acne, acne-removing, and acne-repairing effects and can be widely used in pharmaceutical preparations, skincare products, or cosmetics to improve or treat oily skin, acne, and seborrheic dermatitis.

[0106] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and the present invention also intends to include these modifications and variations.

Claims

1. A method for preparing an epimedium extract, characterized in that, It includes the following steps: S1. Take fresh Epimedium stems and leaves, wash and drain them; S2, place the clean Epimedium stems and leaves in a sealed microwave vacuum container, microwave heat to 30~60℃ under vacuum and depressurization conditions and extract under depressurization for 1~4 hours to obtain steam; S3, the steam is extracted and condensed, and the condensate is collected to obtain Epimedium extract.

2. The method for preparing an Epimedium extract according to claim 1, characterized in that: In step S2, the vacuum degree of the microwave vacuum tank is -0.03 to -0.095 MPa, the microwave heating temperature is 45 to 55°C, and the vacuum extraction lasts for 2 to 3 hours.

3. A method for preparing an Epimedium extract according to claim 1 or 2, characterized in that: The epimedium stems and leaves mentioned are selected from the plant Epimedium genus of the Berberidaceae family. Epimedium brevicomu Maxim., Epimedium sarcodactylis Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Epimedium pubescens Maxim. or Korean Epimedium Epimedium koreanum One or more of the Nakai plants.

4. An epimedium extract, characterized in that: It is prepared by the preparation method according to any one of claims 1-3.

5. The Epimedium extract according to claim 4, characterized in that: It contains one or more of icariin, icariin A, icariin B, icariin C, phenethyl alcohol, leaf alcohol, and 2,4-di-tert-butylphenol.

6. The application of the Epimedium extract as described in claim 4, characterized in that: The Epimedium extract is used to prepare medicines and / or skin care products for the prevention or treatment of skin problems, and the amount of Epimedium extract added is 0.01~100%.

7. The application of the Epimedium extract according to claim 6, characterized in that: The use of the Epimedium extract in the preparation of pharmaceutical formulations or skin care products that inhibit melanin synthesis.

8. The application of the Epimedium extract according to claim 6, characterized in that: The use of the Epimedium extract in the preparation of anti-wrinkle, skin-firming pharmaceutical preparations and / or cosmetics.

9. The application of the Epimedium extract according to claim 6, characterized in that: The use of the Epimedium extract in the preparation of pharmaceutical formulations or skin care products for skin moisturizing.

10. The application of the Epimedium extract according to claim 6, characterized in that: The use of the Epimedium extract in the preparation of pharmaceutical formulations or skin care products for improving or treating oily skin, acne, and seborrheic dermatitis.

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