Application of gardenia pistil tissue in energy activation, protection, repair and aging resistance

Abstract

The invention discloses application of a gardenia stamen extract in preparation of a composition for resisting oxidation, resisting inflammation, inhibiting light products, protecting, improving granulocyte quality, promoting wound healing, resisting allergy, regulating immune function, regulating and controlling moisturizing and anti-aging factors and promoting cell repair factors. The composition is prepared by the following steps: adding an effective dose of cape jasmine flower stamen extract into a human cell strain; the composition can remove free radicals, inhibit inflammatory factors, protect and repair damaged skin cells, avoid cell damage caused by oxidative pressure, promote cell line regeneration and promote skin wound healing, has the effects of resisting allergy and regulating immune efficacy, improves the expression of moisturizing factors, inhibits the expression of glycation aging factors and promotes the expression of cell repair factors, and can be used for preparing the skin care product. The effects of brightening, moisturizing, elasticity, protection, repair, allergy resistance and immunity regulation are achieved.

Description

Technical Field

[0001] The present invention relates to an application of a gardenia stamen extract, and more particularly to a use of the gardenia stamen extract as an additive to cosmetics, skin care products and other skin care products to achieve the purpose of anti-oxidation, anti-inflammation, inhibition of photoproducts, protection, improvement of mitochondrial quality, promotion of wound healing, anti-allergy, regulation of immune function, regulation of moisturizing and anti-aging factors, and promotion of cell repair factors. Background Art

[0002] It is known that ultraviolet rays can cause wrinkles and pigmentation on the skin, and even cause cell aging, cancer or death. Ultraviolet rays (UV) or reactive oxygen species (ROS) are one of the factors that cause the large-scale production of free radicals. Free radicals are highly active and unstable. Excessive free radicals will produce oxidation reactions with cell tissues in the body. After ultraviolet rays increase the activity of free radicals in the human body, under normal conditions, the human body has the ability to repair. However, excessive ultraviolet rays cause a large number of free radicals to be produced, causing tissue cells to lose their normal functions and even damage DNA, reducing the cell DNA repair ability, causing damage or mutation, and then causing cancer.

[0003] In addition, human skin will age, become rough or develop wrinkles as it ages, due to physiological factors or environmental influences. Since the skin of normal young people has a certain elasticity and tension, when the facial muscles relax, the skin will quickly recover and wrinkles will disappear. However, after entering middle age and old age, the skin will begin to become thinner, dry, and have reduced tension, and other aging phenomena. After the facial muscles relax, the skin cannot recover quickly, causing wrinkles to begin to form. Due to factors such as dryness, ultraviolet rays, detergents or chemicals, or hormonal imbalance, as well as the decrease in the barrier function and moisture content of the skin's stratum corneum, the stratum corneum will become rough, lose elasticity and moisturizing function, and then the skin will wrinkle and lose its luster.

[0004] In view of this, there is an urgent need to provide a substance that has antioxidant, anti-inflammatory, photoproduct inhibition, protection, improvement of mitochondrial quality, promotion of wound healing, anti-allergy, regulation of immune function, regulation of moisturizing and anti-aging factors, and promotion of cell repair factors, in order to provide a cosmetic composition and / or food additive. Summary of the invention

[0005] In order to solve the above-mentioned problems, one of the purposes of the present invention is to provide a use of a gardenia stamen extract for preparing a composition for anti-oxidation, anti-inflammatory, photoproduct inhibition, protection, improvement of mitochondrial quality, promotion of wound healing, anti-allergy, regulation of immune function, regulation of moisturizing and anti-aging factors, and promotion of cell repair factors, wherein the composition is an effective dose of a gardenia stamen extract administered to a human cell line to scavenge free radicals, inhibit inflammatory factors, protect and repair damaged skin cells, avoid oxidative stress-induced cell damage, promote cell mitochondrial regeneration, promote skin wound healing, have anti-allergic and immune regulation effects, enhance the expression of moisturizing factors, inhibit the expression of glycation aging factors, and promote the expression of cell repair factors, thereby achieving the effects of brightening, moisturizing, elasticity, protection, repair, anti-allergy and immunity regulation.

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

[0007] As for the use mentioned above, wherein the gardenia stamen extract is further filtered to obtain a clear filtrate; and the clear filtrate is freeze-dried to obtain the gardenia stamen 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 500 μg / mL.

[0010] As mentioned above, in the step of extracting the gardenia stamen tissue soaking liquid by continuous rapid oscillation with low temperature ultrasonic emission, the gardenia stamen 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 in the above-mentioned use, wherein the free radical is a DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical, the composition has the activity of inhibiting the inflammatory factor nitric oxide to achieve the inhibition of inflammatory response.

[0012] As mentioned above, the composition has the performance of reducing the UVB photoproduct cyclobutane pyrimidine dimer (CPD) to protect and repair damaged skin cells.

[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] or

[0020]

[0021] The gardenia stamen tissue provided by the present invention is a gardenia stamen 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 content of cyclopentadiene ether terpenoid compounds in the gardenia stamen tissue extract is high, so the gardenia stamen tissue extract can be effectively used to prepare a composition for antioxidant, anti-inflammatory, photoproduct inhibition, protection, improving mitochondrial quality, promoting wound healing, anti-allergy, regulating immune function, regulating moisturizing and anti-aging factors, and promoting cell repair factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a test chart of the present invention using a fluorescence microscope to observe the fluorescence performance of a sample.

[0023] Figure 2 This is a test chart of the supercoiled form (SC), linear form (LC) and open circular form (OC) of pUC119 DNA taken with a cold light camera system and analyzed with computer software according to the present invention.

[0024] Figure 3 The photographs of the present invention were obtained by observing the changes of cell intervals in 48 hours using a microscope.

[0025] Figure 4 This is a test diagram of the β-hexosaminidase activity after RBL-2H3 is sensitized with IgE and then DNP-BSA is added to induce cell degranulation.

[0026] Figure 5 The gene analysis test of the present invention is carried out to obtain the test chart for the expression of enhancing the moisturizing factor (Filaggrin), inhibiting the glycosylation aging factor (advanced glycation endproducts receptor, AGER) and promoting cell repair (excision repair cross complementing group 1, ERCC1). DETAILED DESCRIPTION

[0027] To achieve the above-mentioned purpose, one embodiment of the present invention is to provide a use of a gardenia stamen extract for preparing a composition for anti-oxidation, anti-inflammatory, photoproduct inhibition, protection, improvement of mitochondrial quality, promotion of wound healing, anti-allergy, regulation of immune function, regulation of moisturizing and anti-aging factors, and promotion of cell repair factors, wherein the composition is an effective dose of about 100 to 500 μg / mL of a gardenia stamen extract administered to a human cell line to scavenge free radicals, inhibit inflammatory factors, protect and repair damaged skin cells, avoid oxidative stress-induced cell damage, promote cell mitochondrial regeneration, promote skin wound healing, have anti-allergic and immune regulation effects, enhance the expression of moisturizing factors, inhibit the expression of glycation aging factors, and promote the expression of cell repair factors, thereby achieving the effects of brightening, moisturizing, elasticity, protection, repair, anti-allergy and immune regulation.

[0028] In this embodiment, the preparation method of the gardenia stamen extract includes: using liquid nitrogen at about -195.79°C (77K) to rapidly freeze and solidify at least the gardenia stamen tissue; grinding the gardenia stamen tissue with a freezing grinder to grind the gardenia stamen tissue into powder; covering and soaking the gardenia stamen tissue with pure water or ethanol solvent to form a gardenia stamen tissue soaking liquid; using a low-temperature ultrasonic emission continuous rapid oscillation extraction device to continuously and rapidly oscillate the gardenia stamen tissue soaking liquid at a low temperature with a total energy of 500-800w, shaking for 5 minutes, resting for 2 minutes, and the continuous oscillation time is between 60 minutes and 120 minutes, and the continuous oscillation time is between 60 minutes and 120 minutes by the low-temperature ultrasonic emission continuous rapid oscillation extraction device. The invention relates to a method for extracting the gardenia tissue soaking liquid by high-speed oscillation to obtain a gardenia tissue extract; treating the gardenia stamen tissue extract by high-speed centrifugation at a centrifugal force of 15,000 g for 10 minutes at a low temperature (4° C.) to remove the sediment at the bottom of the gardenia stamen tissue extract and collect the gardenia stamen extract at the top of the gardenia stamen tissue extract; filtering the supernatant 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 stamen tissue water extract or a gardenia stamen tissue wine extract.

[0029] The following specific examples further illustrate the practical application scope of the present invention, but are not intended to limit the scope of the present invention in any form.

[0030] Experiment 1: Free radical scavenging ability test

[0031] This experiment explored the antioxidant and free radical scavenging effects of the water extract (GJSW) and wine extract (GJSE) of the tissue extract of Gardenia jasminoides. The antioxidant and free radical scavenging abilities of the free radical DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) were tested. DPPH is a stable free radical, dark purple prismatic crystals, with a melting point of 127-129°C (decomposition). It can be used as an analytical reagent for the brightness determination of tocopherols, aliphatic thiols, and reducing substances, and is also a commonly used inhibitor. The dark purple DPPH ethanol solution has a specific absorbance value at a wavelength of 517nm. If the DPPH free radical reacts with the sample, its absorbance value will decrease. The sample's ability to scavenge DPPH free radicals can be judged by the change in absorbance value. The lower the absorbance value, the stronger the sample's ability to scavenge DPPH free radicals. The sample extracts were prepared into different concentrations, and 90 μL of freshly prepared 100 μM DPPH ethanol solution was added to the 96-well plate for reaction, and the absorbance at 517 nm was detected by enzyme immunoassay. The control group was vitamin C.

[0032] The test results are shown in Table 1. The free radical scavenging abilities of the water extract (GJSW) and wine extract (GJSE) (50, 100, 500, 1000 μg / mL) of Gardenia jasminoides tissue were 35.2%, 44.8%, 75.5% and 92.1%; 16.3%, 30.9%, 67.0% and 83.2%, respectively. The free radical scavenging ability of the control group vitamin C (20 μg / mL) was 94.4%, and the free radical scavenging ability of the water extract (GJSW) was better than that of the wine extract (GJSE). The results showed that the extract of Gardenia jasminoides tissue had a good ability to scavenge free radicals.

[0033]

[0034] Table 1

[0035] Experiment 2: Anti-inflammatory test

[0036] Nitric oxide (NO.) is one of the substances that mainly participate in inflammatory reactions. This experiment tested the anti-inflammatory and inhibitory activity of nitric oxide on the water extract (GJSW) and wine extract (GJSE) of gardenia stamen tissue extract. Sodium nitroprusside (SNP) itself is a type of NO donor. It reacts with oxygen to form nitrite. Nitrite reacts with Griess reagent to form 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 stamen extract (GJS) and add 98μL of 25

[0037] mM SNP was reacted for 120 mins, then 100 μL Griess reagent was added for 10 mins, and the absorbance at 546 nm was measured by enzyme immunoassay. The control group was rutin.

[0038] The test results are shown in Table 2. The water extract (GJSW) and wine extract (GJSE) of Gardenia jasminoides (100,

[0039] 500, 1000 μg / mL) had an inhibitory effect on the inflammatory factor nitric oxide by 47.9%, 49.6% and

[0040] 51.4%; 38.9%, 41.9% and 44.6%, while the ability of rutin (500μg / mL) in the control group to inhibit the activity of nitric oxide, a inflammatory factor, was 54.5%, and the ability of the water extract (GJSW) to inhibit the production of nitric oxide was also better than that of the wine extract (GJSE). The results showed that the extract of Gardenia stamen tissue can achieve the activity of inhibiting inflammatory response by inhibiting the activity of NO, and has an anti-inflammatory effect.

[0041]

[0042] Table 2

[0043] Experiment 3: Cell safety test

[0044] This experiment investigates the safety assessment and cell viability test of water extract (GJSW) and wine extract (GJSE) 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 the cell cryopreservation solution (cell culture medium containing 10% DMSO) was added to the cell culture medium. The cells were broken up and transferred to a 25cm2 cell culture flask and grown in a 37°C, 5% CO2 cell culture incubator. The culture medium was changed every 2 days on average. Human skin keratinocytes and human skin fibroblasts (HaCaT and Hs68 cells) were cultured in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for at least 24 hours. To evaluate whether Gardenia jasminoides extract (GJS) affects cell viability in skin cells: different concentrations of Gardenia jasminoides extract (GJS) were added and exposed for a specified time. 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-diphenyltetrazolium bromide) solution was added for reaction. After 4 hours of reaction at 37°C and 5% CO2, 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).

[0045] The test results are shown in Table 3. After the human skin keratinocytes (HaCaT cells) and human skin fibroblasts (HaCaT and Hs68 cells) were treated with the water extract of gardenia stamen tissue (GJSW) (50, 100, 250, 500 μg / mL) for 24 hours, the cell viability of the human skin keratinocytes was 102.5%, 103.6%, 108.2% and 112.5% ​​respectively; the cell viability of the human skin fibroblasts was 101.1%, 101.5%, 102.5% and 105.5% respectively. From the above results, it can be seen that the water extract of gardenia stamen tissue is not toxic in skin cells, so it is safe and will not have adverse effects on the viability of skin cells.

[0046]

[0047] Table 3

[0048] Experiment 4: UVB photoproduct inhibition test

[0049] CPD (cyclobutane pyrimidine dimmer) is a photoproduct produced by DNA irradiated by UVB. Detecting the CPD content in DNA after UVB irradiation in cells can assess the degree of cell damage. This study evaluated whether the water extract and wine extract of Gardenia jasminoides tissue can reduce the expression of CPD to confirm whether it has the function of protecting and repairing damaged skin cells to achieve skin health. Human skin keratinocytes (HaCaT cells) (1.5×10 5 After culturing cells in a 24-well plate for 24 hours, samples were added and cultured for 4 hours. The supernatant was drained and UVB (50 mJ / cm 2 ) irradiation, then add serum-free culture medium containing samples to react in the incubator for 4 hours. After the reaction time, remove the supernatant, wash with PBS, add 500μL 4% formalin, and place at room temperature for 10 minutes for fixation. Then remove formalin, wash twice with PBS, add 500μL 0.5% triton-X100, react on ice for 5 minutes, remove, and wash twice with PBS. Add 1mL 2M HCl at room temperature for 30 minutes to denature the DNA in the cells, and then wash five times with PBS. Then add 1mL 20% FBS at 37℃ for 30 minutes for blocking to prevent non-specific protein binding. After the time, wash five times with PBS. Add 70μL CPD (1:100) and shake evenly at 37℃ for 30 minutes, and then wash five times with PBS. Finally, add an equal amount of PBS and measure its absorbance with an enzyme immunoassay at Excitation: 530nm, Emission: 630nm. Then, 70 μL of Hoechst 33342 staining solution (10 μg / mL) at a ratio of 1:250 was added and shaking was continued for 5 minutes. The cells were immediately washed with PBS for 5 times and the fluorescence performance was observed using a fluorescence microscope.

[0050] The test results are shown in Table 4 and Figure 1 As shown, CPD is a photochemical product. The test was conducted using the characteristics of fluorescence detection. After UVB irradiation, the cells were damaged and fragmented, and photochemical products accumulated. The CPD induction was 152.3%. After the skin cells were treated with 100μg / mL of Gardenia jasminoides tissue water extract (GJSW) and then irradiated with UVB, the photochemical product CPD induction was 79.3%. The results show that the Gardenia jasminoides extract (GJS) can reduce the performance of UVB photoproduct CPD, and has the function of inhibiting photoproducts by 73.0%, so it can protect and repair damaged skin cells, thereby achieving skin photohealth function.

[0051]

[0052] Table 4

[0053] Experiment 5: DNA protection test

[0054] 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, then pour it into a horizontal gel-making mold, 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.

[0055] The test results are shown in Table 5 and Figure 2 As shown in the results, the intact DNA presents a supercoiled structure, but under the influence of 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. The DNA treated with Gardenia jasminoides water extract (GJSW) (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 treatment is 1.9%, and the SC of DNA treated with Gardenia jasminoides water extract (GJSW) (100 and 500μg / mL) is 98.5% and 98.0%, respectively. The results show that Gardenia extract has the ability to protect DNA from DNA damage caused by oxidative stress.

[0056]

[0057] Table 5

[0058] Experiment 6: Promoting mitochondrial regeneration test

[0059] Mitochondria are the power plants of animal and human cells. Each tissue cell has hundreds to thousands of mitochondria, which are organelles responsible for performing many important biochemical functions. They mainly carry out energy metabolism pathways and supply more than 90% of the cell's ATP energy needs. Mitochondria are also involved in the synthesis of pyrimidine nucleotides, the regulation of intracellular calcium ion homeostasis, and the control of cell life and death through the translocation of apoptotic molecules. Mitochondria gradually decrease with age, so mitochondrial mass is closely related to health and aging. To evaluate the ability of Gardenia jasminoides tissue extract (GJS) to promote mitochondrial regeneration. Human skin keratinocytes (HaCaT cells) (1×10 4 / well) were cultured in a 24-well plate and incubated at 37°C and 5% CO2 for at least 24 hours. Samples of different concentrations were added and placed in the incubator for 24 hours. After the reaction time, the culture medium and samples were removed, PBS was added for washing, paraforaldehyde was added to fix the cells, 1% Triton X100 was used for 5 minutes, and then PBS was used to wash the cells, mitoview dye was added, and Hoechst 33342 staining solution was used to stain the cell nucleus (quantitative cell number), and the fluorescence immunoassay was performed using a fluorescent immunoassay analyzer (BioTek, Synergy TM 2, USA) were used to measure the expression of mitochondria (green) (Excitation: 490nm, Emission: 523nm) and nuclear fluorescence (blue) (Excitation: 346nm, Emission: 460nm). The formula for calculating mitochondrial expression was: Mitoview intensity / Hoechst33342intensity×100.

[0060] The test results are shown in Table 6. After the human skin keratinocytes (HaCaT cells) were treated with Gardenia jasminoides stamens tissue water extract (GJSW) (100, 250, 500 μg / mL) for 24 hours, the mitochondrial mass was 109.1%, 110.2% and 118.6% respectively. The results show that Gardenia jasminoides stamens tissue extract (GJS) can promote the mitochondrial mass of human skin cells and promote mitochondrial regeneration function.

[0061]

[0062] Table 6

[0063] Experiment 7: Cell wound healing ability test

[0064] The wound healing test was used to evaluate whether Gardenia jasminoides extract (GJS) has the ability to promote cell repair and skin wound healing. The Culture-Insert cell spacer frame was placed in a 24-well plate to create a uniform cell gap (500±50μm). 3×10 5 Cells with the same cell number of cell / mL were cultured in inserts and cultured in a 37°C and 5% CO2 incubator for at least 24 hours. After the inserts were removed and different concentrations of Gardenia jasminoides tissue extract (GJS) were added for reaction, the changes were observed under a microscope for 48 hours and photographed, and finally quantitative analysis was performed. This was to evaluate whether the addition of Gardenia jasminoides tissue extract (GJS) could promote cell growth and reduce the cell gap. The smaller the cell gap, the more it promotes cell growth.

[0065] The test results are shown in Table 7 and Figure 3 As shown, after 48 hours of cell culture, when the control group (control) was 100.0%, after adding Gardenia jasminoides tissue water extract (GJSW) (100, 250, 500, 1000 μg / mL), the cell spacing became smaller, and the wound healing efficiency was 108.2%, 116.1% and 128.7%. The results show that the Gardenia jasminoides tissue extract has the ability to promote cell growth and reduce the cell spacing, which is speculated to have the ability to promote wound healing.

[0066]

[0067] Table 7

[0068] Experiment 8: Anti-allergy test

[0069] 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. There are hundreds of allergens, including chemicals, ultraviolet rays, pollen, dust, foreign proteins, 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. Mast cell lines (RBL-2H3 cells) can release a large amount of β-glucuronidase (β-hexosaminidase) through degranulation when stimulated by IgE antibodies or calcium ion carriers. The amount released can be used to determine whether the Gardenia jasminoides stamen water extract (GJSW) has the potential to inhibit allergic reactions. Mast cells (2×10 4 cells / well) were cultured in 96-well plates with 10% FBS / DMEM, and pretreated with Gardenia jasminoides tissue water extract (GJSW) prepared in 0.5% FBS / DMEM for 24 hours, followed by the addition of anti-DNP IgE Ab (1 μg / mL) and incubation for 6-12 hours to sensitize mast cells. After washing away the supernatant, DNP-BSA (100 ng / mL) was added and incubated for 1 hour to activate mast cells. The supernatant was collected and frozen for storage. An equal volume of supernatant and p-NAG substrate (1 μM) were added to the 96-well plate, and the absorbance at 405 nm was measured after incubation at 37°C for 1 hour (BioTek, Synergy TM 2, USA).

[0070] The test results are shown in Table 8: Calicimycin A23187 (1 μM) was used to induce allergic reactions, and the inhibition rate of β-hexosaminidase release was 0.0%. After mast cells were treated with Gardenia jasminoides tissue water extract (GJSW) (100, 250, 500 μg / mL), the β-hexosaminidase release was inhibited to 10.5%, 30.3% and 49.6%. The results showed that Gardenia jasminoides tissue water extract (GJSW) inhibited the release of β-hexosaminidase, had anti-allergic effects and immune regulation function.

[0071]

[0072] Table 8

[0073] In addition, 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 jasminoides tissue water extract (GJSW) 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 4 As shown in the figure, after RBL-2H3 was sensitized by IgE, the β-hexosaminidase activity was expressed after adding DNP-BSA to induce cell degranulation. The β-hexosaminidase activity of the group without Gardenia jasminoides extract (GJSW) was 55.4%, while the addition of 100, 250 and 500 μg / mL GJSW decreased to 53.2%, 51.9% and 49.4% respectively. The results showed that Gardenia jasminoides extract (GJSW) has anti-allergic and immune-regulating functions.

[0075] Experiment 9: Regulating moisturizing, anti-aging factors, and promoting cell repair

[0076] Real-time quantitative qPCR was used to analyze the gene expression of Gardenia jasminoides tissue extract (GJS) in regulating the moisturizing factor Filaggrin, inhibiting the anti-glycation receptor AGER, and promoting cell repair ERCC1. Moisturizing factor Filaggrin: Natural moisturizing factor is mainly produced by the disintegration of keratin filaments, a protein present in the human epidermis, in the keratinocytes of the stratum corneum. Filaggrin is 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, compact shape with elasticity, helps the skin resist ultraviolet rays, adapts to changes in pH, but also retains water in the cells to maintain skin moisture. Anti-glycation, inhibiting AGER performance: AGE Receptor for advanced glycation endproducts (AGEs). Glycation reaction: Sugar and collagen will combine under certain conditions to generate AGEs irreversible proteins, and AGEs will destroy collagen and elastic fibers in the skin, making the skin fragile, yellow, and loose. In addition, sugar will also stimulate the production of androgens, leading to excessive secretion of sebaceous glands and causing acne. Cell repair ERCC1: nucleotide excision repair cross complementing group 1. It is an important member of the exonuclease repair family and a key member of the nucleotide excision repair (NER) system, participating 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.

[0077] Primer:

[0078] Filaggrin:5′-GCT GAA GGA ACT TCT GGA AAA GG-3′; 5′-GTTGTGGTCTATATCCAAGT ATC-3′

[0079] AGER:5′-GGCTGGTGTTCCCAATAAGG-3′; 5′-CTTGGCATTGGGACTGGACACT-3′

[0080] ERCC1:5′-ACC CCTCGACGAGGATGAG-3′;5′;-GGGAATTTGGCGACGTAATTC-3′

[0081] 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 extract (GJS) 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 of REzol was added TM C&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 and 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 incubate in 70μL for 2 minutes, quickly put on ice, then add 4μL 5×MMLV-RT buffer solution, 1μL 10mM Each dNTP, 0.5μL RNase inhibitor and 1μL MMLV RTase, incubate at 42℃ for 1 hour, then incubate at 94℃ for 5 minutes, then 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 the cycle threshold (CT) (ΔΔCT) method to interpret the data, 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. 10μL cDNA was taken into a microcentrifuge tube, 5μL 10× reaction buffer, 2μL 25mM MgCl2, 0.8μL 10mM dNTP and 1μL each of 50μM upstream and downstream primers, Taq DNA polymerase (5unit / μL) were added, and finally deionized water was added to make the total volume 50μL, and the PCR reaction was carried out in an automatic temperature cycler (MyGo Pro real-time PCR).

[0082] The test results are as follows Figure 4 As shown, the results of the gene analysis experiment showed that compared with the control group, the water extract of Gardenia jasminoides tissue (GJSW) can effectively regulate the expression of moisturizing factor Filaggrin, regulate the expression of glycosylation aging factor AGER, and promote the expression of cell repair ERCC1.

[0083] Experiment 10: Active ingredient composition analysis and calibration

[0084] The Gardenia stamen tissue extract was made into a concentration of 50ppm and then put into LC-MS for data analysis. Chromatographic fingerprint of Gardenia stamen tissue extract: In the calibration of the active ingredients of Gardenia stamen 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.

[0085] Signal 1 is Deacetylasperulosidic acid (molecular formula: 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:

[0086]

[0087] 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:

[0088]

[0090] 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:

[0091]

[0092] 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:

[0093]

[0095] 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:

[0096]

[0097] 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:

[0098]

[0099] 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):

[0100]

[0101]

[0102] 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 petals and gardenia fruits to extract the same low-temperature extraction method as the gardenia stamen tissue extract to obtain gardenia flower water extract (GJW) and gardenia fruit water extract (GJFW), and performed a tyrosinase activity inhibition test. Tyrosinase is an important enzyme in the melanin production pathway. The tyrosinase activity test can be used to evaluate the whitening effect of the sample. Take 2μL of the sample to a 96-well plate, add 110μL phosphate buffer (pH6.5) to mix, then add 5μL 50unit mushroom tyrosinase, and react at room temperature in the dark for 10 minutes. Then add 83μL 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, SynergyTM2, USA). Tyrosinase activity inhibition rate calculation method (Inhibition of tyrosinase activity%): [1-(OD sample -OD sample background ) / (OD control -OD control background) ]*100%. The control group was vitamin C.

[0103] The experimental results are shown in Table 9 below. From the results shown in Table 9, it can be seen that the ability of the Gardenia flower water extract (concentrations of 100, 500, 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, 1000 μg / mL) to inhibit tyrosinase activity is 15.2%, 14.1%, 16.9%. The ability of the control group of vitamin C (500 μg / mL) to inhibit tyrosinase activity is 95.8%. The results show that the ability of the Gardenia flower water extract to inhibit tyrosinase activity is significantly better than that of the Gardenia fruit water extract.

[0104]

[0105] Table 9

[0106] Furthermore, this experiment used gardenia petals and gardenia fruits to extract with the same low-temperature extraction method as the gardenia stamen tissue extract to obtain gardenia flower water extract (GJW) and gardenia fruit water extract (GJFW), and carried out a moisturizing test, wherein the moisturizing test was to prepare the gardenia flower water extract (GJW) and gardenia fruit water extract (GJFW) into 1% aqueous solutions, place them in a closed hot air oven, evaluate the water evaporation of the samples, and thereby evaluate the moisturizing effect of the samples. 3.0g of GJ was placed in a porcelain crucible and dried in a hot air oven at 75-80℃. The initial weight and the final weight after 2 hours of action were measured. The water content of the gardenia flower extract was determined using a formula to evaluate the moisturizing effect of the gardenia flower extract. The positive control group was hyaluronic acid (1%); the negative control group was pure water. Moisturizing efficacy (%)=[(initial weight of filled crucible)-(final weight of filled crucible)] / [(initial weight of filled crucible)-(initial weight of empty crucible)]×100.

[0107] The test results are shown in Table 10. From the results shown in Table 10, it can be seen that the moisturizing rate (%) of the water extract of gardenia jasminoides is 100.0% and 73.5% at 0 and 2 hours respectively. The water extract of gardenia jasminoides is 100.0% and 7.1% at 0 and 2 hours respectively. The results show that the water extract of gardenia jasminoides has a significantly better moisturizing effect than the water extract of gardenia jasminoides.

[0108]

[0109] Table 10

[0110] In summary, the present invention confirms that the water extract and wine extract of gardenia stamen tissue have the effects of anti-oxidation, anti-inflammation, inhibition of photoproducts, protection, improvement of mitochondrial mass, promotion of wound healing, anti-allergy, regulation of immune function, regulation of moisturizing and anti-aging factors, and promotion of cell repair factors. It has the potential to be developed into a cosmetic material composition, skin care product, food additive or pharmaceutical composition that can achieve the effects of brightening, moisturizing, elasticity, protection, repair, anti-allergy and regulation of immunity, and can indeed achieve the expected use effect through the embodiments disclosed above. The gardenia stamen tissue provided by the present invention is a gardenia stamen 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 content of cyclopentadiene compounds in the gardenia stamen tissue extract is high, so the gardenia stamen tissue extract can be effectively used to prepare a composition for antioxidant, anti-inflammatory, photoproduct inhibition, protection, improving mitochondrial quality, promoting wound healing, anti-allergic, regulating immune function, regulating moisturizing and anti-aging factors, and promoting cell repair factors.

[0111] 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 based on the definition of the accompanying application claims.

Claims

1. A use of a gardenia stamen extract for preparing a composition for anti-oxidation, anti-inflammation, inhibition of photoproducts, protection, improvement of mitochondrial quality, promotion of wound healing, anti-allergy, regulation of immune function, regulation of moisturizing and anti-aging factors, and promotion of cell repair factors, characterized in that: The composition is prepared by administering an effective dose of gardenia stamen extract to a human cell line to remove free radicals, inhibit inflammatory factors, protect and repair damaged skin cells, avoid cell damage caused by oxidative stress, promote cell mitochondrial regeneration, promote skin wound healing, have anti-allergic and immune regulation effects, enhance the expression of moisturizing factors, inhibit the expression of glycation aging factors, and promote the expression of cell repair factors, thereby achieving the effects of brightening, moisturizing, elasticity, protection, repair, anti-allergy and immune regulation.

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

3. The use according to claim 2, characterized in that: The gardenia stamen extract is further filtered to obtain a clear filtrate; and the clear filtrate is freeze-dried to obtain a gardenia stamen 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 500 μg / mL.

6. The use according to claim 2, characterized in that: In the step of extracting the gardenia stamen tissue soaking liquid by continuous rapid oscillation with low temperature emission of ultrasonic waves, the gardenia stamen 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.

7. The use according to claim 1, characterized in that The free radical is DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical, the composition has the activity of inhibiting inflammatory factor nitric oxide to achieve the inhibition of inflammatory response.

8. The use according to claim 1, characterized in that The composition has the performance of reducing the UVB light product cyclobutane pyrimidine dimer (CPD) to protect and repair damaged skin cells.

9. The use according to claim 1, 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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