TWI818223B - Peony extract for anti-inflammation, anti-allergy, anti-oxidation, anti-glycation and anti-wrinkle - Google Patents

Abstract

The present invention provides a peony extract and a method of preparation thereof. The peony extract is a water extract. To obtain the peony extract, complete stools of peonies are immersed in water and subjected to continuous ultrasonication and microwave extraction. The complete stools of peonies comprise: flowers, stalks, leaves and root cortex of peonies. The peony extract comprises monoterpenoid glucosides, flavonoids, tannins, stilbenes, triterpenoids, paeonols and phenols, and demonstrates biological activities including anti-inflammation, anti-allergy, anti-oxidation, anti-glycation and anti-wrinkle.

Description

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

The present invention relates to a plant extract, in particular to a plant extract used for preparing anti-inflammatory, anti-allergic, antioxidant, anti-glycation and anti-wrinkle compositions.

With the improvement of national living standards, 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 about health products is quite popular nowadays. In order to avoid cosmetics and skin care products with chemical ingredients from damaging the skin or even being deposited in the body, people attach great importance to the application of natural products such as health food and beauty products, Chinese herbal medicines, especially Chinese herbal extracts. Its application in cosmetics has become a global trend and has also been the focus of active development in the domestic cosmetics and biotechnology industries in recent years.

Aging is an irreversible state of the human body. When human cells divide for many generations, they will be over-stimulated by the outside world, resulting in growth stagnation and aging. Many research literature points 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 adverse environmental factors, such as long-term exposure to the sun, unbalanced diet, tissue damage, microbial infection, excessive fatigue, and high life pressure. and other exogenous aging factors.

Among them, the reactive oxygen species (ROS) in the human body, such as superoxide radicals (‧O ₂ ^- ) and hydrogen peroxide (H ₂ O ₂ ), will be converted into hydrogen and oxygen free radicals in an environment containing free metals. (‧OH), its high activity will cause the most direct damage to human tissues. Excessive free radical accumulation will not only cause lipid peroxidation in the epidermis and dermal connective tissue, but also cause collagen protein denaturation. Studies have pointed out that peroxidation also indirectly promotes the expression of inflammation-related genes, leading to chronic inflammation.

In addition, because there are many high-sugar foods hidden in the daily diet, modern people's habit of eating out can easily lead to excessive intake of sugar. After excess sugar combines with proteins in the body, it destroys the original structure of the protein. The accumulation of advanced glycation end-products (AGEs) not only causes skin aging, causing sagging, wrinkles, and dullness, but may also Can cause arteriosclerosis, diabetes and other diseases.

In addition to the aging phenomenon caused by the above-mentioned mechanisms, hyaluronic acid in human tissues will also gradually lose with age. Hyaluronic acid, also known as hyaluronic acid and uronic acid, has a transparent and sticky appearance and attracts a large amount of sodium ions. More than 50% of hyaluronic acid contained in the human body is the main component of the extracellular matrix and plays an important role in maintaining skin's moisture content and elasticity. 1/3 of the hyaluronic acid will be degraded by self-generated enzymes every day. As a result, the skin gradually loses its basic moisturizing function and forms gaps. This eventually causes the skin to lose its elasticity and luster, causing sagging and aging wrinkles. Therefore, if the decomposition of hyaluronic acid can be inhibited, it can be expected to improve skin sagging, reduce wrinkles and slow down aging.

In view of this, there is an urgent need to propose a plant-based substance with multiple biological activities such as anti-inflammation, antioxidant, anti-glycation, and anti-wrinkle, in order to provide cosmetic compositions and/or food additives.

The present invention is based on an unexpected discovery. The aqueous extract of peony extracted with water contains monoterpene glycosides (glucosides), flavonoids (flavonoids), tannins (tannins), stilbenes (stilbenes), and triterpenes. Terpenoids, paeonols and phenols are a variety of ingredients with anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle biological activities. Therefore, one object of the present invention is to provide a peony extract, which is obtained by soaking the whole peony plant in water.

Preferably, the water and the whole peony plant are vibrated with continuous ultrasonic waves.

Preferably, the soaking temperature is 50~80˚C.

Preferably, the soaking time is 5 to 60 minutes.

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

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.

Preferably, the peony extract is an extract refined by concentration under reduced pressure or freeze-drying.

According to the present invention, a method for preparing the above-mentioned peony extract is further proposed, which includes: soaking the whole peony plant in water and extracting it.

Preferably, the water and the whole peony plant are vibrated with continuous ultrasonic waves.

Preferably, the soaking temperature is 50~80˚C.

Preferably, the soaking time is 5 to 60 minutes.

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

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.

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

According to the present invention, the peony extract contains monoterpene glycosides (glucosides), flavonoids (flavonoids), tannins (tannins), stilbenes (stilbenes), triterpenoids (triterpenoids), paeonols (paeonols) and phenols ( phenols) and many other ingredients with anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle biological activities. Based on the aforementioned properties, peony extract can be used to prepare anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle compositions for use in cosmetic compositions and/or food additives. This composition is formulated with an effective dose. Peony extract is administered to the desired individual to achieve anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle effects.

Preferably, anti-inflammatory is a biological activity that inhibits the release of proinflammatory cytokine IL-1 (Proinflammatory cytokine Interleukin-1) from cells.

Preferably, the anti-allergy is a biological activity that inhibits the release of cellular β-glucuronidase (β-hexosaminidase).

Preferably, the antioxidant is a cellular activity that promotes autophagy.

Preferably, anti-glycation is a biological activity that inhibits the activation of cell senescence-associated β-galactosidase.

Preferably, anti-wrinkle is a biological activity that inhibits the decomposition of hyaluronic acid (Hyaluronan, HA) by hyaluronidase.

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

In order to make the above and/or other objects, effects, and features of the present invention more obvious and understandable, so that the review committee can understand the content of the present invention, the preferred embodiments and examples are analyzed, and the details are as follows:

One embodiment of the present invention provides a peony extract, which is obtained by soaking the whole peony plant in water.

Preferably, the water and the whole peony plant are vibrated with continuous ultrasonic waves.

Preferably, the soaking temperature is 50~80 ˚C.

Preferably, the soaking time is 5 to 60 minutes.

Among them, the whole peony plant is Paenoina Suffruticosa from the genus Paeonia of the Ranunculaceae family.

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.

The peony extract obtained by soaking the whole peony plant in water is an aqueous extract of the whole peony plant, which contains monoterpene glycosides (glucosides), flavonoids (flavonoids), tannins (tannins), and stilbenes (stilbenes). , triterpenoids, paeonols and phenols, and many other bioactive ingredients with anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle properties.

Preferably, the peony extract is refined by concentration under reduced pressure or freeze-drying.

Another embodiment of the present invention provides a method for preparing the above-mentioned peony extract, which includes: soaking peony materials in water and extracting the peony extract.

Please refer to Figure 1 to illustrate the method 100 for preparing the above peony extract, but it is not limited thereto.

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 to 45 parts by weight of peony flowers, peony stems and leaves 46 to 53 parts by weight and 2 to 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.

Next, as shown in step 102, the whole peony plant 101 is soaked in water and subjected to an extraction step to obtain a crude extract. Wherein, the weight ratio of the whole peony plant to water is 1:7~1:13; in a preferred embodiment, the weight ratio is 1:10.

Preferably, the water and the whole peony plant are vibrated with continuous ultrasonic waves. In a preferred embodiment, the ultrasonic power is between 500 and 800W. The ultrasonic wave is vibrated for 3 minutes and then paused for 1 minute for 30 consecutive seconds. ~60 minutes.

Preferably, the soaking temperature is 50~80 ˚C.

Preferably, the soaking time is 5 to 60 minutes.

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

In a preferred embodiment, the crude extract is further refined to obtain an extraction refinement: please refer to Figure 1. As shown in step 103, the crude extract is processed through a centrifugal separation to obtain a first extraction liquid. Next, as shown in step 104, the first extraction liquid is subjected to a filtration separation process to obtain a second extraction liquid. The filtration separation process uses a multi-microporous structure filter membrane with a pore size between 0.22-0.45 μm, and uses a vacuum sieve. The detector filters the first extraction liquid and removes fine impurities contained in the first extraction liquid to obtain the second extraction liquid. Finally, as shown in step 105, the second extraction liquid is concentrated and freeze-dried by a decompression concentrator and a vacuum freeze dryer to obtain an extraction product; the extraction product contains the plurality of organisms contained in the aforementioned peony extract. The active ingredients are concentrated to a higher concentration and freeze-dried to obtain pellets, which are more conducive to the preparation of multiple cosmetic compositions and/or food additives. The cosmetic compositions can be solutions, suspensions, and gels. jelly, gel, lotion, cream, ointment, powder, foam, capsule or cleaning product.

It should be added here that anyone with ordinary knowledge in the technical field to which the present invention belongs should understand that through the microwave intensification-assisted process, high-frequency electromagnetic waves can penetrate the extraction medium and reach the microtubule bundles and glandular cell systems inside the material. As cells absorb microwave energy, their internal temperature rises rapidly, causing the pressure inside the cells to exceed the capacity of the cell wall to expand, causing the cells to rupture and release the active ingredients within them. When water is used as the solvent, under the action of the microwave field, the water molecules change from a high-speed rotation state to an excited state. At this time, the water molecules vaporize, causing the driving force of the extracted components to increase, or release their excess energy and return 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 interior of the solid to the solid-liquid interface, increasing the extraction rate several times, and lowering the extraction temperature. And dissolve in the extraction medium at a lower temperature, thereby preventing the molecules of the extracted components from being damaged by high temperature.

The peony extract according to the present invention has monoterpene glycosides (glucosides), flavonoids (flavonoids), tannins (tannins), stilbenes (stilbenes), triterpenoids (triterpenoids), paeonols (paeonols) and phenols ( phenols) and other bioactive ingredients with anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle properties. Based on the foregoing characteristics, another embodiment of the present invention proposes a peony extract for use in preparing anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle compositions as beauty or food additives, by adding an effective dose to the Individual use is required to achieve anti-inflammatory, anti-allergic, antioxidant, anti-glycation or anti-wrinkle effects.

The so-called "anti-inflammatory" biological activity here means that the peony extract obtained above can effectively inhibit the inflammatory response of cells. In a preferred embodiment of the present invention, the aforementioned biological activity of inhibiting inflammatory response can be evaluated by stimulating cells with lipopolysaccharide (LPS) and inducing the release of proinflammatory cytokine interleukin-1 (IL-1).

The so-called "anti-allergic" biological activity here means that the peony extract obtained above can effectively inhibit the allergic reaction of cells. In a preferred embodiment of the present invention, the aforementioned biological activity of inhibiting allergies can be stimulated by immunoglobulin E (IgE) to induce degranulation and release of β-glucuronidase (β-hexosaminidase). to evaluate.

The so-called "antioxidant" biological activity here means that the peony extract obtained above can effectively enhance the antioxidant mechanism of cells. In a preferred embodiment of the present invention, the aforementioned antioxidant biological activity can be evaluated by stimulating cells with ultraviolet rays and oxidative stress to induce the number of autophagosomes produced during autophagy.

The so-called "anti-glycation" biological activity here means that the peony extract obtained above can effectively inhibit the glycation of cells. In a preferred embodiment of the present invention, the aforementioned anti-glycation biological activity can stimulate cells through ultraviolet rays to induce the activity of cell senescence-associated β-galactosidase (SA-β-gal). to evaluate.

The so-called "anti-wrinkle" biological activity here refers to the fact that the peony extract obtained above can effectively inhibit the activity of hyaluronidase in cells. In other words, hyaluronic acid is the main component of the extracellular matrix to maintain the moisture content and elasticity of the skin. Hyaluronic acid will be degraded by self-generated enzymes. As a result, the skin loses its basic function of moisturizing, forms gaps, loses elasticity, and relaxes, causing wrinkles. In a preferred embodiment of the present invention, the aforementioned anti-wrinkle biological activity can be evaluated by the extent to which the peony extract inhibits the activity of hyaluronidase to decompose hyaluronic acid.

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

The following examples illustrate the preparation of the peony extract, and evaluate the biological activities of the peony extract such as anti-inflammation, anti-allergy, antioxidant, anti-glycation or anti-wrinkle.

Example 1: Preparation of Peony Extract

Use the whole peony plant, including peony flowers, peony stems and leaves, and peony root bark. The weight ratio of peony flowers, peony stems and leaves, and peony root bark is 43:50:7; soak the whole peony plant in water, and first use the power 500 The water and the whole peony plant were vibrated with ~800W ultrasonic waves, and the vibration was continued for 30~60 minutes at a frequency of 3 minutes followed by a 1 minute pause, and then heated to 50~80˚C by microwave and soaked for 5~60 minutes. To obtain a crude extract, the weight ratio of the whole peony plant to water is 1:10.

Then, the crude extract is further refined to obtain the refined product: the crude extract is subjected to a centrifugal separation process (-20°C/15,000 g/10 minutes) to remove the bottom sediment to obtain the first extract. The first extraction liquid is separated by filtration and becomes a multi-microporous structure filter membrane with a pore size between 0.22-0.45 μm. It is filtered through a vacuum screen to remove the fine impurities contained in the first extraction liquid, and the second extraction liquid is obtained. liquid. Finally, the second extract is passed through a vacuum concentrator and a vacuum freeze dryer to concentrate the filtrate and freeze-dry it to obtain the refined extract.

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

To test whether peony extract inhibits the release of pro-inflammatory cytokine IL-1 from human skin keratinocytes (HaCaT) to achieve anti-inflammatory effects. The aforementioned HaCaT cells were dispersed with cell culture medium, cultured in a 24-well multi-well cell culture plate at a cell number of 3 × 10 ⁵ /mL, and cultured in a 37 ºC and 5% CO ₂ incubator for 24 hours. The peony extract was added to the cell culture medium and allowed to react for 30 minutes, and then 1 μg/mL lipopolysaccharide (LPS) was added for reaction. After reaching the reaction time, collect the cell culture medium, centrifuge at 1200 rpm for 5 minutes, and transfer the supernatant to a sterilized 1.5 mL centrifuge tube and store it frozen at -20 °C. Table 1 Inflammatory factor IL-1 content test LPS - + + + + Peony extract concentration (μg/mL) 0 0 50 100 500 IL-1 content 1.0±0.3 3.7±0.4 1.9±0.2 1.6±0.4 1.2±0.1

Use the BD OptEIA ^TM Set to measure the IL-1 content in the supernatant: Coat the bottom of a 96-well micro-porous plate with capture antibody (capture Ab), and add 100 μL of diluted capture antibody to each plate well, where the capture antibody The dilution factor is 250 times, then seal the plate with plastic wrap and place it at 4 ºC overnight. Wash the aforementioned 96-well microporous plate three times with wash buffer (PBST). Then, add 200 μL assay diluent to each plate well and react at room temperature for 1 hour. Repeat the aforementioned cleaning steps. Add 100 μL of cell supernatant, seal the plate with plastic wrap, react at room temperature for 2 hours, and then wash 5 times with washing solution (PBST). Add 100 μL working detector, seal the plate and react at room temperature for 1 hour, then wash 7 times with PBST. The working detector used here is a 500-fold diluted detection antibody mixed with a 250-fold diluted enzyme reagent. 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 H ₃ PO ₄ or 2N H ₂ SO ₄ ), and measure the absorbance value at 450 nm (BioTek , Synergy ^TM 2, USA).

In this example, LPS was added to the cell culture medium but the peony extract was not added as a positive control group; LPS was not added as a negative control group, and the IL-1 content measured in the cell supernatant was defined as 1.0. Please refer to Table 1. After treatment with LPS, the IL-1 content in the cell supernatant of the positive control group was 3.7, while after treatment with peony extract (50, 100, 500 μg/mL), the IL-1 content in the cell supernatant was 3.7. 1 contents were 1.9, 1.6 and 1.2 respectively. The results showed that peony extract has the ability to inhibit the release of cellular pro-inflammatory factor IL-1 and reduce cellular inflammatory response. Table 2 Beta-glucuronidase inhibition test A23187 inducer - + + + + Peony extract concentration (μg/mL) 0 0 50 100 500 β-glucuronidase inhibition rate (%) 100.0±2.3 0.0±5.6 4.8±3.7 21.2±2.7 48.4±3.1

Example 3: Evaluating the anti-allergic application of peony extract

This experiment measured the amount of β-glucuronidase released by mast cells (RBL-2H3) after IgE stimulation to evaluate whether peony extract has the effect of inhibiting allergic reactions. The aforementioned mast cells were cultured in a 96-well multi-well cell culture dish at a cell density of 2×10 ⁴ cells/well in 10% FBS/DMEM culture medium. The cells were pretreated with peony extract in DMEM cell culture medium with 0.5% FBS for 24 hours. Next, anti-DNP IgE Ab (1 μg/mL) was added and incubated for 6-12 hours. Wash away the aforementioned cell culture medium containing anti-DNP IgE Ab, add DNP-BSA (100 ng/mL) and incubate for 1 hour to activate mast cells, then collect the cell supernatant and freeze it for storage. Add cell supernatant and p-NAG substrate (1 μM) to another 96-well micro-well plate at a volume ratio of 1:1, and incubate at 37°C for 1 hour. Then measure the absorbance at 405 nm. (BioTek, Synergy ^™ 2, USA).

In this example, Cassithromycin A23187 (1 µM) was used to induce allergic reactions as a positive control group, and its β-glucuronidase inhibition rate was defined as 0.0%; untreated cells were used as a negative control group, and its β-glucuronidase inhibition rate was defined as 0.0%. Glucuronidase inhibition rate 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 β-glucuronidase inhibition rate dropped to 4.8%, 21.2% and 48.4%. The results showed that the peony extract The extract has the ability to inhibit the release of β-glucuronidase and reduce cellular allergic reactions caused by IgE. table 3 Skin cell autophagy test UVB + H ₂ O ₂ - + + + + Peony extract concentration (μg/mL) 0 0 50 100 500 Skin cell autophagy (%) 100.0±0.6 105.5±0.7 107.5±0.6 112.2±1.5 116.7±1.1

Example 4: Evaluation of the antioxidant application of peony extract.

The quantitative change of autophagosomes in human skin keratinocytes (HaCaT) is used as a quantitative standard for autophagy to evaluate whether the peony extract can enhance autophagy to improve UV resistance. and oxidative stress effects. The aforementioned human HaCaT cells were cultured in a 96-well multi-well cell culture plate at a cell density of 1.5×10 ⁵ cell/mL, and grown in a 37˚C, 5% CO ₂ incubator for more than 24 hours, and then the peony extract was added reaction. After the reaction time is reached, remove the cell culture medium and wash with PBS. The cells were irradiated with UVB at a dose of 20 mJ/cm ² to induce autophagy, and then fresh culture medium containing 0.1mM H ₂ O ₂ was added and allowed to act for 1 hour. Add Autophagosome detection reagent working solution, place in the incubator to react for 15 minutes to 1 hour, and then wash the cells with washing solution (PBST). Add quantitative PBS and measure the absorbance value (BioTek, Synergy ^TM 2, USA) at the setting of Excitation: 360 nm/Emission: 550 nm.

In this example, HaCaT cells that have not been exposed to ultraviolet light and H ₂ O ₂ are used as the control group, and their autophagy is defined as 100%. Please see Table 3. Under the action of UVB and H ₂ O ₂ , the autophagy of HaCaT cells increased to 105.5%. After the action of peony extract (50, 100 and 500 μg/mL), the autophagy of HaCaT cells increased. to 107.5%, 112.2% and 116.7%. The results show that the peony extract can promote the autophagy of cells, enhance the ability of cells to eliminate free radicals and resist ultraviolet rays.

Example 5: Evaluating the application of peony extract against glycation

The peony was evaluated by testing the senescence-associated β-galactosidase (SA-β-gal) activity in human skin keratinocytes (HaCaT) or human skin fibroblasts (Hs68). Whether the extract has the ability to inhibit SA-β-gal activity in skin cells to achieve anti-glycation and anti-aging effects. The aforementioned HaCaT cells and Hs68 cells were cultured in a 24-well multi-well cell culture dish at a cell density of 1 × 10 ⁵ /mL, and cultured for 24 hours to wait for cell attachment. After replacing the fresh culture medium, the peony extract was added and treated for 24 hours. Next, the cells were irradiated with UVB at a dose of 50 mJ/ ^cm2 . Then, the original culture medium was replaced with serum-free culture medium and cultured for 24 hours. After the reaction time has elapsed, senescence β-galactosidase staining kit (Cell Signaling Technology, Danvers, MA, USA) was used for staining, and aged cells were observed and counted under a microscope. The aged cells appeared blue under the action of the dye.

In this example, untreated HaCaT cells and Hs68 cells were used as control groups, and the intracellular SA-β-gal activity was defined as 100%. Please refer to Table 4. After UVB irradiation, the activity of SA-β-gal in cells increased to 456.3%. After treatment with peony extract (50, 100 and 500 μg/mL), the activity of SA-β-gal dropped to 392.7%, 325.1% and 276.5% respectively. The results showed that the peony extract can inhibit UVB-induced activation of SA- The activity of β-gal reduces the glycation of cells. Table 4 Test to inhibit SA-β-gal activation UVB - + + + + Peony extract concentration (μg/mL) 0 0 50 100 500 SA-β-gal activation (%) 100.0±2.3 456.3±5.6 392.7±3.7 325.1±2.7 276.5±3.1

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

In this example, the protein colloidal electrophoresis (SDS-PAGE) experimental method was used to test whether the purified hyaluronidase activity is affected by peony extract to evaluate its anti-aging effect. The principle is: hyaluronic acid (Hyaluronan, HA) is added during the gel making process. ), after reacting the peony extract with hyaluronidase (HAase), it was sequentially dropped into the film for SDS-PAGE. After completion, remove the film and stain it with alcian blue stain dissolved in 3% acetic acid solution to observe whether the peony extract inhibits HAase activity. If a transparent band appears clearly in the dark blue film background, it means that the peony extract cannot inhibit HAase activity; conversely, if the color of the dark blue film remains unchanged, it means that the peony extract successfully inhibits HAase activity. Among them, the concentration of HAase tested in this example is 3000 units/mL.

(1) Prepare the sample to be tested in this test: epigallocatechin gallate (EGCG) is known in the art to have the ability to inhibit HAase activity. Mix 4 μL of EGCG with a concentration of 100ug/ml and 2 μL of HAase diluted 200X. Prepare a positive control group sample; mix 11.5 μL 0.15M sodium chloride-sodium formate buffer (NaCl-sodium formate buffer, pH5.7) with 2 μL diluted 200X HAase to prepare a negative control group; use 11.5 μL 25 mg/mL The peony extract was mixed with 2 μL of HAase diluted 200X to prepare the experimental group. Then, the samples to be tested, including the positive and negative control groups and the experimental group, were mixed evenly, placed in a constant temperature water bath, and reacted at 37°C for 18 hours.

(2) Make SDS-polyacrylamide gels: SDS-polyacrylamide gels include running gel and stacking gel, of which the separating colloid is 10% (v/ v) Polyacrylamide and add 1.7mL of 0.1% hyaluronic acid; the focal gel is formulated with 5% (v/v) polyacrylamide. After reacting for 18 hours, the samples to be tested were evenly mixed with 4.5 μL of four-fold electrophoresis indicator (4X loading dye) and allowed to stand at room temperature. Prepare a protein marker (Mid-Range Protein Marker, GM47, GeneMark) as a control group. Mix 4 μL of protein marker with 4 μL of twice the electrophoresis indicator, heat it in boiling water at 100 °C for 10 minutes, and let it stand at room temperature. .

(3) Electrophoresis analysis of the sample to be tested: Set up the SDS-polyacrylamide film on the protein electrophoresis tank, and inject the control group and the sample to be tested, including the positive control group, negative control group and experimental group, into a small amount of focus colloid in sequence. into the well, then inject an appropriate amount of running buffer into the protein electrophoresis tank, and set the voltage to 80 volts. After 3 hours, remove the separation colloid film from the protein electrophoresis tank and wash it with HEPES (pH7.4) buffer. Separate the colloidal film three times, each time with gentle shaking at room temperature for 10 minutes.

(4) Dyeing analysis and separation of colloid film: Soak the separation colloid film in sodium chloride-sodium formate buffer (0.15M NaCl-0.1M sodium formate, pH5.7), place it in a constant temperature water bath, and react at 37 °C for 18 hours. ; Shake the separation colloid film in Airson's blue staining solution for 20 minutes, then stain it with Coomassie brilliant blue R-250 for 30 minutes, destain and photograph the separation colloid film image, and then use Image J Perform quantitative image analysis and interpret results.

Please refer to Figure 2A, which shows the image of the staining of the separated colloid film. The positive control group was stained blue with Elson's blue stain, showing that its hyaluronic acid was not degraded by hyaluronidase because EGCG inhibited the activity of HAase; the negative control group The ones in the experimental group that were not stained by Airson's blue stain were transparent, indicating that the hyaluronic acid had been degraded by hyaluronidase; the experimental group was stained by Airson's blue dye and appeared blue, indicating that the hyaluronic acid had not been degraded by hyaluronidase. Please refer to Figure 2B. After quantitative analysis of stained images, the inhibition of hyaluronidase activity in the negative control group was defined as 0.0%. The peony extract can inhibit hyaluronidase activity by 59.0%. The results show that the peony extract can effectively inhibit hyaluronidase. active.

It can be further concluded from the above embodiments that compared with the prior art, the present invention has the following advantages:

1. The peony extract of the present invention achieves the effect of promoting anti-inflammatory effects of cells by inhibiting the pro-inflammatory factor IL-1 in cells.

2. The peony extract of the present invention reduces the allergic reaction of cells stimulated by immunoglobulin IgE by inhibiting the release of β-glucuronidase, thereby achieving anti-allergic effects.

3. The peony extract of the present invention can effectively promote cell autophagy and eliminate excessive free radicals, so as to achieve the effect of resisting ultraviolet rays and oxidative stress.

4. The peony extract of the present invention has the activity of effectively inhibiting the aging-related β-galactosidase activated by ultraviolet rays, so as to achieve the effects of anti-ultraviolet rays and anti-glycation.

5. The peony extract of the present invention can inhibit the activity of hyaluronidase, thereby slowing down the degradation and loss of hyaluronic acid and improving moisturizing and anti-wrinkle effects.

Although the present invention discloses several embodiments as above, they are not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can make various modifications and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the listed patent application scope.

100:Method 101: Provide whole peony plant 102: Soak the whole peony plant in water and obtain the crude extract using continuous ultrasonic and microwave extraction. 103: Centrifuge the crude extract to obtain the first extraction liquid 104: Perform a filtration and separation process on the first extraction liquid to obtain the second extraction liquid 105: Concentrate and dry the second extract under reduced pressure to obtain the refined extract.

Figure 1 is a flow chart of peony extraction technology; Figure 2A is a protein colloid electrophoresis pattern showing the inhibition of hyaluronidase activity by the peony extract of Example 6; Figure 2B is a histogram showing the inhibition of hyaluronidase activity by the peony extract of Example 6.

100:Method

101: Provide whole peony plant

102: Soak the whole peony plant in water and obtain the crude extract using continuous ultrasonic and microwave extraction.

103: Centrifuge the crude extract to obtain the first extraction liquid

104: Perform a filtration and separation process on the first extraction liquid to obtain the second extraction liquid

105: Concentrate and dry the second extract under reduced pressure to obtain the refined extract.

Claims (5)

A use of peony extract for preparing an anti-inflammatory, anti-allergic, antioxidant, anti-glycation and anti-wrinkle composition. The composition is administered to a desired individual with an effective dose of the peony extract to achieve anti-inflammation. , anti-allergic, antioxidant, anti-glycation and anti-wrinkle effects, wherein the preparation method of the peony extract includes: soaking the whole peony plant in water; and first using continuous ultrasonic vibration and then heating to assist the water and the whole peony plant. Extract to obtain a crude extract, wherein the water and the whole peony plant are continuously shaken with ultrasonic power of 500 to 800W for 30 to 60 minutes, and then heated to 50 to 80°C with microwaves and soaked for 5 to 60 minutes; wherein, the 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. The use as described in claim 1, wherein the preparation method of the peony extract further includes: refining the crude extract to obtain a refined product. The use as described in claim 2, wherein the refining step is completed by concentration under reduced pressure or freeze-drying. The use as described in any one of claims 1 to 3, wherein the weight ratio of the whole peony plant to water is 1:7~1:13. The use as described in any one of claims 1 to 3, wherein the effective dose is 50-500 μg/mL; wherein the peony extract is administered to the skin cells of the desired individual.