Peony extract for anti-inflammatory, anti-allergic, antioxidant, anti-glycation and anti-wrinkle uses

Abstract

The present invention discloses a peony extract and a manufacturing method thereof. It is a water extract, and the whole peony plant is soaked in water and obtained by a continuous ultrasonic microwave extraction method. The whole peony plant involved here includes peony flowers, peony stems and leaves, and peony root bark. This peony extract contains monoterpenoid glucosides, flavonoids, tannins, stilbenes, triterpenoids, paeonols, phenols, etc., and has multiple biological activities such as anti-inflammatory, anti-allergic, antioxidant, anti-glycation, and anti-wrinkle.

Description

Technical Field

[0001] The present invention relates to a plant extract, in particular to a plant extract for preparing an anti-inflammatory, anti-allergic, anti-oxidative, anti-glycation and anti-wrinkle composition. Background Art

[0002] As people's living standards improve, modern people pay more and more attention to their appearance, and fighting skin aging has gradually become the main theme of the market. People's demand for products related to promoting cell repair, anti-wrinkle, and anti-aging has increased significantly. In addition, knowledge of health products is quite popular nowadays. In order to avoid chemical cosmetics from damaging the skin or even depositing in the body, people pay attention to the application of natural products such as Chinese herbal medicine in beauty products. In particular, the use of Chinese herbal extracts in cosmetics has become a global trend and has also been the focus of active development of the domestic cosmetics and biotechnology industries in recent years.

[0003] Aging is an irreversible state of the human body. When human cells undergo multiple generations of division, they will be overstimulated by the outside world, leading to growth stagnation and aging. Many research papers point out that aging is closely related to chronic inflammation, oxidative stress and glycation. In addition to aging caused by age, the human body will also accelerate or prematurely age under the influence of many unfavorable environmental factors, such as long-term exposure to sunlight, unbalanced diet, tissue damage, microbial infection, excessive fatigue, high life pressure and other exogenous aging factors.

[0004] Among them, reactive oxygen species (ROS) in the human body, such as superoxide free radicals (·O2 - ) and hydrogen peroxide (H2O2) will be converted into hydroxyl radicals (·OH) in an environment containing free metals. The high activity of free radicals will cause the most direct damage to human tissues. In addition to causing lipid peroxidation in the epidermis and dermis connective tissue, excessive free radical accumulation will also cause collagen denaturation. Studies have shown that peroxidation reactions also indirectly promote the expression of inflammation-related genes, leading to chronic inflammation.

[0005] In addition, since there are many high-sugar foods hidden in our daily diet, modern people are accustomed to eating out, which can easily lead to excessive intake of sugar. After the excess sugar combines with the protein in the body, it destroys the original structure of the protein, and the accumulation of advanced glycation end-products (AGEs) produced by it not only causes skin aging, sagging, wrinkles, and dullness, but may also cause diseases such as arteriosclerosis and diabetes.

[0006] In addition to the aging phenomenon caused by the above mechanisms, hyaluronic acid in human tissues also gradually decreases with age. Hyaluronic acid, also known as hyaluronic acid and glucuronic acid, is transparent and viscous in appearance and attracts a large number of sodium ions. More than 50% of the hyaluronic acid contained in the human body is the main component of the extracellular matrix, playing an important role in maintaining functions such as the water content and elasticity of the skin. One-third of the hyaluronic acid is degraded by autogenous enzymes every day, and the skin gradually loses its basic moisturizing function and forms voids, ultimately resulting in the loss of skin elasticity, luster, relaxation, and the appearance of aging wrinkles. Therefore, if the decomposition of hyaluronic acid can be inhibited, it is expected to achieve the purpose of improving skin relaxation, reducing wrinkles, and slowing down aging.

[0007] In view of this, there is an urgent need to propose a plant-based substance with multiple biological activities such as anti-inflammatory, antioxidant, anti-glycation, and anti-wrinkle effects, in order to provide a cosmetic composition. Summary of the Invention

[0008] The present invention is completed based on unexpected findings. The water extract of peony obtained by water extraction has various components with anti-inflammatory, anti-allergic, antioxidant, anti-glycation, or anti-wrinkle biological activities, such as glucosides, flavonoids, tannins, stilbenes, triterpenoids, paeonols, and phenols. Therefore, one object of the present invention is to provide a peony extract obtained by soaking the whole peony plant in water.

[0009] Preferably, the water and the whole peony plant are subjected to continuous ultrasonic oscillation.

[0010] Preferably, the soaking temperature is 50-80°C.

[0011] Preferably, the soaking time is 5-60 minutes.

[0012] Preferably, the whole peony plant includes peony flowers, peony stems and leaves, and peony root bark.

[0013] Preferably, the whole peony plant contains the following raw materials in parts by weight: 38-45 parts by weight of peony flowers, 46-53 parts by weight of peony stems and leaves, and 2-9 parts by weight of peony root bark.

[0014] Preferably, the peony extract is an extraction refined product obtained by vacuum concentration treatment or freeze-drying treatment.

[0015] According to the present invention, a preparation method of the above-mentioned peony extract is further provided, including: obtaining by soaking the whole peony plant in water.

[0016] Preferably, the water and the whole peony plant are subjected to continuous ultrasonic oscillation.

[0017] Preferably, the soaking temperature is 50 to 80 °C.

[0018] Preferably, the soaking time is 5 to 60 minutes.

[0019] Preferably, the whole peony plant includes peony flowers, peony stems and leaves, and peony root bark.

[0020] Preferably, the whole peony plant contains the following raw materials in parts by weight: 38 to 45 parts by weight of peony flowers, 46 to 53 parts by weight of peony stems and leaves, and 2 to 9 parts by weight of peony root bark.

[0021] Preferably, the preparation method further includes: refining the crude extract obtained by soaking peony materials in water through vacuum concentration treatment or freeze-drying treatment.

[0022] According to the present invention, the peony extract has various components with anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle biological activities, such as monoterpene glycosides, flavonoids, tannins, stilbenes, triterpenoids, paeonols, and phenols. Based on the foregoing characteristics, the peony extract can be used for the preparation of an anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle composition for use in a cosmetic composition. This composition is administered to a desired individual in an effective dose of the peony extract to achieve anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle effects.

[0023] Preferably, the anti-inflammatory is the biological activity of inhibiting the release of the proinflammatory cytokine IL-1 (Interleukin-1).

[0024] Preferably, the anti-allergy is the biological activity of inhibiting the release of cell β-hexosaminidase.

[0025] Preferably, the antioxidant is the cell activity of enhancing autophagy in cells.

[0026] Preferably, the anti-glycation is the biological activity of inhibiting the activation of senescence-associated β-galactosidase related to cell aging.

[0027] Preferably, the anti-wrinkle is the biological activity of inhibiting the activity of hyaluronidase to decompose hyaluronan (HA).

[0028] Preferably, the effective dose is 50 - 500 μg / mL. Description of the Drawings

[0029] Figure 1 is a flow chart of the peony extraction method;

[0030] Figure 2A is a protein gel electrophoresis diagram showing the inhibition of hyaluronidase activity by the peony extract of Example VI;

[0031] Figure 2B is a histogram showing the inhibition of hyaluronidase activity by the peony extract of Example VI. Detailed Description of the Invention

[0032] To make the above and / or other objects, effects, and features of the present invention more obvious, preferred embodiments and examples are hereby given and analyzed in detail as follows:

[0033] One embodiment of the present invention provides a peony extract, which is obtained by extracting the whole peony plant with water.

[0034] Preferably, the water and the whole peony plant are continuously ultrasonically oscillated.

[0035] Preferably, the soaking temperature is 50 - 80°C.

[0036] Preferably, the soaking time is 5 - 60 minutes.

[0037] Among them, the whole peony plant uses Paeonia Suffruticosa of the genus Paeonia in the family Ranunculaceae.

[0038] Preferably, the whole peony plant contains the following raw materials in parts by weight: 38 - 45 parts by weight of peony flowers, 46 - 53 parts by weight of peony stems and leaves, and 2 - 9 parts by weight of peony root bark.

[0039] The peony extract obtained by extracting the whole peony plant with water is a water extract of the whole peony plant, which has various bioactive components with anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle effects, such as glucosides, flavonoids, tannins, stilbenes, triterpenoids, paeonols, and phenols.

[0040] Preferably, the peony extract is refined by vacuum concentration treatment or freeze-drying treatment.

[0041] Another embodiment of the present invention provides a method for preparing the above-mentioned peony extract, which includes: extracting with water-soaked peony materials.

[0042] Please refer to Figure 1 , which exemplifies the method 100 for preparing the above-mentioned peony extract, but is not limited thereto.

[0043] First, as shown in step 101, the whole peony plant is used, including peony flowers, peony stems and leaves, and peony root bark; wherein, the whole peony plant contains the following raw materials in parts by weight: 38-45 parts by weight of peony flowers, 46-53 parts by weight of peony stems and leaves, and 2-9 parts by weight of peony root bark; in a preferred embodiment, the weight ratio of peony flowers, peony stems and leaves, and peony root bark is 43:50:7.

[0044] Next, as shown in step 102, extraction is carried out on the above-mentioned whole peony plant 101 by soaking with water to obtain a crude extract. Among them, the weight ratio of the whole peony plant to water is 1:7 to 1:13; in a preferred embodiment, the weight ratio is 1:10.

[0045] Preferably, the water and the whole peony plant are continuously ultrasonically oscillated. In a preferred embodiment, the ultrasonic power is between 500 and 800 W, and the oscillation is continuous for 30 to 60 minutes at a frequency of pausing for 1 minute after oscillating for 3 minutes.

[0046] Preferably, the soaking temperature is 50-80 °C.

[0047] Preferably, the soaking time is 5-60 minutes.

[0048] Preferably, heating is used to assist in reaching the soaking temperature during soaking for extraction. In a preferred embodiment, electromagnetic wave heating is used, and the electromagnetic wave frequency is between 300 MHz and 300 GHz.

[0049] In a preferred embodiment, the crude extract is further refined to obtain a refined extract: Please refer to Figure 1, as shown in step 103, the crude extract is subjected to centrifugal separation to obtain a first extract. Then, as shown in step 104, the first extract is subjected to filtration separation to obtain a second extract. The filtration separation is carried out using a microporous membrane with a pore size between 0.22 - 0.45 μm. The first extract is filtered using a vacuum sieve to remove the fine impurities contained in the first extract, thereby obtaining the second extract. Finally, as shown in step 105, the second extract is concentrated and freeze-dried by a vacuum concentrator and a vacuum freeze dryer to obtain an extraction refined product. The extraction refined product has the plural bioactive components contained in the aforementioned peony extract. After concentration treatment, it reaches a higher concentration, and the pellets obtained after freeze-drying treatment are more conducive to the preparation of various cosmetic compositions, where the cosmetic composition can be a solution, suspension, jelly, gel, emulsion, cream, ointment, powder, foam, capsule, or cleaning product.

[0050] Here, it should be added that any person with ordinary knowledge in the technical field to which the present invention pertains should understand that through the microwave-enhanced assisted process, high-frequency electromagnetic waves can penetrate the extraction medium and reach the microvascular bundles and alveolar systems inside the material. Since the cells absorb microwave energy, their internal temperature rises rapidly, thereby causing the pressure inside the cells to exceed the capacity that the cell wall expansion can withstand, and then causing the cells to rupture and release the active ingredients inside. When water is used as the solvent, under the action of the microwave field, water molecules change from a high-speed rotation state to an excited state. At this time, the vaporization of water molecules causes an increase in the driving force of the extraction components, or releases their excess energy and returns to the ground state. The released energy will be transferred to the molecules of other substances to accelerate their thermal motion, thereby shortening the time for the molecules of the extraction components to diffuse from the inside of the solid to the solid-liquid interface, increasing the extraction rate by several times, and being able to reduce the extraction temperature and dissolve in the extraction medium at a lower temperature, thereby avoiding the destruction of the molecules of the extraction components by high temperature.

[0051] The peony extract according to the present invention has various bioactive components such as monoterpene glycosides, flavonoids, tannins, stilbenes, triterpenoids, paeonols, and phenols, which have anti-inflammatory, anti-allergic, antioxidant, anti-glycation, or anti-wrinkle effects. Based on the aforementioned characteristics, another embodiment of the present invention proposes a use of the peony extract for the preparation of anti-inflammatory, anti-allergic, antioxidant, anti-glycation, or anti-wrinkle compositions for beauty purposes, which is to administer an effective dose to the required individual to achieve anti-inflammatory, anti-allergic, antioxidant, anti-glycation, or anti-wrinkle effects.

[0052] The so-called "anti-inflammatory" biological activity herein refers to the ability of the aforementioned peony extract to effectively inhibit the inflammatory response of cells. In a preferred embodiment of the present invention, the biological activity of inhibiting the inflammatory response can be evaluated by stimulating cells with lipopolysaccharide (LPS) and inducing the release of the pro-inflammatory cytokine interleukin-1 (IL-1).

[0053] The so-called "anti-allergic" biological activity herein refers to the ability of the aforementioned peony extract to effectively inhibit the allergic response of cells. In a preferred embodiment of the present invention, the biological activity of inhibiting allergy can be evaluated by stimulating cells with immunoglobulin E (IgE) and inducing the release of β-hexosaminidase during degranulation.

[0054] The so-called "antioxidant" biological activity herein refers to the ability of the aforementioned peony extract to effectively enhance the antioxidant mechanism of cells. In a preferred embodiment of the present invention, the biological activity of antioxidant can be evaluated by stimulating cells with ultraviolet light and oxidative stress and inducing the number of autophagosomes generated during autophagy.

[0055] The so-called "anti-glycation" biological activity herein refers to the ability of the aforementioned peony extract to effectively inhibit the glycation of cells. In a preferred embodiment of the present invention, the biological activity of anti-glycation can be evaluated by stimulating cells with ultraviolet light and inducing the activity of senescence-associated β-galactosidase (SA-β-gal).

[0056] The so-called "anti-wrinkle" biological activity herein refers to the ability of the aforementioned peony extract to effectively inhibit the activity of hyaluronidase in cells. That is to say, hyaluronic acid is the main component of the extracellular matrix to maintain the water content and elasticity of the skin. Hyaluronic acid will be degraded by the enzymes generated by the body itself, so the skin loses its basic moisturizing function, forms voids, loses elasticity and then becomes loose and produces wrinkles. In a preferred embodiment of the present invention, the biological activity of anti-wrinkle can be evaluated by the degree to which the peony extract inhibits the activity of hyaluronidase in decomposing hyaluronic acid.

[0057] Preferably, the effective dose is 50 - 500 μg / mL.

[0058] The preparation of the peony extract is illustrated by the following examples, and the anti-inflammatory, anti-allergic, antioxidant, anti-glycation, anti-wrinkle and other biological activities of the peony extract are evaluated.

[0059] Example 1: Preparation of peony extract

[0060] The whole peony plant is used, including peony flowers, peony stems and leaves, and peony root bark, and the weight ratio of peony flowers, peony stems and leaves, and peony root bark is 43:50:7; the whole peony plant is soaked in water, and the water and the whole peony plant are first oscillated by ultrasonic wave at a power of 500-800 W, and at a frequency of pausing for 1 minute after oscillating for 3 minutes, continuously oscillated for 30-60 minutes, and then heated by microwave to 50-80 °C and soaked for 5-60 minutes to obtain a crude extract, where the weight ratio of the whole peony plant to water is 1:10.

[0061] Next, the crude extract is further refined to obtain an extraction refined product: the crude extract is subjected to a centrifugal separation treatment (-20 °C / 15,000 g / 10 minutes) to remove the bottom precipitate to obtain a first extract. The first extract is subjected to a filtration separation treatment, a microporous structure filter membrane with a pore size between 0.22-0.45 μm, and is filtered by a vacuum sieve to remove the fine impurities contained in the first extract to obtain the second extract. Finally, the second extract is concentrated and freeze-dried by a vacuum concentrator and a vacuum freeze dryer to obtain an extraction refined product.

[0062] Example 2: Evaluation of the anti-inflammatory application of peony extract.

[0063] Test whether the peony extract inhibits the release of the pro-inflammatory cytokine IL-1 in human skin keratinocyte (HaCaT) cells to achieve anti-inflammatory efficacy. The aforementioned HaCaT cells are dispersed with cell culture medium and cultured in a 24-well multi-well cell culture plate at a cell number of 3 × 10 5 / mL, and cultured in an incubator at 37 °C and 5% CO2 for 24 hours. After adding the peony extract to the cell culture medium and reacting for 30 minutes, 1 μg / mL lipopolysaccharide (LPS) is added and reacted. After reaching the reaction time, the cell culture medium is collected, centrifuged at 1200 rpm for 5 minutes, and the supernatant is taken into a sterilized 1.5 mL centrifuge tube and stored frozen at -20 °C.

[0064] Table 1

[0065]

[0066] Use BD OptEIA TMSet for determination of IL-1 content in the supernatant: Coat the bottom of a 96-well microplate with capture antibody (capture Ab). Add 100 μL of the diluted capture antibody to each well, with the dilution factor of the capture antibody being 250-fold. Then seal the plate with plastic wrap and place it at 4 °C overnight. Wash the 96-well microplate described above 3 times with wash buffer (PBST). Next, add 200 μL of assay diluent to each well and react at room temperature for 1 hour, and then repeat the above washing step. Add 100 μL of cell supernatant, seal the plate with plastic wrap, and react at room temperature for 2 hours. Then wash it 5 times with wash buffer (PBST). Add 100 μL of working detector. After sealing the plate and reacting at room temperature for 1 hour, wash it 7 times with wash buffer (PBST). The working detector used here is a detection antibody diluted 500-fold mixed with an enzyme reagent diluted 250-fold. Finally, add 50 μL of substrate solution and react at room temperature for 30 minutes, then add 50 μL of stop solution (stop solution: 1M H3PO4 or 2N H2SO4), and measure the absorbance at 450 nm (BioTek, Synergy TM 2, USA).

[0067] In this example, the cell culture medium supplemented with LPS but without the peony extract was used as the positive control group; the group without LPS added was the negative control group, and the IL-1 content measured in its cell supernatant was defined as 1.0. Please refer to Table 1. After the action of LPS, the IL-1 content in the cell supernatant of the positive control group was 3.7, while after the action of the peony extract (50, 100, 500 μg / mL), the IL-1 contents in the cell supernatant were 1.9, 1.6, and 1.2 respectively. The results showed that the peony extract has the ability to inhibit the release of the pro-inflammatory factor IL-1 in cells and reduce the cell inflammatory response.

[0068] Table 2

[0069]

[0070] Example 3: Evaluation of the anti-allergic application of the peony extract

[0071] In this experiment, by measuring the amount of β-glucuronidase released after IgE stimulation of mast cells (RBL-2H3), the inhibitory effect of peony extract on allergic reactions was evaluated. The aforementioned mast cells were cultured in a 96-well multi-well cell culture plate at a cell density of 2×10 4 cells / well in a culture medium of 10% FBS / DMEM. Pretreat with peony extract in DMEM cell culture medium containing 0.5% FBS for 24 hours. Then, add anti-DNP IgE Ab (1 μg / mL) and culture for 6 - 12 hours. Wash away the cell culture medium containing anti-DNP IgE Ab, add DNP-BSA (100 ng / mL) and culture for 1 hour to activate mast cells. Then collect the cell supernatant and freeze it for storage. Add the cell supernatant and p-NAG substrate (1 μM) to another 96-well microplate in a volume ratio of 1:1, and measure the absorbance at 405 nm (BioTek, Synergy TM 2, USA) after culturing at 37°C for 1 hour.

[0072] In this example, calcimycin A23187 (1 µM) was used to induce allergic reactions as a positive control group, and the inhibition rate of β-glucuronidase was defined as 0.0%; untreated cells were used as a negative control group, and the inhibition rate of β-glucuronidase was defined as 100%. Please refer to Table 2. After the mast cells were treated with the peony extract (50, 100, 500 μg / mL), the inhibition rates of β-glucuronidase decreased to 4.8%, 21.2%, and 48.4%, respectively. The results showed that the peony extract had the ability to inhibit the release of β-glucuronidase and reduce the cell allergic reactions induced by IgE.

[0073] Table 3

[0074]

[0075] Example 4: Evaluation of the antioxidant application of peony extract.

[0076] By using the change in the number of autophagosomes in human skin keratinized cells (HaCaT) as a quantitative standard for autophagy in cells, the ability of the peony extract to enhance autophagy in cells and thus enhance the effects of anti-ultraviolet and oxidative stress was evaluated. The aforementioned human HaCaT cells were cultured at a density of 1.5×10 5Cells were cultured at a cell density of 2 in a 96-well multi-well cell culture plate and grown in an incubator at 37°C and 5% CO2 for more than 24 hours, and then the peony extract was added for reaction. After reaching the reaction time, the cell culture medium was removed and washed with PBS. The cells were irradiated with UVB at a dose of 20 mJ / cm TM to induce autophagy, and then fresh culture medium containing 0.1 mM H2O2 was added and allowed to act for 1 hour. Autophagosome detection reagent working solution was added and the reaction was carried out in the incubator for 15 minutes to 1 hour, and then the cells were washed with washing solution (PBST). Quantitative PBS was added, and the absorbance was measured at the settings of Excitation: 360 nm / Emission: 550 nm (BioTek, Synergy

[0077] 2, USA).

[0078] Example 5: Evaluation of the application of peony extract in anti-glycation

[0079] The activity of senescence-associated β-galactosidase (SA-β-gal) in human skin keratinized cells (HaCaT) or human skin fibroblasts (Hs68) was used to evaluate whether the peony extract has the ability to inhibit the activity of SA-β-gal in skin cells, so as to achieve the effects of anti-glycation and anti-aging. The above-mentioned HaCaT cells and Hs68 cells were cultured in a 24-well multi-well cell culture plate at a cell density of 1 × 10 5 / mL and cultured for 24 hours to wait for the cells to adhere. After replacing the fresh culture medium, the peony extract was added and treated for 24 hours. Then, a dose of 50 mJ / cm 2UVB-irradiated cells. Then, the original culture medium was replaced with serum-free culture medium and cultured for 24 hours. After reaching the reaction time, staining was performed using a senescence β-galactosidase staining kit (Cell Signaling Technology, Danvers, MA, USA), and senescent cells were observed and counted under a microscope. The senescent cells appeared blue under the action of the stain.

[0080] In this example, untreated HaCaT cells and Hs68 cells were used as the control group, and the intracellular SA-β-gal activity was defined as 100%. Refer to Table 4. After UVB irradiation, the SA-β-gal activity in the cells increased to 456.3%. After treatment with peony extract (50, 100, and 500 μg / mL), the SA-β-gal activity decreased to 392.7%, 325.1%, and 276.5%, respectively. The results showed that the peony extract could inhibit the activity of SA-β-gal induced by UVB and reduce the glycation of cells.

[0081] Table 4

[0082]

[0083] Example 6: Evaluation of the application of peony extract in inhibiting hyaluronidase activity.

[0084] In this example, the anti-aging effect was evaluated by testing whether the activity of purified hyaluronidase was affected by peony extract using the protein gel electrophoresis (SDS-PAGE) experimental method. The principle is as follows: Hyaluronan (HA) was added during the gel preparation process. After the reaction of the peony extract with hyaluronidase (HAase), it was sequentially dropped into the gel for SDS-PAGE. After completion, the gel was removed and stained with alcian blue stain dissolved in 3% acetic acid solution to observe whether the peony extract inhibited the activity of HAase. If there were obvious transparent bands in the dark blue gel background, it indicated that the peony extract could not inhibit the activity of HAase; conversely, if the color of the dark blue gel remained unchanged, it indicated that the peony extract successfully inhibited the activity of HAase. Among them, the concentration of HAase tested in this example was 3000 units / mL.

[0085] (1) Preparation of the test samples: It is well-known in the art that epigallocatechin gallate (EGCG) has the ability to inhibit the activity of HAase. A positive control group sample was prepared by mixing 4 μL of EGCG at a concentration of 100 μg / ml with 2 μL of HAase diluted 200X; a negative control group was prepared by mixing 11.5 μL of 0.15 M sodium chloride - sodium formate buffer (NaCl - sodium formate buffer, pH 5.7) with 2 μL of HAase diluted 200X; an experimental group was prepared by mixing 11.5 μL of 25 mg / mL peony extract with 2 μL of HAase diluted 200X. Then, after the test samples, including the positive and negative control groups and the experimental group, were mixed evenly, they were respectively placed in a constant temperature water bath and reacted at 37°C for 18 hours.

[0086] (2) Preparation of SDS - polyacrylamide gels: SDS - polyacrylamide gels include a running gel and a stacking gel. The running gel was prepared with 10% (v / v) polyacrylamide and 1.7 mL of 0.1% hyaluronic acid added; the stacking gel was prepared with 5% (v / v) polyacrylamide. After the test samples reacted for 18 hours, each was evenly mixed with 4.5 μL of 4X loading dye and left standing at room temperature. A protein marker (Mid - Range Protein Marker, GM47, GeneMark) was prepared as a control group. After mixing 4 μL of the protein marker with 4 μL of 2X loading dye, it was heated in boiling water at 100 °C for 10 minutes and left standing at room temperature.

[0087] (3) Electrophoresis analysis of the test samples: The SDS - polyacrylamide gel was set up on a protein electrophoresis tank, and the control group and the test samples, including the positive control group, negative control group, and experimental group, were sequentially injected into the small holes of the stacking gel. Then, an appropriate amount of running buffer was injected into the protein electrophoresis tank, and after setting the voltage at 80 volts for 3 hours, the running gel film was removed from the protein electrophoresis tank and washed three times with HEPES (pH 7.4) buffer, gently shaking at room temperature for 10 minutes each time.

[0088] (4) Staining analysis and separation of colloidal film: Immerse the separated colloidal film in sodium chloride-sodium formate buffer (0.15 M NaCl - 0.1 M sodium formate, pH 5.7), and place it in a constant temperature water bath. React at 37 °C for 18 hours; Shake the separated colloidal film in Alcian blue staining solution for 20 minutes, then stain it with Coomassie brilliant blue R-250 for 30 minutes, then decolorize and photograph the image of the separated colloidal film, and then use ImageJ for image quantitative analysis and result interpretation.

[0089] Please refer to Figure 2A , where the image of the stained separated colloidal film is presented. The positive control group is stained blue by the Alcian blue staining agent, indicating that its hyaluronic acid is not degraded by hyaluronidase because EGCG inhibits the activity of HAase; the negative control group is not stained by the Alcian blue staining agent and is transparent, indicating that its hyaluronic acid has been degraded by hyaluronidase; the experimental group is stained blue by the Alcian blue staining agent, indicating that its hyaluronic acid is not degraded by hyaluronidase. Please refer to Figure 2B , after quantitative analysis of the stained image, the inhibition of hyaluronidase activity in the negative control group is defined as 0.0%. After conversion, the peony extract can inhibit hyaluronidase activity up to 59.0%. The results show that the peony extract can effectively inhibit hyaluronidase activity.

[0090] From the above embodiments, it can be further concluded that compared with the prior art, the present invention has the following advantages:

[0091] 1. The peony extract of the present invention can promote the anti-inflammatory effect of cells by inhibiting the pro-inflammatory cytokine IL-1 of cells.

[0092] 2. The peony extract of the present invention can reduce the allergic reaction of cells stimulated by immunoglobulin IgE by inhibiting the release of β-glucuronidase, so as to achieve the anti-allergic effect.

[0093] 3. The peony extract of the present invention can effectively enhance autophagy of cells to eliminate excessive free radicals, so as to achieve the effects of anti-ultraviolet and antioxidant stress.

[0094] 4. The peony extract of the present invention can effectively inhibit the activity of ultraviolet-activated senescence-related β-galactosidase, so as to achieve the effects of anti-ultraviolet and anti-glycation.

[0095] 5. The peony extract of the present invention can inhibit the activity of hyaluronidase, so as to slow down the degradation and loss of hyaluronic acid and improve the effects of moisturizing and anti-wrinkle.

[0096] Although several embodiments of the present invention are disclosed above, they are not intended to limit the present invention. Those of ordinary skill in the art to which the present invention pertains can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be defined by the claims.

Claims

1. Use of a peony extract for preparing a cosmetic composition with anti-inflammatory, anti-allergic, antioxidant, anti-glycation and anti-wrinkle effects, characterized in that, administering the cosmetic composition with an effective dose of the peony extract to a desired individual to achieve the effects of anti - inflammation, anti - allergy, anti - oxidation, anti - glycation and anti - wrinkle, wherein: the peony extract is a water extract, and its preparation method includes: soaking the whole peony plant in water, wherein the weight ratio of the whole peony plant to water is 1:10, and the whole peony plant contains 43 parts by weight of peony flowers, 50 parts by weight of peony stems and leaves, and 7 parts by weight of peony root bark; first, ultrasonic oscillation is carried out at a power of 500 - 800 W, and at a frequency of oscillating for 3 minutes and then pausing for 1 minute, continuously oscillating the water and the whole peony plant for 30 - 60 minutes, then heating the water and the whole peony plant to 50 - 80 °C and soaking the water and the whole peony plant for 5 - 60 minutes to obtain a crude extract; the crude extract is centrifuged to remove the bottom precipitate to obtain a first extract, wherein the centrifugation is carried out at - 20 °C at 15,000 g for 10 minutes; the first extract passes through a microporous structure filter membrane with a pore size between 0.22 - 0.45 μm to remove the fine impurities contained in the first extract to obtain a second extract; and the second extract is concentrated and freeze - dried by a vacuum concentrator and a vacuum freeze - dryer to obtain an extraction refined product as the peony extract.

2. The use according to any one of claims 1, wherein, The effective dose is 50 - 500 μg / mL.

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