Application of gardenia in moisturizing, anti-inflammation, brightening and whitening, anti-wrinkle and anti-aging

Abstract

The invention discloses an application of a gardenia extract in preparation of a composition capable of preserving moisture, resisting oxidation, resisting inflammation, brightening and whitening, promoting collagen generation, promoting cell protection and repairing functions, resisting allergy, regulating immune functions, resisting wrinkles and resisting aging. The composition is prepared by the following steps: adding an effective dose of the gardenia extract into a human cell strain; the traditional Chinese medicine composition is capable of retarding moisture evapotranspiration, scavenging free radicals, inhibiting inflammatory factors, inhibiting tyrosinase activity, avoiding DNA damage caused by oxidation, promoting collagen generation in cells and protecting the cells from being damaged by oxidation pressure, has the effects of resisting allergy and regulating immune efficacy, improves the expression of moisturizing factors, improves the expression of anti-wrinkle factors, and can be used for preparing the anti-wrinkle food. The performance of aging factors is inhibited, the performance of cell repair is promoted, and the effects of moisturizing, brightening, tightening, elasticity, protection, repair and immunity regulation are achieved.

Description

Technical Field

[0001] The present invention relates to an application of a gardenia flower extract, and more particularly to a method of using the gardenia petal extract as an additive for cosmetics, skin care products and other skin care products to achieve the effects of moisturizing, brightening, firming, elasticity, protection and repair, as well as moisturizing, anti-oxidation, anti-inflammatory, whitening, promoting collagen production, promoting cell protection and repair functions, anti-allergy, regulating immune function, anti-wrinkle and anti-aging. Background Art

[0002] In recent years, due to the improvement of people's living standards, modern people pay more and more attention to their appearance. However, the ozone layer is being damaged day by day, which has greatly increased people's demand for moisturizing, whitening and spot-lightening related products, so the market size of cosmetics and skin care products is expanding year by year. In addition, modern people have a growing awareness of health. In order to avoid cosmetics and skin care products containing chemical ingredients that harm the skin, the application of natural Chinese herbal medicines in health foods and beauty has been widely valued. In particular, the application of Chinese herbal extracts in cosmetics has become a global trend and is also the focus of active development of the domestic cosmetics and biotechnology industries.

[0003] Skin aging and diseases are also closely related to free radicals. In addition to the endogenous aging caused by aging, the skin is also affected by many adverse environmental factors, which accelerate or prematurely age. These exogenous causes of aging, such as long-term exposure to sunlight or high-energy radiation, unclean diet, tissue damage, microbial infection, excessive stress in life, excessive fatigue or excessive oil intake, will also increase the production of free radicals and melanin or the loss of metabolic factors. Reactive oxygen species (ROS) in the human body, such as superoxide free radicals (.O2-) and hydrogen peroxide (H2O2), can be converted into highly active hydroxyl free radicals (.OH) in the presence of metal ions, which will cause the most direct damage to human tissues. Excessive free radicals accumulate in the skin, which will cause damage to DNA in the epidermis and dermis connective tissue, lipid peroxidation, and denaturation of various collagen proteins, causing the connective tissue to lose its original function and produce skin aging phenomena, such as wrinkles, loss of elasticity, and dryness.

[0004] In view of this, there is an urgent need to provide a substance that can moisturize, resist oxidation, resist inflammation, whiten, promote collagen production, promote cell protection and repair functions, resist allergies, regulate immune function, and resist wrinkles and aging, so as to provide a cosmetic composition and / or food additive. Summary of the invention

[0005] To solve the above problems, an object of the present invention is to provide a use of a gardenia extract for preparing a composition having moisturizing, anti-oxidation, anti-inflammatory, whitening, promoting collagen production, promoting cell protection and repair functions, anti-allergy, regulating immune function, anti-wrinkle and anti-aging effects, wherein the composition is an effective dose of gardenia extract administered to a human cell line to slow down water evaporation, scavenge free radicals, inhibit inflammatory factors, inhibit tyrosinase activity, avoid oxidation-induced DNA damage, promote collagen production in cells, protect cells from oxidative stress, have anti-allergy and immune regulation effects, enhance the performance of moisturizing factors, enhance the performance of anti-wrinkle factors, inhibit the performance of aging factors and promote cell repair, thereby achieving the effects of moisturizing, brightening, firming, elasticity, protection, repair and immune regulation.

[0006] As for the above-mentioned use, the preparation method of the gardenia extract includes: rapidly freezing and solidifying at least gardenia tissue; grinding the gardenia tissue to grind the gardenia tissue into powder; covering and soaking the gardenia tissue with a solvent to form a gardenia tissue soaking liquid; emitting low-temperature ultrasound to continuously and rapidly oscillate and extract the gardenia tissue soaking liquid to obtain a gardenia tissue extract; centrifuging the gardenia tissue extract to obtain a gardenia extract and a precipitate, removing the precipitate and collecting the gardenia extract.

[0007] As for the use mentioned above, wherein the gardenia extract is further filtered to obtain a clear filtrate; and the clear filtrate is freeze-dried to obtain the gardenia extract powder.

[0008] As for the above-mentioned use, wherein the solvent is water, 95% ethanol or anhydrous ethanol.

[0009] The use as described above, wherein the effective dose is 100 to 1000 μg / mL.

[0010] As mentioned above, in the step of extracting the gardenia tissue soaking liquid by continuous rapid oscillation with low temperature emission ultrasound, the gardenia tissue soaking liquid is continuously and rapidly oscillated with a total energy of 500-800w, and the oscillation time is between 60 minutes and 120 minutes.

[0011] As for the above-mentioned use, wherein the composition is a cosmetic composition, a food additive or a pharmaceutical composition.

[0012] As mentioned above, the gardenia extract contains gallic acid or rutin.

[0013] As in the above-mentioned use, the gardenia stamen extract contains an active ingredient, and the compound structure of the active ingredient is shown in the following structural formula:

[0014]

[0015] Wherein, R is terpene.

[0016] For the above-mentioned use, wherein the structure of R is as shown in the following structural formula (I), structural formula (II), structural formula (III), structural formula (IV) or structural formula (V):

[0017]

[0018]

[0019] The gardenia tissue provided by the present invention is a gardenia tissue extract obtained by freeze solidification, grinding into powder, adding water or ethanol for extraction, ultrasonic vibration, filtration, freeze drying and other extract preparation methods. The gardenia tissue extract has high contents of gallic acid, rutin, deacetylated rutin, jasminoidin, jasminoidin, jasminoidin and deacetylated rutin methyl ester. Therefore, the gardenia tissue extract can be effectively used to prepare a composition for moisturizing, anti-oxidation, anti-inflammatory, whitening, promoting collagen production, promoting cell protection and repair functions, anti-allergy, regulating immune function, anti-wrinkle and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a test chart of the supercoiled form (SC), linear form (LC) and open circular form (OC) of pUC119 DNA taken by a cold light camera system and analyzed by computer software.

[0021] Figure 2 This is a test chart showing the β-hexosaminidase activity after RBL-2H3 is sensitized with IgE and then DNP-BSA is added to induce cell degranulation.

[0022] Figure 3 The present invention is a test chart for conducting a gene analysis test to obtain the expression of enhancing the moisturizing factor (Filaggrin), enhancing the anti-wrinkle factor matrix metalloproteinase inhibitor 1 (tissue inhibitors of metalloproteinase 1, TIMP-1), inhibiting the aging factor mTOR, and promoting cell repair nucleotide excision repair cross complementing group 1 (excision repair cross complementing group 1, ERCC1). DETAILED DESCRIPTION

[0023] To achieve the above-mentioned purpose, one embodiment of the present invention is to provide a use of a gardenia extract for preparing a composition for moisturizing, anti-oxidation, anti-inflammatory, whitening, promoting collagen production, promoting cell protection and repair function, anti-allergy, regulating immune function, anti-wrinkle and anti-aging, wherein the composition is an effective dose of about 100 to 1000 μg / mL of gardenia extract administered to a human cell line to slow down water evaporation, scavenge free radicals, inhibit inflammatory factors, inhibit tyrosinase activity, avoid oxidation-induced DNA damage, promote collagen production in cells, protect cells from oxidative stress, have anti-allergy and immune regulation effects, enhance the performance of moisturizing factors, enhance the performance of anti-wrinkle factors, inhibit the performance of aging factors and promote cell repair, so as to achieve the effects of moisturizing, brightening, firming, elasticity, protection, repair and regulating immunity. In some embodiments, the effective dose is 100 to 1,000 μg / mL. Specifically, the gardenia extract herein is an extract comprising gardenia petals, but not other parts.

[0024] In this embodiment, the preparation method of the gardenia extract includes: using liquid nitrogen at about -195.79°C (77K) to rapidly freeze and solidify at least gardenia tissue; using a cryo-grinder to grind the gardenia tissue into powder; covering and soaking the gardenia tissue with pure water or ethanol solvent to form a gardenia tissue soaking liquid; using a low-temperature ultrasonic continuous rapid oscillation extraction device to continuously and rapidly oscillate the gardenia tissue soaking liquid at a low temperature with a total energy of 500-800w, shaking for 5 minutes, resting for 2 minutes, and continuously oscillating for 60 minutes to 120 minutes, and extracting the gardenia tissue by continuous rapid oscillation of low-temperature ultrasonic emission. The invention relates to a method for preparing a gardenia tissue soaking liquid by centrifuging the gardenia tissue extract at a low temperature (4° C.) at a high speed of 15,000 g for 10 minutes to remove the sediment at the bottom of the gardenia tissue extract and collect the gardenia extract at the top of the gardenia tissue extract; filtering the gardenia extract by a vacuum filter and a microporous filter membrane with a pore size of 0.22-0.45 μm to obtain a clear filtrate; and freeze-drying the clear filtrate by a vacuum concentrator and a vacuum freeze dryer to obtain a gardenia extract powder, which is a gardenia tissue water extract (GJW) or a gardenia tissue wine extract (GJE). In some embodiments, the solvent used to cover the soaked gardenia tissue is water, 95% ethanol or anhydrous ethanol.

[0025] The following specific embodiments can further demonstrate the practical application scope of the present invention, but are not intended to limit the patent protection scope of the present invention in any form.

[0026] Experiment 1: Cationic free radical scavenging ability test

[0027] This experiment investigates the antioxidant efficacy of water extract (GJW) and wine extract (GJE) of Gardenia jasminoides tissue extract in scavenging cationic free radicals. The antioxidant and free radical scavenging abilities of the ABTS cationic free radical were tested. ABTS undergoes an oxidation reaction with H2O2 and peroxidase to form a blue-green aqueous solution of ABTS·+[2,29-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid)] cationic free radical, which is used as a total antioxidant capacity test. It is a reversible redox system. It has a specific absorbance value at a wavelength of 734nm. When ABTS·+ can be reduced by the sample, its absorbance value decreases. The ABTS·+ free radical scavenging ability of the sample can be determined by the change in absorbance value. The lower the absorbance value, the stronger the sample's ability to scavenge ABTS·+ free radicals. Prepare a 7mM ABTS solution and mix it with a 2.45mM K2S2O8 solution for 1h. Add 95.7 μL of 40× diluted ABTS mixture to a 96-well plate, then add samples of different concentrations and react for 20 minutes in the dark, and use an enzyme immunoassay to detect the absorbance at 734 nm. The control group is vitamin C.

[0028] The test results are shown in Table 1. The ABTS·+ free radical scavenging abilities of the gardenia tissue water extract (GJW) and wine extract (GJE) (50, 100, 500, 1000 μg / mL) were 18.1%, 30.2%, 76.1% and 98.4%; 13.3%, 25.3%, 45.2% and 75.2%, respectively. The ABTS·+ free radical scavenging ability of the control group vitamin C (20 μg / mL) was 86.4%, and the free radical scavenging ability of the water extract (GJW) was better than that of the wine extract (GJE). The results showed that the gardenia tissue extract had a good free radical scavenging ability.

[0029]

[0030] Table 1

[0031] Experiment 2: Whitening test

[0032] This experiment investigates the whitening effect of the water extract (GJW) and wine extract (GJE) of Gardenia jasminoides tissue extract, which is achieved by inhibiting the activity of tyrosinase. Tyrosinase is one of the important enzymes in the melanin production pathway. The tyrosinase activity test is used to evaluate the whitening effect of the sample and its concentration of whitening effect. Take 2μL of the sample to a 96-well plate, add 110μL of phosphate buffer (pH6.5) to mix, then add 5μL of 50unit mushroom tyrosinase, and react at room temperature in the dark for 10 minutes. Then add 83μL of 12.5mM L-DOPA and react at room temperature in the dark for 10 minutes. Then measure the absorbance at a wavelength of 492nm (BioTek, Synergy TM 2, USA). Calculation method of tyrosinase activity inhibition rate (Inhibition of tyrosinase activity%): [1–(OD sample -OD sample background ) / (OD control –OD control background )]*100%. The control group was vitamin C.

[0033] The test results are shown in Table 2. The ability of gardenia tissue water extract (GJW) and wine extract (GJE) (100, 500, 1000 μg / mL) to inhibit tyrosinase activity were 31.4%, 35.3% and 58.0%; 29.8%, 40.9% and 50.6%, respectively, while the ability of the control group vitamin C (500 μg / mL) to inhibit tyrosinase activity was 95.8%. Among them, the ability of water extract (GJW) to inhibit tyrosinase activity was better than that of wine extract (GJE). The results showed that gardenia tissue extract has a good inhibitory ability on tyrosinase activity.

[0034] In addition, in order to compare whether different parts of the same plant tissues have different effects on efficacy, in order to avoid the problem of hindsight, this experiment used the same low-temperature extraction method as the gardenia flower tissue extract to obtain the gardenia fruit water extract (GJFW), and the same test method as the gardenia flower water extract (GJW) was used to perform the same experiment, and the test results are shown in Table 2 below. From the results shown in Table 2, it can be seen that the ability of the gardenia flower water extract (concentrations of 100, 500, and 1000 μg / mL) to inhibit tyrosinase activity is 31.4%, 35.3%, and 58.0%, respectively; the ability of the gardenia fruit water extract (concentrations of 100, 500, and 1000 μg / mL) to inhibit tyrosinase activity is 15.2%, 14.1%, and 16.9%. The ability of vitamin C (500 μg / mL) in the control group to inhibit tyrosinase activity was 95.8%. The results showed that the ability of the gardenia flower water extract to inhibit tyrosinase activity was significantly better than that of the gardenia fruit water extract.

[0035]

[0036] Table 2

[0037] Experiment 3: Anti-inflammatory test

[0038] Nitric oxide (NO.) is one of the main substances involved in inflammatory reactions. This experiment tested the anti-inflammatory and inhibitory activity of nitric oxide in the water extract (GJW) and wine extract (GJE) of gardenia tissue extract. Sodium nitroprusside (SNP) itself is a kind of NO donor. It reacts with oxygen to form nitrite. Nitrite reacts with Griess reagent to become a pink solution with a specific absorbance at a wavelength of 546nm. If the sample can inhibit the rate of nitric oxide generation and reduce the production of nitrite, its absorbance will decrease; the lower the absorbance, the stronger the sample's ability to scavenge nitric oxide free radicals. Take 2μL of gardenia extract (GJW) and add 98μL of 25mM SNP to react for 120mins, then add 100μL of Griess reagent to react for 10mins, and use an enzyme immunoassay to detect the absorbance at 546nm. The control group is rutin.

[0039] The test results are shown in Table 3. The inhibitory activity of the inflammatory factor nitric oxide of the gardenia tissue water extract (GJW) (100, 500, 1000 μg / mL) was 14.3%, 24.4% and 31.0%, while the inhibitory activity of the inflammatory factor nitric oxide of the control group rutin (500 μg / mL) was 49.2%. The results show that the gardenia tissue extract can achieve the activity of inhibiting inflammatory response by inhibiting the activity of nitric oxide, so it has an anti-inflammatory effect.

[0040]

[0041] Table 3

[0042] Experiment 4: Moisturizing test

[0043] The experiment was conducted to test the water content and moisturizing efficacy of gardenia extract (GJ). Gardenia water extract (GJW) was prepared into a 1% aqueous solution and placed in a closed hot air oven to evaluate the water evaporation of the sample, thereby evaluating the moisturizing efficacy of the sample. 3.0g of GJ was placed in a porcelain crucible and dried in a hot air oven at 75-80°C. The initial weight and the final weight after 2, 4, 8, 24 and 48 hours of action were measured. The water content of gardenia extract was determined using the formula to evaluate the moisturizing efficacy of gardenia extract. The positive control group was hyaluronic acid (1%); the negative control group was pure water. Moisturizing efficacy (%) = [(initial weight of the filled crucible) - (final weight of the filled crucible)] / [(initial weight of the filled crucible) - (initial weight of the empty crucible)] × 100.

[0044] The test results are shown in Table 4. The moisturizing rates (%) of the Gardenia jasminoides tissue water extract (GJW) were 100.0%, 73.5%, 65.9%, 55.9%, 51.4% and 49.2% at 0, 2, 4, 8, 24 and 48 hours, respectively. The results show that the Gardenia jasminoides tissue extract has a moisturizing effect.

[0045]

[0046] Table 4

[0047] In addition, in order to compare whether different parts of the same plant tissues have different effects on efficacy, in order to avoid the problem of hindsight. Therefore, this experiment used gardenia fruit to extract the same low-temperature extraction method as the gardenia flower tissue extract to obtain gardenia fruit water extract (GJFW), and the same experiment was carried out using the same test method as the gardenia flower water extract (GJW). The test results are shown in Table 5 below. From the results shown in Table 5, it can be seen that the moisturizing rate (%) of the gardenia flower water extract is 100.0% and 73.5% at 0 and 2 hours respectively. The moisture content of the gardenia fruit water extract is 100.0% and 7.1% at 0 and 2 hours respectively. The results show that the gardenia flower water extract has a significantly better moisturizing effect than the gardenia fruit water extract.

[0048]

[0049] Table 5

[0050] Experiment 5: Cell safety test

[0051] This experiment investigates the safety assessment and cell viability test of water extract (GJW) and wine extract (GJE) of Gardenia jasminoides tissue extract on human cells. Human skin keratinocytes (HaCaT cells) were quickly moved to a 37°C water bath to thaw rapidly within 40-60 seconds, and then cell cryopreservation solution (cell culture medium containing 10% DMSO) was added to the cell culture medium, the cells were broken up and moved to a 25 cm 2 Cell culture flasks were grown in a cell culture incubator at 37°C and 5% CO2, and the culture medium was changed every 2 days on average. Human skin keratinocytes (HaCaT cells) were cultured in 96-well plates and incubated at 37°C and 5% CO2 for at least 24 hours. To evaluate whether Gardenia jasminoides extract (GJ) affects cell viability on skin cells: different concentrations of Gardenia jasminoides extract (GJ) were added and acted for a specified time. After the reaction time, the old culture medium was removed, washed once with PBS, and replaced with new culture medium. 10μL of MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution was added for reaction. After 4 hours of reaction at 37°C and 5% CO2, the culture medium was removed, 100μL of DMSO was added to dissolve the formazan precipitate, and finally the absorbance was measured at a wavelength of 570nm (BioTek, Synergy TM 2, USA).

[0052] The test results are shown in Table 6. After the human skin keratinocytes (HaCaT cells) were treated with Gardenia jasminoides tissue water extract (GJW) (50, 100, 250, 500, 1000 μg / mL) for 24 hours, the cell viability of the human skin keratinocytes was 101.8%, 122.8%, 129.6%, 124.7% and 117.7% respectively. From the results, it can be seen that the water extract of Gardenia jasminoides tissue extract is not toxic in skin cells, so it is safe and does not have adverse effects on the viability of skin cells.

[0053]

[0054] Table 6

[0055] Experiment 6: DNA protection test

[0056] Physiological metabolism can increase oxidative stress and cause DNA damage. In this experiment, pUC119 DNA was used to induce DNA damage with H2O2 (1mM) to evaluate whether the water extract and wine extract of Gardenia jasminoides tissue can protect DNA from DNA damage caused by oxidative stress. Prepare 0.8% agarose gel, dissolve the weighed agarose powder in 1×TAE buffer, heat the powder in a microwave oven to completely dissolve it, add 0.5mg / L Ethidium bromide (EtBr) and mix well, pour it into a horizontal gel-making template, insert a tooth comb, remove the tooth comb after solidification, put the prepared agarose gel into a horizontal electrophoresis tank and add 1×TAE buffer. Add pUC119 DNA to the sample, PBS, 0.5mM FeSO4, and 1mM H2O2 in sequence, mix gently and evenly, react at 37℃ for 1h, then add an appropriate amount of 6×Loading dye to the sample after the reaction, add it to the small hole in the gel, and perform electrophoresis at 100V. After the pUC119 DNA had run to an appropriate distance, the gel was taken out and photographed with a cold light camera system (Avegene-SPOT). The expression of the pUC119 DNA supercoiled form (SC), linear form (LC) and open circular form (OC) was analyzed with Image J computer software.

[0057] The test results are shown in Table 7 and Figure 1 As shown in the results, the intact DNA presents a supercoiled structure, but after being affected by oxidative stress, the supercoiled structure will be destroyed and form a linear structure. Therefore, the pUC119 DNA was induced with H2O2 (1mM) to induce DNA damage by taking advantage of the different speeds of nucleic acids with different molecular weights moving in the pores of the colloid. However, the DNA treated with Gardenia jasminoides (GJW) (100 and 500μg / mL) can maintain the intact DNA form and present a supercoiled structure. The SC structure of DNA without H2O2 treatment is 100.0%, the SC structure of DNA treated with H2O2 is 1.9%, and the SC structure of DNA treated with Gardenia jasminoides (GJW) (100 and 500μg / mL) is 98.5% and 98.0%, respectively. The results show that Gardenia jasminoides extract has the ability to protect DNA from DNA damage caused by oxidative stress.

[0058]

[0059] Table 7

[0060] Experiment 7: Test to promote collagen production content

[0061] Collagen is a biological macromolecule and the most abundant protein in the human body, accounting for more than 30% of the total protein in the body. 70% of the human skin is made up of collagen. When collagen is insufficient, not only will there be problems with the skin and bones, but it will also have an adverse effect on the internal organs. In other words, collagen is an important component that is indispensable for maintaining normal body activities. It is also a substance that keeps the body young and prevents aging. In addition, collagen can prevent diseases, improve physical fitness, and is very helpful for beauty and health. Collagen in the skin is an important factor in maintaining skin elasticity and anti-wrinkle. Its synthesis is affected by age. Therefore, if the synthesis of collagen can be promoted in time, wrinkles and loss of elasticity and luster can be avoided.

[0062] Skin fibroblasts (3T3L-1) were cultured at 2×10 5 After culturing cells in a 3-cm dish for 24 hours, the supernatant was removed and serum-free culture medium containing different concentrations of samples was added for 48 hours. After washing with PBS, the cells were scraped off and centrifuged at 1200rpm for 5 minutes, and the supernatant was removed. This experiment uses Sircol soluble collagen assaykit for collagen determination. First, 100μL of cell solution was mixed with 1mL Sircol dye reagent, then shaken evenly at room temperature for 30 minutes, centrifuged at 12000rpm for 10 minutes, and the supernatant was directly poured out. Then 750μL ice-coldacid-salt wash reagent was added, and centrifuged at 12,000rpm for 10 minutes to completely remove the dye reagent. Then 250μL alkali reagent was added and mixed evenly, 100μL was taken to a 96-well dish, and the absorbance was measured at 555nm using an enzyme immunoassay analyzer.

[0063] The test results are shown in Table 8. After the gardenia water extract (GJW) acted on skin fibroblasts, the production of collagen in the cells was promoted by 113.8%, 116.9% and 122.0% respectively. The results showed that the gardenia extract (GJ) has the effect of promoting the production of collagen in skin fibroblasts.

[0064]

[0065] Table 8

[0066] Experiment 8: Test to promote cell protection function

[0067] Ultraviolet rays affect the body mainly on epidermal cells. When exposed to excessive ultraviolet rays, the cell's repair ability will be damaged, causing cell aging. To evaluate whether Gardenia jasminoides extract (GJ) can protect skin cells from damage by environmental ultraviolet rays. Human skin keratinocytes (1×10 4 / well) were cultured in 96-well plates and cultured in a 37°C and 5% CO2 incubator for at least 24 hours. Control group: The cell supernatant was removed and replaced with fresh serum-free culture medium. After 4 hours of culture, the cells were washed with PBS and placed in fresh serum-free culture medium. The culture was continued for 4 hours and survival analysis was performed. UV irradiation group: The cell supernatant was removed and replaced with fresh serum-free culture medium. After 4 hours of culture in a cell culture incubator, the supernatant was removed and washed with PBS and dried. After UV irradiation, fresh serum-free culture medium was immediately added. The culture was continued for 4 hours and survival analysis was performed. UV irradiation + gardenia extract (GJ) group: The cell supernatant was removed, and the extracts were mixed with fresh serum-free culture medium. After 4 hours of culture, the supernatant was removed, washed with PBS and dried. After UV irradiation, fresh serum-free culture medium containing the extract was immediately added. The culture was continued for 4 hours and survival analysis was performed. After the reaction time, the old culture medium was removed, the cells were washed once with PBS, and replaced with new culture medium. 10 μL of MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution was added for reaction. The cells were reacted at 37°C and 5% CO2 for 4 hours. After that, the culture medium was removed, 100 μL of DMSO was added to dissolve the formazan precipitate, and the absorbance was measured at a wavelength of 570 nm (BioTek, Synergy TM 2, USA).

[0068] The test results are shown in Table 9. 2 ) irradiated, the cell viability was about 75.8%, while after being treated with Gardenia jasminoides extract (GJW) (100, 250 and 500 μg / mL), the cell viability was 81.8%, 86.7% and 97.5%. The results showed that Gardenia jasminoides extract (GJ) has the ability to protect cells from UV damage.

[0069]

[0070] Table 9

[0071] Experiment 9: Anti-allergy test

[0072] Allergy is an abnormal reaction of the body, which is an abnormal reaction to allergens. Allergies will occur when allergens come into contact with people with allergic constitutions. Allergens include hundreds of chemicals, ultraviolet rays, pollen, dust, isoproteins, etc. In the process of allergic reactions, allergic mediators play a direct role. Allergens are the external cause of allergic symptoms, while low immunity and the oxidative damage of mast cells and basophils by a large number of free radicals are the internal causes of allergies. There are two types of cells in the human body, namely mast cells and basophils, which are widely distributed in the nasal mucosa, bronchial mucosa, gastrointestinal mucosa, and around the microvessels in the connective tissue under the skin and in the capsule of the internal organs. These two types of cells contain allergic mediators such as histamine, leukotrienes, 5-hydroxytryptamine, and kinase. When stimulated by IgE antibodies or calcium ion carriers, mast cell lines (RBL-2H3 cells) can release a large amount of β-glucuronidase (β-hexosaminidase) through degranulation. The amount released can be used to determine whether the Gardenia jasminoides water extract (GJW) to be tested has the potential to inhibit allergic reactions.

[0073] When mast cells bind to IgE on their surface, the cells become sensitized. Therefore, when allergens are present, the triggers of allergic reactions in the intracellular granules are released, resulting in an allergic reaction, which is called degranulation. β-glucuronidase (β-hexosaminidase) is an enzyme released when mast cells are activated by allergens. It can be used to quantify the degree of mast cell degranulation. Mast cells are a type of immune cell widely present in tissues, and the RBL-2H3 cell line is often used to study the mechanism of mast cell-mediated allergic reactions. RBL-2H3 was cultured at 2×10 4 After cells / well were cultured in 96-well, anti-DNP IgE Ab (1μg / ml) was added and cultured for 12 hours, and then the Gardenia extract (GJW) sample was added for 4 hours, the supernatant was removed and PBS was added to wash the cells, 100ng / mL dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) was added and cultured for 1 hour, and the supernatant was collected. The cells were lysed by adding 0.5% triton-X100 for 30 minutes. An equal volume of cell supernatant and 1μM p-nitrophenyl-N-acetyl-D-glucosaminide (p-NAG) were added to the 96-well and cultured at 37℃ for 1 hour, and the absorbance was measured at a wavelength of 405nm.

[0074] The test results are as follows Figure 2As shown in the figure, after RBL-2H3 was sensitized by IgE, the β-hexosaminidase activity was expressed after the addition of DNP-BSA to induce cell degranulation. The β-hexosaminidase activity of the group without Gardenia jasminoides extract (GJW) was 58.2%, while the addition of 100, 250 and 500 μg / mL GJW decreased to 57.6%, 56.3% and 52.9% respectively. The results showed that Gardenia jasminoides extract (GJW) has anti-allergic effect and immune regulation function.

[0075] Experiment 10: Regulating moisturizing factors, anti-wrinkle factors, anti-aging factors, and promoting cell repair

[0076] Real-time quantitative qPCR was used to analyze the gene expression of the moisturizing factor Filaggrin, the anti-wrinkle factor TIMP-1, the anti-aging factor mTOR, and the cell repair promoting ERCC1 regulated by Gardenia jasminoides tissue water extract (GJW).

[0077] Moisturizing factor Filaggrin: Natural moisturizing factor is mainly produced by the disintegration of keratin filaments, a protein existing in the human epidermis, in the keratinized cells of the stratum corneum to form a hydrophilic hygroscopic substance. Filaggrin filaments are a key protein in the formation of the skin barrier on the skin surface. Its precursor is profilaggrin (a large protein molecule composed of Filaggrin in series), which is located in the keratin granules contained in the cells of the granular layer of the skin. The protein monomer produced by enzymatic hydrolysis of this macromolecule is Filaggrin. After Filaggrin is produced, it enters the junction of the granular layer and the stratum corneum. Through the aggregation with keratin, it not only makes the skin cell skeleton form a strong and compact shape, helps the skin resist ultraviolet rays, adapts to changes in pH, but also retains water in the cells to maintain skin moisture.

[0078] Anti-wrinkle factor TIMP-1: Matrix metalloproteinase inhibitor 1 (TIMP-1) is a tissue inhibitor of metalloproteinases. It can specifically inhibit the activity of matrix metalloproteinases (MMPs) and plays an important role in cell proliferation and differentiation. Increased TIMP-1 expression can inhibit the activity of MMPs, thereby preventing collagen from being decomposed and fighting against wrinkles on the skin.

[0079] Anti-aging factor mTOR: mTOR is a serine-threonine protein kinase and an important factor causing aging and cancer. It promotes cells to absorb heat, shuts down the autophagy mechanism and prevents cell renewal. Therefore, in recent years, studies on inhibiting the expression of mTOR have pointed out that by inhibiting the expression of mTOR in aging cells, cells can have the potential for regeneration.

[0080] Cell repair ERCC1: nucleotide excision repair cross-complementing group 1. It is an important member of the exonucleoside repair family and a key member of the nucleotide excision repair (NE) system, involved in DNA chain cutting and damage recognition. The ERCC1 gene is also the first cloned human DNA damage repair gene. The expression level of ERCC1 directly affects the physiological process of DNA repair.

[0081] Primer:

[0082] Filaggrin:5′-GCT GAA GGA ACT TCT GGA AAA GG-3′; 5′-GTTGTGGTCTATATCCAAGT ATC-3′

[0083] TIMP-1:5′-AAGGCTCTGAAAAGGGCTTC-3′; 5′-GAAAGATGGGAGTGGGAACA-3′

[0084] mTOR:5′-ACTCGCTTCTATGACCAACTGA-3′; 5′-TTTCCATGACAACTGGGTCATTG-3′

[0085] ERCC1:5′-ACC CCTCGACGAGGATGAG-3′; 5′-GGGAATTTGGCGACGTAATTC-3′

[0086] Human keratinocytes (HaCaT cells) (1.5×10 5 cell / mL) were inoculated in a 6-well plate and cultured for 24 hours. After the Gardenia jasminoides tissue water extract (GJW) was mixed with serum-free culture medium for 24 hours, the RNA of the cells was extracted. The supernatant was collected into a 15 mL centrifuge tube, the cells were removed with Trypsin, and the cells were collected with PBS and centrifuged to remove the supernatant. 1 mL REzol was added TMC&T was mixed evenly at room temperature for 5 minutes to dissolve the cells. 200μL chloroform was added and mixed evenly for 15 seconds. Incubate at 4℃ for 5 minutes to purify and isolate Total RNA. Centrifuge at 12,000rpm at 4℃ for 10 minutes. The top transparent layer in the centrifuge tube was taken into a 1.5mL microcentrifuge tube soaked in DEPC-H2O and sterilized. An equal volume of isopropanol was added and mixed evenly and allowed to stand at 4℃ for 5 minutes to precipitate RNA. Centrifuge at 12,000rpm at 4℃ for 10 minutes. The supernatant was removed. 200μL 75% ice alcohol was added to clean the RNA. Centrifuge at 12,000rpm at 4℃ for 10 minutes. The supernatant was completely removed and inverted in the air in a sterile operating table to dry in the shade to completely evaporate the moisture in the centrifuge tube. 100μL 0.1% DEPC-H2O was added to disperse the RNA and stored in a -80℃ refrigerator. Quantitative RNA: Take 2 μL RNA in 198 μL 0.1% DEPC-H2O, mix well, take 100 μL in a micro-quartz tube, and measure the absorbance at OD260 nm and OD280 nm using a spectrophotometer (BioTek, Synergy TM2, USA). Reverse transcription (RT): Add 0.1% DEPC-H2O to adjust the concentration to 2mg RNA, make up the volume to 12.5μL, add 1μL Oligo dT primer and mix well, then add 70μL for 2 minutes, quickly put on ice, add 4μL 5×MMLV-RT buffer solution, 1μL 10mM Each dNTP, 0.5μL RNase inhibitor and 1μL MMLV RTase, and make it at 42℃ for 1 hour, then at 94℃ for 5 minutes, add ice ddH2O to make up the volume to 100μL, and the cDNA preparation is completed and stored in a -80℃ refrigerator. The first step of qRT-PCR was completed using a fluorescence detection system (prism 7700 sequencing detection system) and Qiagen's Quantitect SYBR Green RT-PCR kit according to the manufacturer's procedures. The primer sequence is the same as that used to generate arrayed cDNAs. The gene amplification conditions were as follows: reverse transcription at 50°C for 30 minutes, followed by 95°C for 15 minutes to complete the PCR initial activation step. Melting temperature was 95°C for 15 seconds, and annealing / extension was at 60°C for 1 minute, for a total of 40 cycles. Finally, the size of the amplified product was confirmed by interpreting the data using the cycle threshold (CT) (ΔΔCT) method, and the CT values ​​of the control group and the laboratory were compared. The β-actin gene was used as a quantitative analysis. The fold change of gene expression was calculated. 10 μL of cDNA was taken into a microcentrifuge tube, and 5 μL of 10× reaction buffer, 2 μL of 25 mM MgCl2, 0.8 μL of 10 mM dNTP, and 1 μL of each 50 μM upstream and downstream primer, Taq DNA polymerase (5 unit / μL) were added. Finally, deionized water was added to make the total volume 50 μL, and the PCR reaction was carried out in an automatic temperature cycler (MyGo Pro real-time PCR).

[0087] The test results are as follows Figure 3 As shown, the results of the gene analysis test showed that Gardenia jasminoides tissue water extract (GJW) can effectively regulate and enhance the expression of moisturizing factor Filaggrin, enhance the expression of anti-wrinkle factor TIMP-1, inhibit the expression of aging gene mTOR in human skin keratinocytes, and promote the expression of cell repair ERCC1 compared with the control group (Control).

[0088] Experiment 10: Active ingredient composition analysis and calibration

[0089] Determination of total phenol content in Gardenia jasminoides extract (GJ): Phenolic compounds are antioxidants and phytonutrients found in plants. Phenolic antioxidants can resist oxidative stress that causes neurodegenerative diseases and some cardiovascular diseases. The phosphomolybdic acid complex in Folin-Ciocalteu'sphenolreagent will be reduced to molybdenum by phenol, and the reagent will change from golden yellow to blue-green, which can be used to quantify the phenol content in the sample. Take 1μL of Gardenia jasminoides extract (GJ) and add 25μL of 10X diluted Folin-Ciocalteu'sphenolreagent to react for 5mins, then add 20μL Na2CO3 and 50μL secondary water to react for 1h, and detect the absorbance at 700nm with an enzyme immunoassay. Different concentrations of gallic acid (0-1280μg / mL) were used as standards to prepare calibration curves.

[0090] The test results are shown in Table 10. 1 g of gardenia jasmine water extract (GJW) is equivalent to 38.2 mg of gallic acid. In other words, the total phenol content of gardenia jasmine water extract (GJW) is equivalent to 38.2 mg / g of gallic acid equivalent.

[0091]

[0092] Table 10

[0093] Determination of total flavonoid content in Gardenia jasminoides extract (GJ): Flavonoids exist in fruits and vegetables and have antioxidant, inflammation control, cell damage prevention, and anti-aging effects. Flavonoids can chelate with aluminum chloride in an alkaline environment to form a red chelate with a specific absorbance at a wavelength of 510nm. Take 1μL of Gardenia jasminoides extract (GJ) and add 3μL of 5% NaNO2 to react for 6mins, then add 3μL of 10% AlCl3 to react for 6mins, and finally add 40μL of 4% NaOH and 4μL of secondary water to react for 15mins. The absorbance at 510nm is detected by enzyme immunoassay. Different concentrations of rutin (0-1280μg / mL) are used as standards to make calibration curves.

[0094] The test results are shown in Table 11. 1 g of Gardenia jasminoides water extract (GJW) is equivalent to 42.8 mg of rutin. In other words, the total flavonoid content of Gardenia jasminoides water extract (GJW) is equivalent to 42.8 mg / g of rutin equivalent.

[0095]

[0096] Table 11

[0097] The Gardenia tissue extract was made into a concentration of 50ppm and then put into LC-MS for data analysis. Chromatographic fingerprint of Gardenia tissue extract: In the calibration of the active ingredients of Gardenia tissue extract, a total of 5 signals were calibrated as the analyzed compounds. After comparing with the database data, signals 1 to 5 were all matched to the corresponding molecular weights, and the compounds matched by signals 1 to 5 are described in detail as follows.

[0098] Signal 1 is Deacetylasperulosidic acid (molecular formula:

[0099] C 16 H 22 O 11 , MW=390.1162) is an iridoid compound found in some medicinal plants, which increases superoxide dismutase activity and has antioxidant activity. Atopic dermatitis (AD) is a disease characterized by severe itching, immune imbalance and skin barrier dysfunction, and its prevalence is increasing rapidly worldwide. Deacetylasperulosidic acid has anti-atopic activity in keratinocytes, mast cells and eosinophils, the three main cell types associated with atopic dermatitis, and has anti-allergic activity. Deacetylasperulosidic acid has antigenic activity and inhibits the induction of chromosomal aberrations in hamster ovary cells and mice. It can prevent 4-nitroquinoline 1-oxide (4NQO)-induced DNA damage in vitro, inhibit IL-2 production and natural killer cell activation. The structure of the Deacetylasperulosidic acid compound is shown below:

[0100]

[0101] Signal 2 is Shanzhiside (molecular formula: C 16 H 22 O 11, MW=392.1319) is a terpene glycoside. Shanzhiside has immunosuppressive activity. It co-stimulated activation of peripheral blood T cells in vitro through phorbolmyristate acetate and anti-CD28 monoclonal antibody, thereby significantly inhibiting IL-2 secretion. The structure of Shanzhiside compound is shown below:

[0102]

[0103] Signal 3 is Geniposide (molecular formula: C 17 H 24 O 10 , MW = 388.1369) is the glycoside form of genipin, a bioactive iridoid glycoside found in a variety of medicinal materials, such as Gardenia. Gardenia glycosides show a variety of pharmacological effects, including neuroprotective, antidiabetic, hepatoprotective, anti-inflammatory, analgesic, antidepressant-like, cardioprotective, antioxidant, immunomodulatory, antithrombotic and antitumor activities. These pharmacological benefits are produced by the regulatory effects of Gardenia glycosides on several proteins and genes associated with inflammatory and oxidative stress processes. The structure of the Geniposide Gardenia glycoside compound is shown below:

[0104]

[0105] Signal 4 is Gardenoside (C 17 H 24 O 11 , MW=404.1319), and has a hepatoprotective effect. Gardenoside inhibits the activation of inflammatory factors TNF-α, IL-1β, IL-6 and NF-κB. Gardenoside also inhibits cell steatosis induced by free fatty acids (FFA). Gardenoside inhibits pain in the rat chronic contraction injury model by regulating P2X3 and P2X7 purine receptors. The structure of the Gardenoside compound is shown below:

[0106]

[0107] Signal 5 is Deacetylasperulosidic acid methyl ester (molecular formula: C 18 H 26 O 11 , MW = 418.3950) is also a cycloterpenoid compound, present in some medicinal plants, with antioxidant activity. The structure of the deacetylasperulosidic acid methyl ester compound is shown below:

[0108]

[0109] From the compound results detected by the above signals 1 to 5, it is known that the Gardenia stamen extract of the present invention contains an active ingredient of iridoid compounds, and the compound structure of the active ingredient is shown in the following structural formula:

[0110]

[0111] Wherein, R is an cyclopentadiene. In some embodiments, the structure of R is as shown in the following structural formula (I), structural formula (II), structural formula (III), structural formula (IV) or structural formula (V):

[0112]

[0113]

[0114] In summary, the present invention confirms that the water extract and wine extract of gardenia tissue have the effects of moisturizing, anti-oxidation, anti-inflammation, whitening, promoting collagen production, promoting cell protection and repair function, anti-allergy, regulating immune function, anti-wrinkle and anti-aging. It has the potential to be developed into a cosmetic material composition, skin care product, food additive or pharmaceutical composition with the effects of moisturizing, brightening, firming, elasticity, protection, repair and immune regulation of the skin, and can indeed achieve the expected use effect through the embodiments disclosed above. The gardenia tissue provided by the present invention is a gardenia tissue extract obtained by freeze-solidification, grinding into powder, adding water or ethanol for extraction, ultrasonic vibration, filtration, freeze-drying and other extract preparation methods. The gardenia tissue extract has high contents of gallic acid, rutin, deacetylated rutin, jasminoidin, jasminoidin, jasminoidin and deacetylated rutin methyl ester. Therefore, the gardenia tissue extract can be effectively used to prepare a composition for moisturizing, anti-oxidation, anti-inflammatory, whitening, promoting collagen production, promoting cell protection and repair functions, anti-allergy, regulating immune function, anti-wrinkle and anti-aging.

[0115] It should be understood that the foregoing description of the embodiments is given only by way of example, and various modifications can be made by those with ordinary knowledge in the technical field to which the present invention belongs. The above description and examples provide a complete description of the process and use of the exemplary embodiments of the present invention. Although specific embodiments of the present invention are disclosed in the above embodiments, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs can make various changes and modifications to it without departing from the principles and spirit of the present invention. Therefore, the scope of patent protection of the present invention shall be subject to the attached claims.

Claims

1. A composition comprising a gardenia extract for moisturizing, anti-oxidation, anti-inflammation, whitening, promoting collagen production, promoting cell protection and repair functions, anti-allergy, regulating immune function, anti-wrinkle and anti-aging, characterized in that: The composition is prepared by administering an effective dose of gardenia extract to a human cell line to slow down water evaporation, eliminate free radicals, inhibit inflammatory factors, inhibit tyrosinase activity, avoid oxidation-induced DNA damage, promote collagen production in cells, protect cells from oxidative stress, have anti-allergic and immune-regulating effects, enhance the performance of moisturizing factors, enhance the performance of anti-wrinkle factors, inhibit the performance of aging factors, and promote cell repair, thereby achieving the effects of moisturizing, brightening, firming, elasticity, protection, repair, and immune-regulating.

2. The use according to claim 1, characterized in that The preparation method of the gardenia flower extract comprises: rapidly freezing and solidifying at least gardenia flower tissue; grinding the gardenia flower tissue to grind the gardenia flower tissue into powder; covering and soaking the gardenia flower tissue with a solvent to form a gardenia flower tissue soaking liquid; emitting low-temperature ultrasound to continuously and rapidly oscillate and extract the gardenia flower tissue soaking liquid to obtain a gardenia flower tissue extract; centrifuging the gardenia flower tissue extract to obtain a gardenia flower extract and a precipitate, removing the precipitate and collecting the gardenia flower extract.

3. The use according to claim 2, characterized in that: The gardenia extract is further filtered to obtain a clear filtrate; and the clear filtrate is freeze-dried to obtain a gardenia extract powder.

4. The use according to claim 2, characterized in that: The solvent is water, 95% ethanol or anhydrous ethanol.

5. The use according to claim 1, characterized in that: The effective dose is 100 to 1,000 μg / mL.

6. The use according to claim 2, characterized in that: In the step of extracting the gardenia tissue soaking liquid by continuous rapid oscillation with low temperature emission ultrasound, the gardenia tissue soaking liquid is subjected to continuous rapid oscillation extraction with a total energy of 500-800w, and the oscillation time is between 60 minutes and 120 minutes.

7. The use according to claim 1, characterized in that The composition is a cosmetic material composition, a food additive or a medicine composition.

8. The use according to claim 1, characterized in that The gardenia flower extract contains gallic acid or rutin.

9. The use according to any one of claims 1 to 8, characterized in that: The gardenia stamen extract contains an active ingredient, and the compound structure of the active ingredient is shown in the following structural formula: Wherein, R is terpene.

10. The use according to claim 9, characterized in that The structure of R is shown in the following structural formula (I), structural formula (II), structural formula (III), structural formula (IV) or structural formula (V):

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