Terminalia chebula extract as well as preparation method and application thereof

Abstract

The invention provides a fructus chebulae extract as well as a preparation method and application thereof. Specifically, the medicine terminalia fruit extract is obtained through a reflux extraction method such as ethanol extraction, the medicine terminalia fruit extract has remarkable biological activity, can inhibit secretion of inflammatory factors, regulate expression of AKT1 and TRPV1 and / or inhibit loss of type I collagen, and the medicine terminalia fruit extract is safe, free of irritation and free of toxic and side effects. The compound can be used for developing daily or daily chemical products for resisting allergy, relieving, removing redness and easing pain of nerves. The preparation method of the fructus chebulae extract is simple, low in equipment requirement, low in cost and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of daily cosmetics, and in particular to a Terminalia chebula extract, a preparation method thereof and an application thereof. Background Art

[0002] Terminalia chebula (Terminalia chebula Retz.), also known as Terminalia chebula and Terminalia chebula fruit, is a deciduous tree of the genus Terminalia (Terminalia L.) in the Combretaceae family. Its fruit is a traditional medicine included in the Chinese Pharmacopoeia. It is native to India and Myanmar. Currently, its wild populations in China are primarily distributed in the low-temperature valleys of the Nujiang River basin in southwestern Yunnan, at altitudes of 800-1100 meters.

[0003] Terminalia chebula is widely used as a crude drug in various traditional medicinal systems. Reported chemical constituents include tannins, phenolic acids, triterpenes, flavonoids, aliphatic compounds, and other bioactive compounds. In recent years, in-depth research on terminalia chebula has revealed its antioxidant, antitumor, antiviral, antibacterial, and neuroprotective properties.

[0004] Existing processes for preparing Terminalia chebula extracts primarily involve simple extraction and purification. For example, CN102526136B discloses an antioxidant active ingredient from Terminalia chebula and a method for preparing the same. The antioxidant active ingredient is extracted using a solvent, then extracted with an organic solvent, and then freeze-dried after purification. The antioxidant is then used in oral foods, health supplements, and pharmaceuticals. There is an urgent need to develop a new Terminalia chebula extract, its preparation method, and its application. Summary of the Invention

[0005] The invention aims to provide a novel Terminalia chebula extract and a preparation method and application thereof.

[0006] In a first aspect of the present invention, a use of a Terminalia chebula extract is provided for preparing a product having efficacy selected from the group consisting of:

[0007] (a) Inhibit inflammatory factors;

[0008] (b) upregulate AKT1 expression;

[0009] (c) downregulation of TRPV1 expression;

[0010] (d) Inhibit the loss of type I collagen.

[0011] In another preferred embodiment, the gallic glucoside content in the Terminalia chebula extract is ≥50 wt%.

[0012] In another preferred embodiment, the gallic glucoside content in the Terminalia chebula extract is ≥65 wt%.

[0013] In another preferred example, the up-regulation of AKT1 expression has one or more characteristics selected from the following group:

[0014] (1) Up-regulating the expression of the AKT1 gene in cells;

[0015] (2) Increasing the p-AKT1 signal by up-regulating the expression of the AKT1 protein in cells.

[0016] In another preferred example, the up-regulation rate of the AKT1 gene expression in human skin fibroblasts HSF by the Terminalia chebula extract is 60-150%, preferably 120-140%.

[0017] In another preferred example, the up-regulation rate of the AKT1 gene expression in mouse monocyte-macrophage leukemia cells RAW264.7 by the Terminalia chebula extract is 100-600%, preferably 400-500%.

[0018] In another preferred example, when at a high concentration such as 6.25 μg / mL, the Terminalia chebula extract up-regulates the expression of the AKT1 gene in human skin fibroblasts HSF by 100-150%, preferably 120-140%.

[0019] In another preferred example, when at a high concentration such as 12.5 μg / mL, the Terminalia chebula extract up-regulates the expression of the AKT1 gene in mouse monocyte-macrophage leukemia cells RAW264.7 by 300-600%, preferably 400-500%.

[0020] In another preferred example, the up-regulation ratio of the AKT1 protein expression in human skin fibroblasts HSF by the Terminalia chebula extract is 0.5-3 (relative to the negative control group), preferably 1-2 (relative to the negative control group).

[0021] In another preferred example, the up-regulation ratio of the p-AKT1 protein expression in human skin fibroblasts HSF by the Terminalia chebula extract is 1-5 (relative to the negative control group), preferably 1-2 (relative to the negative control group).

[0022] In another preferred example, when at a high concentration such as 6.25 μg / mL, the Terminalia chebula extract up-regulates the expression of the AKT1 protein in human skin fibroblasts HSF by a ratio of 1.5-3 (relative to the negative control group), preferably 1.6-2.4 (relative to the negative control group).

[0023] In another preferred example, when at a high concentration such as 6.25 μg / mL, the Terminalia chebula extract up-regulates the expression of the p-AKT1 protein in human skin fibroblasts HSF by a ratio of 1.5-3 (relative to the negative control group), preferably 1.6-2.4 (relative to the negative control group).

[0024] In another preferred example, the up-regulation ratio of the expression of AKT1 protein in mouse monocyte macrophage leukemia cell RAW264.7 by the Terminalia chebula extract is 1-6 (relative to the negative control group), preferably 1-4 (relative to the negative control group).

[0025] In another preferred example, the up-regulation ratio of the expression of p-AKT1 protein in mouse monocyte macrophage leukemia cell RAW264.7 by the Terminalia chebula extract is 1-5 (relative to the negative control group), preferably 1-3 (relative to the negative control group).

[0026] In another preferred example, when the concentration of the Terminalia chebula extract is high, such as 12.5 μg / mL, the up-regulation ratio of the expression of cell AKT1 protein in mouse monocyte macrophage leukemia cell RAW264.7 is 2-6 (relative to the negative control group), preferably 3-4 (relative to the negative control group).

[0027] In another preferred example, when the concentration of the Terminalia chebula extract is high, such as 12.5 μg / mL, the up-regulation ratio of the expression of cell p-AKT1 protein in mouse monocyte macrophage leukemia cell RAW264.7 is 1-4 (relative to the negative control group), preferably 2-3 (relative to the negative control group).

[0028] In another preferred example, the inflammatory factor is selected from the group consisting of: TNF-a, IL-6, or a combination thereof.

[0029] In another preferred example, the inflammatory factor is IL-6.

[0030] In another preferred example, when the concentration of the Terminalia chebula extract is low, such as 0.234 μg / mL, the inhibition rate of IL-6 is 50-70%, preferably 55-65%.

[0031] In another preferred example, when the concentration of the Terminalia chebula extract is high, such as 0.938 μg / mL, the inhibition rate of TNF-a is 15-20%, preferably 16-18%.

[0032] In another preferred example, the inhibition rate of the expression level of TRPV1 by the Terminalia chebula extract is 10-60%.

[0033] In another preferred example, when the concentration of the Terminalia chebula extract is high, such as 0.078 mg / mL, the inhibition rate of the expression level of TRPV1 is 45-60%.

[0034] In another preferred example, the Terminalia chebula extract is obtained by extraction with 50-70 (v / v)% ethanol.

[0035] In another preferred embodiment, the method for preparing the Terminalia chebula extract comprises the steps of:

[0036] Weigh an appropriate amount of Terminalia chebula, add ethanol at 50 - 70 (v / v)%, and reflux for extraction at 70 - 80 °C;

[0037] Filter the obtained extract to get a filtrate, add ethanol at 50 - 70 (v / v)% to the remaining filter residue, and reflux for extraction at 70 - 80 °C;

[0038] Combine the filtrates and concentrate under reduced pressure to obtain an extract, thus obtaining the Terminalia chebula extract.

[0039] In another preferred embodiment, the method for preparing the Terminalia chebula extract comprises the steps of: Weigh an appropriate amount of Terminalia chebula, add 5 - 12 times the amount of raw material of ethanol at 50 - 70 (v / v)%; reflux for extraction at 70 - 80 °C for 1.0 - 3.0 hours; filter the obtained extract to get a filtrate; for the remaining filter residue, add 5 - 12 times the amount of raw material of ethanol at 5 ...

[0040] In another preferred embodiment, the product further has one or more features selected from the following group:

[0041] (1) No skin irritation;

[0042] (2) Anti - inflammatory;

[0043] (3) Anti - allergic, soothing and analgesic; <s

[0044] (4) Reducing redness.

[0045] In another preferred embodiment, the dosage of the product containing the Terminalia chebula extract preparation is 0.05 - 99 wt%.

[0046] In the second aspect of the present invention, a method for up - regulating AKT1 expression in vitro is provided, and the method comprises:

[0047] Cultivate the cells under the condition of adding the Terminalia chebula extract as described in the first aspect of the present invention, wherein the concentration of the Terminalia chebula extract is 1 - 20 μg / mL.

[0048] In another preferred embodiment, the cells are selected from the following group: human skin fibroblasts HSF, mouse mononuclear macrophage leukemia cells RAW264.7.

[0049] In another preferred embodiment, the up - regulation of AKT1 expression has one or more features selected from the following group:

[0050] (1) Up - regulating the expression of the AKT1 gene in cells;

[0051] (2) Up-regulate the expression of AKT1 protein in cells;

[0052] (3) Increase the p-AKT1 signal by up-regulating the expression of AKT1 protein in cells.

[0053] In another aspect of the present invention, a method for inhibiting the content of TRPV1 in vitro is also provided. The method includes:

[0054] Culturing cells under the condition of adding the Terminalia Chebula extract as described above, wherein the concentration of the Terminalia Chebula extract is 1-20 μg / mL.

[0055] In another preferred example, the cells are selected from the following group: human skin fibroblasts HSF, mouse mononuclear macrophage leukemia cells RAW264.7.

[0056] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Results of the in vitro skin irritation test of Terminalia Chebula extract (abbreviated as TCE).

[0058] Figure 2 Experimental results of the inhibition of TNF-α secretion by Terminalia Chebula extract TCE. "*" indicates a significant difference compared with the control group, "***": p < 0.001.

[0059] Figure 3 Experimental results of the inhibition of IL-6 secretion by Terminalia Chebula extract TCE. "*" indicates a significant difference compared with the control group, "***": p < 0.001.

[0060] Figure 4 Effect of Terminalia Chebula extract TCE on the expression of AKT1 protein in human skin fibroblasts HSF (using β-actin as an internal reference). "*" indicates a significant difference compared with the negative control group, "****": p < 0.0001.

[0061] Figure 5 Effect of Terminalia Chebula extract TCE on the expression of p-AKT1 protein in human skin fibroblasts HSF (using β-actin as an internal reference). "*" indicates a significant difference compared with the negative control group, "****": p < 0.0001.

[0062] Figure 6 Effect of Terminalia chebula extract TCE on the expression of cell AKT1 protein in mouse monocyte macrophage leukemia cell RAW264.7 (using β-actin as an internal reference). "*" indicates significant difference compared with the negative control group, "****": p < 0.0001.

[0063] Figure 7 Effect of Terminalia chebula extract TCE on the expression of cell p-AKT1 protein in mouse monocyte macrophage leukemia cell RAW264.7 (using β-actin as an internal reference). "*" indicates significant difference compared with the negative control group, "****": p < 0.0001.

[0064] Figure 8 Experimental results of the inhibitory rate of Terminalia chebula extract TCE on TRPV1. "*" indicates significant difference compared with the negative control group, "****": p < 0.0005.

[0065] Figure 9 Experimental results of the effect of Terminalia chebula extract on type I collagen (blue fluorescence is nuclear staining, green fluorescence is type I collagen staining).

[0066] Figure 10 Experimental results of the effect of Terminalia chebula extract TCE on type I collagen.

[0067] Figure 11 Photo of Subject No. 14 in Efficacy Example 5.

[0068] Figure 12 Red area map of Subject No. 14 in Efficacy Example 5. Detailed implementation manner

[0069] Through extensive and in-depth research, and through a large number of screenings and tests, the present inventor provides a Terminalia chebula extract and its preparation and application. The Terminalia chebula extract has significant biological activity, can inhibit the secretion of inflammatory factors, regulate the expression of AKT1 and TRPV1, and / or inhibit the loss of type I collagen. The Terminalia chebula extract is safe and non-irritating, and can be used to develop daily or daily chemical products with anti-allergy and soothing, anti-glycation, anti-aging and nerve analgesia effects. And the preparation method of the Terminalia chebula extract is simple, has low equipment requirements and low cost, and is suitable for industrial production. On this basis, the present invention is completed.

[0070] Terms

[0071] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0072] As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0073] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of", or "consisting of".

[0074] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4 - 40 °C, preferably, 25 ± 5 °C.

[0075] Preparation of Terminalia chebula Retz. extract

[0076] The Terminalia chebula Retz. extract of the present invention can be prepared using conventional methods. Preferably, the method includes the steps of:

[0077] Weigh an appropriate amount of Terminalia chebula Retz., add 5 - 12 times the amount of the raw material of 50 - 70 (v / v)% ethanol; reflux and extract at 70 - 80 °C for 1.0 - 3.0 hours; naturally filter the obtained extract through a 300 - mesh sieve to obtain a filtrate; for the remaining filter residue, add 5 - 12 times the amount of the raw material of 50 - 70 (v / v)% ethanol, and reflux and extract at 70 - 80 °C for 1.0 - 3.0 hours. After combining the filtrates, concentrate under reduced pressure (50 °C - 70 °C, vacuum pressure 0.060 - 0.08 Mpa) into an extract, and concentrate to 0.5 - 2.2 times the feeding amount. Filter the above - concentrated liquid through a 0.45 μm membrane to obtain a filtrate.

[0078] Lyophilize the above - mentioned filtrate (pre - freezing: temperature - 40 °C to - 45 °C, time 60 - 90 min; sublimation drying: temperature - 30 °C to - 10 °C, vacuum degree 5 Pa to 20 Pa, time 8 - 12 h; desorption drying: temperature 35 °C to 40 °C, vacuum degree < 5 Pa, time 12 - 20 h; cold trap temperature: - 60 °C to - 50 °C). After pulverizing with a pulverizer and passing through an 80 - mesh sieve, obtain the freeze - dried powder of Terminalia chebula Retz. extract, or dry it into a powder to obtain the powder of Terminalia chebula Retz. extract.

[0079] The main advantages of the present invention include:

[0080] 1. The Terminalia chebula Retz. extract of the present invention is non - irritating when used transdermally.

[0081] 2. The Terminalia chebula Retz. extract of the present invention can significantly inhibit the expression of TRPV1 content.

[0082] 3. The Terminalia chebula Retz. extract of the present invention can significantly up - regulate the expression of AKT1.

[0083] 4. The Terminalia chebula extract of the present invention also has the efficacy of neural analgesia and can be applied to new daily chemical products.

[0084] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0085] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0086] Example 1

[0087] Weigh 10 g of Terminalia chebula, add 100 ml of 65% ethanol; reflux and extract at 80 °C for 2.0 hours; naturally filter the obtained extract through a 300-mesh sieve to obtain a filtrate; for the remaining filter residue, add 100 ml of 65% ethanol and reflux and extract at 80 °C for 2.0 hours. After combining the filtrates, concentrate under reduced pressure (50 °C - 70 °C, vacuum pressure 0.060 - 0.08 Mpa) to obtain an extract, and concentrate to 10 g. Filter the above concentrated solution through a 0.45 μm membrane to obtain a filtrate. Freeze-dry the above filtrate, and use gallic acid as a reference substance to test the content of galloglucoside components to be 75.12% (calculated based on the mass of the freeze-dried powder).

[0088] Example 2

[0089] Weigh 10 g of Terminalia chebula, add 100 mL of water; extract at 80 °C for 2.0 hours; naturally filter the obtained extract through a 300-mesh sieve to obtain a filtrate; for the remaining filter residue, add 100 ml of water and reflux and extract at 80 °C for 2.0 hours. After combining the filtrates, concentrate under reduced pressure (50 °C - 70 °C, vacuum pressure 0.060 - 0.08 Mpa) to obtain an extract, and concentrate to 10 g. Filter the above concentrated solution through a 0.45 μm membrane to obtain a filtrate. Freeze-dry the above filtrate, and use gallic acid as a reference substance to test the content of galloglucoside components to be 46.98% (calculated based on the mass of the freeze-dried powder).

[0090] Example 3

[0091] Weigh 10 g of Terminalia chebula, add 100 mL of 75% ethanol, extract at 80 °C for 2.0 hours, and naturally filter the obtained extract through a 300-mesh sieve to obtain a filtrate. For the remaining filter residue, add 100 mL of 75% ethanol and reflux extract at 80 °C for 2.0 hours. Combine the filtrates and concentrate under reduced pressure (50 °C - 70 °C, vacuum pressure 0.060 - 0.08 Mpa) to obtain an extract, and concentrate it to 10 g. Filter the above concentrated solution through a 0.45 μm membrane to obtain a filtrate. Freeze-dry the above filtrate, and use gallic acid as a reference substance to test the content of galloylglucosides, which is 50.12% (calculated based on the mass of the freeze-dried powder).

[0092] From the results of Examples 1 - 3, it can be seen that in the heating reflux extraction method, 65% ethanol is the optimal extraction solvent, and the highest content of galloylglucosides in the extract is 75.12%. When the ethanol concentration increases, the content of galloylglucosides in the extract will instead decrease to 50.12%. Increasing the polarity of the extraction solvent will also reduce the yield of galloylglucosides. For example, when water is used as the extraction solvent, the content of galloylglucosides in the extract decreases to 46.98%.

[0093] Safety implementation case 1

[0094] Evaluate the skin irritation of Terminalia chebula extract and encapsulated Terminalia chebula extract respectively, referring to OECD TG439 "In vitro skin irritation: Reconstructed human epidermis test method".

[0095] 1 Skin model preparation: Add the pre-warmed maintenance culture medium to a 12-well culture plate at 2 mL / well, transfer the skin model to the 12-well plate containing the maintenance culture medium, and place it in an incubator to culture overnight;

[0096] 2 Reaction of the test sample with MTT: Add 2 mL of 0.3 mg / mL MTT solution and 10 μL of the test sample (weigh 0.0220 g of the freeze-dried powder of Terminalia chebula extract, add 0.528 mL of DPBS buffer solution, mix well, and prepare a 4% concentration) to the 12-well plate, mix well, and incubate in an incubator for 3 h;

[0097] 3 Sample addition: Take 10 μL of the test sample (including NC and PC), evenly apply it on the surface of the skin model, after standing at room temperature for 15 min, thoroughly rinse the sample with DPBS until there is no residue, and then place it in a well containing fresh

[0098] maintenance culture medium and incubate for 42 h. Set 3 parallels for each group;

[0099] 4 MTT Reaction: Transfer the skin model to a well containing 2 mL of 0.3 mg / mL MTT solution and incubate in an incubator for 3 h. After the MTT reaction is completed, use a punch to remove the epidermal tissue, transfer the tissue to a 2 mL centrifuge tube, add 500 μL of isopropyl alcohol hydrochloride to each tube, mix well, and place in the dark at room temperature

[0100] overnight;

[0101] 5 Detection: Take 200 μL of the solution from each tube and place it in a 96-well plate. Use isopropyl alcohol hydrochloride as the blank control and measure the absorbance at a wavelength of 570 nm.

[0102] 6 Data Arrangement and Analysis

[0103] Using isopropyl alcohol hydrochloride as the blank control (Blank), the tissue activity of the negative control group (NC) is 100%. Calculate the relative tissue activities of the experimental group and the positive control group (PC):

[0104] Relative tissue activity (%) = [(OD 实验 / PC - OD Blank ) / (OD NC - OD Blank )] * 100%

[0105] OD 实验 : OD of the sample to be tested

[0106] OD PC : OD value of the positive control group

[0107] OD NC : OD value of the negative control group

[0108] OD Blank : OD value of the blank control (isopropyl alcohol hydrochloride) [[ID=4�]]

[0109] Classify skin irritation according to Table 1:

[0110] Table 1 Skin Irritation Classification

[0111]

[0112] 7 Experimental Results

[0113] The experimental results are as Figure 1 shown in and Table 2.

[0114] Table 2 Experimental Results

[0115]

[0116] According to OECD TG 439 (2021) "In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method", under the conditions of this test, the Terminalia chebula extract does not have skin irritation at a concentration of 4%, and the GHS classification is unclassified.

[0117] Effect Experiment of Terminalia chebula Extract on the Secretion of TNF-α and IL-6 by LPS-induced Macrophage Raw264.7 Cells in Efficacy Example 1

[0118] 1 Experimental Method

[0119] 1.1 Inoculation: When the cell density > 70%, discard the culture medium, add 5 mL of PBS to wash the cells, then add 4 mL of DMEM basal medium again. Use a pipette to blow and beat the cells until they are resuspended, transfer them to a 2 mL centrifuge tube, centrifuge at 1000 rpm for 4 minutes, and discard the supernatant; add 1 mL of complete medium again, blow and mix well, dilute by an appropriate multiple and count, dilute the cells to an appropriate concentration, and inoculate the cells into a 96-well plate at a volume of 200 μL per well, and place it back in the incubator for incubation (37 °C, 5% CO2).

[0120] 1.2 Experimental Grouping: Group according to Table 3, and set another 3 cell-free wells as the zero-adjustment group, and plate according to 4 replicates for each concentration in each group;

[0121] 1.3 Solution Preparation: According to the obtained cell viability test results, select an appropriate sample concentration as the test concentration and prepare the sample solution to be tested;

[0122] Table 3 Experimental Grouping

[0123]

[0124] 1.4 Administration: Take out the 96-well plate after 24 h, discard the old culture medium, add samples according to 4 replicates for each concentration in each group, add 180 μL of the corresponding concentration sample to each well, and then place it back in the incubator for culture (37 °C, 5% CO2); take out the 96-well plate after 1 h, add 20 μL of basal medium to each well in the zero-adjustment well and the blank control group (BC), and add 20 μL of LPS (1 μg / mL) to each well in the negative control group (NC), positive control group (PC) and experimental group, and then place it back in the incubator for incubation.

[0125] 1.5 Collection of Cell Supernatant: Take out the 96-well plate after administration for 24 h, collect the corresponding cell supernatant of each sample group into a centrifuge tube, centrifuge at 1000 rpm for 10 min, collect the supernatant and place it in a 1.5 mL centrifuge tube, and store it at -20 °C for standby.

[0126] 1.6 Determination of the Concentrations of TNF-α and IL-6 in the Cell Supernatant by ELISA Kit

[0127] 2 Experimental Results

[0128] The experimental results are as follows Figure 2 and Figure 3 shown, indicating that the Terminalia chebula Retz. extract has the efficacy of inhibiting the secretion of TNF-α and IL-6. Among them, the inhibitory effect on IL-6 is better. At a low concentration (0.234 μg / mL), the inhibition rate is about 60%.

[0129] Example of Efficacy 2: Experiment on the Effect of Terminalia chebula Retz. Extract on AKT1

[0130] 1. Cell Viability Detection Test

[0131] 1.1 Detection Method:

[0132] Refer to "GB / T 16886.5-2017 In Vitro Cytotoxicity Test" for experimental design: Take out the cells with a confluence of 60% from the incubator, remove the original culture medium, and add 100 μL of blank control solution and Terminalia chebula Retz. extract samples with different concentrations (specific concentrations are shown in Table 4) respectively. After adding the samples, place the cells in the incubator and continue to culture for a specified time. After the culture is completed, observe the cell morphology and detect it using MTT dye at a wavelength of 570 nm in an enzyme-labeling instrument, and set 650 nm as the reference wavelength.

[0133] Cell viability (%) = OD 570e / OD 570b × 100%;

[0134] OD 570e : The average absorbance value of the test sample or control after blank correction;

[0135] OD 570b : The average absorbance value of the blank control after blank correction.

[0136] 1.2 Detection Results

[0137] Under normal culture conditions, the effects of Terminalia chebula Retz. extract on the cell viability of human skin fibroblasts HSF and mouse mononuclear macrophage leukemia cells RAW264.7 are shown in Table 4.

[0138] Table 4 Effects of Samples on the Cell Viability of Human Skin Fibroblasts HSF and Mouse Mononuclear Macrophage Leukemia Cells RAW264.7

[0139]

[0140] The experimental results show that the Terminalia chebula Retz. extract has high safety, and the cell viability is higher than 95% in the concentration range of 0.78125 μg / mL to 12.500 μg / mL.

[0141] 2. Detection of Gene Expression Levels of Terminalia chebula Extract in Human Skin Fibroblasts HSF and Mouse Mononuclear Macrophage Leukemia Cells RAW264.7

[0142] 2.1 Preparation of test samples:

[0143] 2.1.1 Experimental controls: Blank control, resveratrol, PP1 inhibitor, UVA stimulation (5 J / cm 2 , perpendicular to the cell plate, UVA is 4 cm away from the cell plate, and the irradiation intensity is 1400 μw / cm2), LPS (5 μg / mL).

[0144] 2.1.2 Sample settings:

[0145] For human skin fibroblasts HSF: Sample group 1: 6.2500 μg / mL Terminalia chebula extract, sample group 2: 3.1250 μg / mL Terminalia chebula extract; sample group 3: 1.5626 μg / mL Terminalia chebula extract.

[0146] For mouse mononuclear macrophage leukemia cells RAW264.7: Sample group 1: 12.500 μg / mL Terminalia chebula extract, sample group 2: 6.2500 μg / mL Terminalia chebula extract; sample group 3: 3.1250 μg / mL Terminalia chebula extract

[0147] 2.2 Detection methods

[0148] Digest the cells in the logarithmic growth phase and prepare them into a density of 2.0×10 5 cells / mL and inoculate them into 6-well plates, 2 mL per well. After about 24 h until the cells grow into a monolayer, human skin fibroblasts are first stimulated with UVA (5 J / cm 2 , perpendicular to the cell plate, UVA is 4 cm away from the cell plate, and the irradiation intensity is 1400 μw / cm 2 ) for 60 min, and then different concentrations of sample solutions are added. At the same time, a blank group, a model group, and a positive control group (resveratrol 10 μM) are set, and the cells are treated for 48 h. After the treatment time ends, the cells are lysed for gene expression testing.

[0149] Mouse mononuclear macrophage leukemia cells are first stimulated with LPS (5 μg / mL) for 24 h, and then different concentrations of sample solutions are added. At the same time, a blank group, a model group, and a positive control group (PP1 inhibitor 1 nM) are set, and the cells are treated for 24 h. After the treatment time ends, the cells are lysed for gene expression testing.

[0150] 2.2.4. RNA extraction:

[0151] 2.2.5 Reverse transcription of cDNA: The extracted RNA is used according to the following steps with PrimeScript TMcDNA was synthesized using the RT reagent Kit (Perfect Real Time). The prepared reaction system was placed in a PCR instrument. The reverse transcription reaction conditions were as follows: incubated at 37 °C for 15 min, then maintained at 85 °C for 2 min, and finally stored at 4 °C. The synthesized cDNA was used for the next q-PCR experiment after the reaction was completed.

[0152] 2.2.6 Detection of gene expression levels: The reaction system was configured according to the requirements in the q-PCR detection kit, and gene amplification was performed in a fluorescence quantitative PCR instrument. The reaction settings were as follows: pre-denaturation at 95 °C for 30 s, 40 cycles of reaction at 95 °C for 15 s + 60 °C for 30 s, and finally the melting curve analysis reaction at 95 °C for 15 s + 60 °C for 30 s + 95 °C for 15 s. Finally, the 2 -ΔΔCt analysis method was used to detect the differences in gene expression among groups. (Note: The Ct value represents the cycle threshold of the sample, which means the number of cycles corresponding to when the fluorescence signal of the product amplification in the PCR process reaches the set threshold. The higher the concentration, the lower the Ct value appears. The 2 -ΔΔCt method gives the fold change in the expression of the target gene in the experimental group relative to the target gene in the control group.

[0153] 2.3 Detection results

[0154] Under normal culture conditions, the effects of Terminalia chebula extract on the expression of AKT1 gene in human skin fibroblasts HSF and mouse mononuclear macrophage leukemia cells RAW264.7 are shown in Table 5 and Table 6.

[0155] (1) In human skin fibroblasts HSF, compared with the negative control group, the expression of the AKT1 gene in cells treated with 6.2500 μg / mL Terminalia chebula extract was upregulated by  128.28%, the expression of the AKT1 gene in cells treated with 3.1250 μg / mL Terminalia chebula extract was upregulated by 77.18%, and the expression of the AKT1 gene in cells treated with 1.5626 μg / mL Terminalia chebula extract was upregulated by 85.57%.

[0156] (2) In mouse mononuclear macrophage leukemia cells RAW264.7, compared with the negative control group, the expression of the AKT1 gene in cells treated with 12.500 μg / mL Terminalia chebula extract was upregulated by 464.53%, the expression of the AKT1 gene in cells treated with 6.2500 μg / mL Terminalia chebula extract was upregulated by 167.76%, and the expression of the AKT1 gene in cells treated with 3.1250 μg / mL Terminalia chebula extract was upregulated by 154.39%.

[0157] Table 5 Effects of Terminalia chebula extract on the expression of AKT1 gene in human skin fibroblasts HSF

[0158] Experimental grouping AKT1 gene expression Up-regulation rate (%) Blank control group 1±0.02 - Negative control (UVA stimulation) group 0.65±0.05 - Positive control: 10 μM resveratrol 1.71±0.09 161.83 6.25 μg / ml Terminalia chebula extract 1.49±0.02 128.28 3.125 μg / ml Terminalia chebula extract 1.15±0.11 77.18 1.5626 μg / ml Terminalia chebula extract 1.21±0.18 85.57

[0159] Table 6 Effects of Terminalia chebula Retz. extract on the expression of AKT1 gene in mouse monocyte-macrophage leukemia cell line RAW264.7

[0160]

[0161]

[0162] 3. Effects of Terminalia chebula Retz. extract on the expression of AKT1 protein and p-AKT1 protein in human skin fibroblasts HSF and mouse monocyte-macrophage leukemia cell line RAW264.7

[0163] 3.1 Test method

[0164] 3.1.1. Digest the cells in the logarithmic growth phase and prepare a cell suspension with a density of 2.0×10 5 cells / mL. Inoculate 2 mL of the cell suspension into each well of a 6-well plate;

[0165] 3.1.2. Approximately 24 h after the cells grow into a monolayer, human skin fibroblasts are first stimulated with UVA (5 J / cm 2 , perpendicular to the cell plate, with the UVA source 4 cm away from the cell plate and an irradiation intensity of 1400 μW / cm 2 ) for 60 min. Then, different concentrations of the sample solution are added. At the same time, a blank group, a model group, and a positive control group (resveratrol 10 μM) are set. Incubate for 48 h, and then lyse the cells to perform protein expression tests after the incubation time ends.

[0166] 3.1.3. Mouse monocyte-macrophage leukemia cells are first stimulated with LPS (5 μg / mL) for 24 h. Then, different concentrations of the sample solution are added. At the same time, a blank group, a model group, and a positive control group (PP1 inhibitor 1 nM) are set. Incubate for 24 h, and then lyse the cells to perform protein expression tests after the incubation time ends.

[0167] 3.1.4. Protein extraction: Total protein extraction, membrane protein extraction, and nuclear protein extraction. Take 5 μL of the protein solution for protein concentration determination (BCA method). For the remaining samples, add 1 / 4 volume of 5×SDS gel loading buffer according to the volume, denature at 95°C in a metal bath for 5 min, aliquot, and store at -80°C in a refrigerator.

[0168] 3.1.5. Protein electrophoresis and membrane transfer: Place the electrophoresis tank in an ice bath. Electrophorese at a voltage of 70 V for 30 min to compress the loaded protein samples into a narrow band, and then change the voltage to 120 V for electrophoresis until the blue bromophenol blue runs to the bottom of the gel, which takes about 90 min. According to the principle of "black gel, white membrane", place the PVDF membrane in the membrane transfer clamp and use a rapid membrane transfer instrument for protein transfer.[[ID= thirty-five]] [[ID= thirty-six]]

[0169] 3.1.6. Blocking Incubation: Incubate for 1 h with shaking on a decolorizing shaker at room temperature. Select the incubation conditions and time according to the properties of the primary antibody. After incubation, recover the antibody working solution and wash 3 - 5 times with TBST on a decolorizing shaker at room temperature, 10 - 15 minutes each time. Select the incubation conditions and time according to the properties of the secondary antibody. After incubation, recover the antibody working solution and wash 3 - 5 times with TBST on a decolorizing shaker at room temperature, 10 - 15 minutes each time.

[0170] 3.1.7. Color Development and Exposure: Turn on the instrument to pre - cool, prepare the ECL luminescent solution, adjust the camera focus, take a white - background photo, add the ECL luminescent solution, adjust the photo - taking time node according to the signal strength, and save the photo.

[0171] 3.2 Detection Results:

[0172] Under normal culture conditions, the effects of Terminalia chebula extract on the expression of AKT1 protein and p - AKT1 protein in human skin fibroblasts HSF and mouse mononuclear macrophage leukemia cells RAW264.7 are as Figures 4 - 7 shown. The relative ratios are shown in Table 7 and Table 8.

[0173] Table 7 Effects of Terminalia chebula extract on the expression of AKT1 and p - AKT1 proteins in human skin fibroblasts HSF (using β - actin as an internal reference)

[0174]

[0175] Table 8 Effects of Terminalia chebula extract on the expression of AKT1 protein and p - AKT1 protein in mouse mononuclear macrophage leukemia cells RAW264.7 (using β - actin as an internal reference)

[0176]

[0177] "*" indicates a significant difference compared with the negative control group, "****": p < 0.0001.

[0178] (1) In human skin fibroblasts HSF, the relative ratio of the expression of cell AKT1 protein to the negative control group for Terminalia chebula extract at 6.2500 μg / mL was 1.88, showing a significant upregulation; the relative ratio of the expression of cell p-AKT1 protein to the negative control group was 1.81, showing a significant upregulation; the relative ratio of the expression of cell AKT1 protein to the negative control group for Terminalia chebula extract at 3.1250 μg / mL was 1.38, showing a significant upregulation; the relative ratio of the expression of cell p-AKT1 protein to the negative control group was 1.18, showing a significant upregulation; the relative ratio of the expression of cell AKT1 protein to the negative control group for Terminalia chebula extract at 1.5626 μg / mL was 0.99, showing no significant difference, and the relative ratio of the expression of cell p-AKT1 protein to the negative control group was 1.10, showing a significant upregulation.

[0179] (2) In mouse mononuclear macrophage leukemia cells RAW264.7, the relative ratio of the expression of cell AKT1 protein to the negative control group for Terminalia chebula extract at 12.5000 μg / mL was 3.23, showing a significant upregulation; the relative ratio of the expression of cell p-AKT1 protein to the negative control group was 2.21, showing a significant upregulation; the relative ratio of the expression of cell AKT1 protein to the negative control group for Terminalia chebula extract at 6.2500 μg / mL was 1.68, showing a significant upregulation; the relative ratio of the expression of cell p-AKT1 protein to the negative control group was 1.39, showing a significant upregulation; the relative ratio of the expression of cell AKT1 protein to the negative control group for Terminalia chebula extract at 3.1250 μg / mL was 1.19, showing no significant difference, and the relative ratio of the expression of cell p-AKT1 protein to the negative control group was 1.42, showing a significant upregulation.

[0180] Effect of Terminalia chebula extract on transient receptor potential vanilloid 1 (TRPV1) in Efficacy Example 3

[0181] 1 Experimental method:

[0182] 1.1 Cell viability assay: Collect cells in the logarithmic growth phase, adjust the cell density and inoculate them into a 96-well plate, with 100 μL of culture medium in each well. After culturing in a CO2 cell incubator for 24 h, aspirate the culture medium, wash the cells twice with PBS buffer, prepare the sample according to the sample usage method, and then group and administer the drug according to the final concentration prepared in Table 9.

[0183] Table 9 Concentration grouping

[0184]

[0185] 1.2 Administration: Discard the culture medium in each well of the microplate. Add normal culture medium to the blank well and negative control well, add capsazepine to the positive control well, and add samples at different concentrations in Table 9 to the sample groups. After adding the drugs, continue to culture in a CO2 cell incubator for 24 h, and then perform the MTT cell cytotoxicity test. Read the absorbance OD value at 490 nm. Calculate the cell survival rate. Only culture medium is added to the blank well. Select the sample concentration with cell viability ≥ 90% for subsequent efficacy detection.

[0186] 1.3 Detection: Adjust cells at a certain concentration (see Table 10 for details), re-seed them in a 96-well microplate. After 24 h ± 1 h, when the cells adhere to the wall, perform grouped drug administration and proceed to the next experiment. Set 3 replicate wells for each group.

[0187] Table 10 Experimental grouping

[0188]

[0189] 1.4 Sample collection: At the end of 24 h of culture, collect the supernatant for ELISA detection. Collect 150 μL of supernatant from each well into a 1.5 mL centrifuge tube, centrifuge at 10000 rPm for 10 min before ELISA detection, and collect the supernatant for the experiment.

[0190] 1.5 ELISA detection: Select TRPV1 for detection in this experiment. Perform the experiment according to the kit instructions.

[0191] 2 Data processing

[0192] 2.1 Cell viability (%) = OD490 of the sample group / OD490 of the negative control group * 100%

[0193] Note: Deduct the blank well from each group of data.

[0194] 2.2 Inhibition rate (%) = (1 - sample group / model group) * 100% [[ID=!28]]

[0195] 3 Experimental results

[0196] Table 11 TRPV1 inhibition rate results

[0197]

[0198] It can be seen from the above experimental results that at the concentrations of 0.078 mg / mL, 0.039 mg / mL, and 0.020 mg / mL, the inhibition rates of Terminalia chebula Retz. extract on TRPV1 are 50.35% ± 1.65%, 34.76% ± 1.51%, and 21.05% ± 0.47% respectively. Compared with the model group, P < 0.05, showing significant differences; indicating that Terminalia chebula Retz. extract has the effect of inhibiting the expression of TRPV1 content, with an inhibition rate of 20 - 55%, and has anti-allergic, soothing, and analgesic effects.

[0199] Effect Example 4: Influence of Terminalia chebula Retz. Extract on Type I Collagen

[0200] 1 Experimental Method

[0201] 1.1 Seeding: When the cell density ≥ 70%, collect the cells and count them, adjust the cell density to a certain concentration, and seed the cells into a 6-well plate at a volume of 2000 μL per well, then put it back into the incubator.

[0202] 1.2 Experimental Grouping: Plate according to 2 replicates per concentration in each group in Table 12; among them, the Terminalia chebula Retz. extract is 6.25 μg / mL (calculated as the lyophilized powder).

[0203] 1.3 Solution Preparation: According to the obtained cell viability test results, select an appropriate sample concentration as the test concentration and prepare the sample solution to be tested: Use vitamin E as the positive control for the experiment.

[0204] Table 12 Experimental Grouping

[0205]

[0206] 1.4 Drug Administration: After 24 h, take out the 6-well plate, discard the old medium, add samples according to 2 replicates per concentration in each group. Add 2000 μL of basal medium to each well of the blank control group (BC) and the negative control group (NC), add 200 μL of the positive control sample to each well of the positive control group, and add 200 μL of the corresponding concentration sample to each of the 2 replicates of each concentration in the experimental group, then put it back into the incubator for culture (37 °C, 5% CO2);

[0207] 1.5 UVA Irradiation: After 1 h, take out the 24-well plate, and perform UVA irradiation of 20 J / cm 2 on the negative control group (NC), the positive control group (PC) and the experimental group according to the experimental grouping. The blank control group (BC) is not irradiated. Incubate overnight in the incubator.

[0208] 1.6 Fluorescent Staining of Type I Collagen

[0209] 1.7 Data Processing and Analysis: Analyze the fluorescence expression intensity of type I collagen in each group using Image J, plot the graph using GraphPad Prism software, and perform statistical analysis using T-test between groups. # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference. ### P < 0.001 indicates an extremely significant difference.

[0210] 2 Experimental Results

[0211] The experimental results are shown in Figure 9 and Figure 10, compared with the UVA irradiation group, the Terminalia chebula extract had a significant inhibitory effect on the decrease of type I collagen in UVA-induced 3T3 fibroblasts. This indicates that under the experimental conditions, the Terminalia chebula extract has an inhibitory effect on the loss of type I collagen in the UVA-induced 3T3 fibroblast photoaging model and has an anti-wrinkle effect.

[0212] Efficacy Example 5 Human Clinical Experiment of Terminalia chebula Extract

[0213] In this experiment, laboratory technicians screened eligible subjects meeting the test requirements according to the test requirements and recorded the Fitzpatrick type and skin type (neutral, dry, oily, combination dry or combination oily) of the subjects' faces. By having adult subjects continuously use the essence cream sample containing Terminalia chebula extract for 56 days according to the instructions, it was evaluated whether the sample had effects such as moisturizing, repairing, soothing, etc., and whether the product was mild (non-irritating) and suitable for sensitive skin.

[0214] The total number of valid subjects completing the test was 30, including healthy Chinese men and women with sensitive skin (screened through the Baumann questionnaire, including medical aesthetic population), 3 men and 27 women, aged 36 to 60, with an average age of 52.83 ± 6.95 years, meeting the volunteer inclusion and exclusion criteria for subjects.

[0215] 1 Evaluation of the mildness of Terminalia chebula extract

[0216] Table 13 Experimental results

[0217]

[0218] Experimental results: It can be seen from the data in Table 13 that the measured values of various irritating effects of the essence cream containing Terminalia chebula extract changed very little during use, indicating high mildness.

[0219] 2 Evaluation of the redness reduction effect

[0220] During the experiment, the facial skin condition of the subjects was observed, and VISIA CR was used to analyze the facial images in combination with IPP for skin red area analysis. The photographed image of subject No. 14 is as Figure 11 shown, and the red area map is as Figure 12 shown.

[0221] Table 14 Experimental results of redness reduction

[0222]

[0223] Among them, the change rate = (analysis value after using the product - analysis value before using the product) + analysis value before using the product x 100%

[0224] The experimental results are shown in Table 14. After using the product for 14 days, the a* value of the red area of the skin in the cheek test area decreased significantly by 17.16%, and the proportion of the area of the red area of the skin decreased significantly by 38.42%.

[0225] After using the product for 28 days, the a* value of the red area of the skin in the cheek test area decreased significantly by 19.66%, and the proportion of the area of the red area of the skin decreased significantly by 52.65%.

[0226] After using the product for 56 days, the a* value of the red area of the skin in the cheek test area decreased significantly by 21.22%, and the proportion of the area of the red area of the skin decreased significantly by 59.17%. The decrease in the analyzed value of the a* value of the red area of the skin indicates an improvement in skin flushing, and the decrease in the proportion of the area of the red area of the skin indicates an improvement in skin flushing, indicating that the product containing Terminalia chebula Retz. extract can significantly improve skin flushing and soothe the skin.

[0227] All documents mentioned in this invention are cited in this application for reference as if each document was cited separately for reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of Terminalia chebula extract, characterized in that, For preparing a product having an efficacy selected from the following group: (a) Inhibiting inflammatory factors; (b) Upregulating AKT1 expression; (c) Downregulating TRPV1 expression; (d) Inhibiting the loss of type I collagen.

2. The use according to claim 1, characterized in that, The content of galloyl glucosides in the Terminalia chebula extract is ≥ 50 wt%.

3. The use according to claim 1, characterized in that, The upregulation of AKT1 expression has one or more characteristics selected from the following group: (1) Upregulating the expression of the AKT1 gene in cells; (2) Increasing the p-AKT1 signal by upregulating the expression of the AKT1 protein in cells.

4. The use according to claim 1, wherein The inflammatory factors are selected from the following group: TNF-a, IL-6, or a combination thereof.

5. The use according to claim 1, wherein The inhibition rate of the Terminalia chebula extract on the expression level of TRPV1 is 10-60%.

6. The use according to claim 1, wherein, The Terminalia chebula extract is obtained by extraction with 50-70 (v / v)% ethanol.

7. The use according to claim 1, characterized in that, The product further has one or more characteristics selected from the following group: (1) Without skin irritation; (2) Anti-inflammatory; (3) Anti-allergic, soothing and analgesic; (4) Reducing redness.

8. The use according to claim 1, wherein, The dosage of the product containing the Terminalia chebula extract preparation is 0.05-99 wt%.

9. A method for upregulating AKT1 expression in vitro, characterized in that, The method includes: Culturing cells under the condition of adding the Terminalia chebula extract as described in claim 1, wherein the concentration of the Terminalia chebula extract is 1-20 μg / mL.

10. The method according to claim 9, wherein The upregulation of AKT1 expression has one or more characteristics selected from the following group: (1) Upregulating the expression of the AKT1 gene in cells; (2) Upregulating the expression of the AKT1 protein in cells; (3) Increasing the p-AKT1 signal by upregulating the expression of the AKT1 protein in cells.

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