Humulus lupulus extract with anti-inflammatory, bacteriostatic, soothing and skin barrier repairing effects and preparation process thereof

Abstract

The invention discloses a humulus lupulus extract with anti-inflammatory, bacteriostatic, soothing and skin barrier repairing effects and a preparation process thereof, and relates to the technical field of cosmetic raw materials. The preparation process of the humulus lupulus extract comprises the following steps: mixing humulus lupulus and an ethanol water solution, performing hot reflux extraction for 1-3 times, performing vacuum concentration on an extracting solution, and drying to obtain a crude humulus lupulus extract; and dissolving the crude humulus lupulus extract with water to obtain a solution, performing column chromatography separation with AB-8 resin, performing elution with water and an ethanol aqueous solution, collecting an eluent, performing vacuum concentration on the eluent, and drying to obtain the humulus lupulus extract. In the preparation process, the effect of high content of total flavonoids can be achieved by only adopting one-time column chromatography separation. Under the optimal preparation process conditions, the anti-inflammatory, bacteriostatic, soothing and skin barrier repairing effects of the humulus lupulus extract are verified, and it is indicated that the humulus lupulus extract has the bacteriostatic, anti-inflammatory, soothing and repairing effects and can be applied to skin care products.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic raw materials, and in particular to a hop extract having anti-inflammatory, antibacterial, soothing and skin barrier repairing effects and a preparation process thereof. Background Art

[0002] Hops (Humulus lupulus L.) is a perennial climbing herbaceous plant of the genus Humulus in the Cannabaceae family. It is mainly distributed in northern Xinjiang, my country, and is also cultivated in North China, Northeast China, Zhejiang, Shandong, etc. Hops has multiple pharmacological activities such as antibacterial, anti-tumor, antioxidant, sedative, hypnotic, hypoglycemic, hypotensive, and estrogen-like effects.

[0003] Hops is a plant that is both a medicine and food. It is widely used in the brewing process of beer and is the basic raw material for beer production. Nowadays, more experimental studies have shown that hops contain a variety of chemical components, such as polysaccharides, resins, polyphenols, volatile oils, flavonoids, etc. Among them, flavonoids are an important class of active ingredients in hops. Among them, the most common research is on xanthohumol in hops. It is a natural isopentenyl flavonoid compound secreted by the hop glands of hops and has significant anti-cancer effects. As one of the chemical components in hops, polysaccharides have good pharmacological activity, but so far, there is still little research on polysaccharides in hops.

[0004] At present, the research on hops is more focused on improving the flavor of beer in beer brewing. There is little research on the active flavonoids and polysaccharides contained in hop extracts in terms of anti-inflammatory, soothing, antibacterial and barrier repair. There is a lack of scientific basis and technical support for the research and development of raw materials for skin care products. With the rise of cosmetic plant raw materials, hops is a plant raw material with great potential, and its efficacy in skin care is also worthy of further development and exploration.

[0005] Chinese patent CN104586945A discloses a hop total flavonoid extract, a preparation method and its use as a drug for the prevention and treatment of liver damage and cancer. The preparation method of the hop total flavonoid comprises the following steps: (1) drying the hops, crushing them, extracting them with ethanol or methanol, and recovering the extract under reduced pressure to obtain a crude extract; (2) treating the crude extract with a non-polar macroporous adsorption resin, gradient eluting with a solvent, collecting and combining the eluates containing the hop total flavonoids, and concentrating them under reduced pressure to obtain crude hop total flavonoids; or treating the extract with petroleum sulphate in turn; Ether or cyclohexane, dichloromethane or chloroform, ethyl acetate extraction, dichloromethane or chloroform, ethyl acetate extract concentrated under reduced pressure to obtain crude hop total flavonoids; (3) the crude hop total flavonoids obtained in step (2) are subjected to polyamide column chromatography, gradient elution is performed with a solvent, the eluate containing hop total flavonoids is collected and combined, concentrated under reduced pressure, and dried to obtain refined hop total flavonoids; or the crude hop total flavonoids are subjected to silica gel column chromatography, gradient elution is performed with an organic solvent, the eluate containing hop total flavonoids is collected and combined, concentrated under reduced pressure, and dried to obtain refined hop total flavonoids. The above preparation method uses two column chromatography treatments, the process is cumbersome, not conducive to industrial application, and the efficacy of hop total flavonoids extract in skin care is not studied.

[0006] Chinese patent CN108017604B discloses an α-acid derivative and a preparation method and use thereof. The preparation method of the α-acid derivative comprises the following steps: (1) hops (Humulus lupulus L.) extracting with 50%-100% ethanol, and recovering the extract to obtain a crude extract; (2) separating the crude extract obtained in step (1) by silica gel column chromatography, and gradient eluting with a mixed solvent of petroleum ether and ethyl acetate, a mixed solvent of petroleum ether and acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and acetone, a mixed solvent of chloroform and methanol, or a mixed solvent of dichloromethane and methanol; (3) separating the fraction 100:1-100:25 obtained in the above step (2) by ODS column chromatography, and gradient eluting with a mixed solvent of methanol and water, or a mixed solvent of acetonitrile and water as the mobile phase; (4) separating the fraction 1:9-7:3 obtained in the above step (3) by HPLC-RID10A, and gradient eluting with a mixed solvent of methanol and water, or a mixed solvent of acetonitrile and water as the mobile phase, to obtain α-acid derivatives 1-6. The above preparation method uses three column chromatography treatments, which is cumbersome and not conducive to industrial application, and the efficacy of α-acid derivatives in skin care is not studied.

[0007] The literature "Sun Shikun. Research on the extraction and purification process of flavonoids in hops [D]. Lanzhou University, 2017." studied the extraction process of flavonoids in hops. The results showed that ethanol was the best extraction solvent and ultrasound was the best extraction method. The best extraction conditions were: 60% ethanol solution concentration, 15 minutes extraction time, 1:30 (g / mL) solid-liquid ratio, 80W power, and the content of flavonoids extracted from hops was 3.013%. Macroporous resin was used to separate and purify the flavonoids extracted from hops. According to the analysis, 75% ethanol solution was the best eluent. The speed of loading the adsorption stock solution (flavonoid crude extract) was 3.0mL / min, and the adsorbed flavonoids were eluted with 75% ethanol solution, the elution rate was 1.5mL / min, the concentration of the separated eluent was 0.018mg / mL, and the extraction rate of flavonoids was 81.82%. According to the structure confirmation of the collected flavonoids, this method has a better extraction effect on flavonoids in hops. The above-mentioned optimal extraction process uses macroporous resin to separate and purify flavonoids, and the separation effect is not as good as the present invention.

[0008] In view of this, in order to solve the deficiencies of the prior art preparation methods, the present invention provides a hop extract having anti-inflammatory, antibacterial, soothing and skin barrier repairing effects and a preparation process thereof. Summary of the invention

[0009] The purpose of the present invention is to provide a hop extract having anti-inflammatory, antibacterial, soothing and skin barrier repairing effects and a preparation process thereof, which greatly improves the total flavonoid content in the hop extract.

[0010] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0011] In one aspect, the present invention provides a process for preparing a hop extract, comprising the following steps:

[0012] Step 1, mixing hops and ethanol aqueous solution, performing heat reflux extraction for 1-3 times, concentrating the extract under reduced pressure and drying to obtain a crude hop extract;

[0013] Step 2: Dissolve the crude hop extract in water to obtain a solution, perform column chromatography separation using AB-8 resin, elute with water and ethanol aqueous solution, collect the eluate, concentrate the eluate under reduced pressure, and then dry it to obtain the hop extract.

[0014] Preferably, in step 1, the ratio of hops to ethanol aqueous solution is 15g:600-900mL.

[0015] Further preferably, in step 1, the ratio of hops to ethanol aqueous solution is 15 g:750 mL.

[0016] Preferably, in step 1, the concentration of the ethanol aqueous solution is 60%-90%.

[0017] Further preferably, in step 1, the concentration of the ethanol aqueous solution is 75%.

[0018] Preferably, in step 1, each thermal reflux extraction lasts for 120-210 min.

[0019] Further preferably, in step 1, each thermal reflux extraction is performed for 210 min.

[0020] Preferably, in step 2, the concentration of the solution is 0.5-5 g / L.

[0021] Further preferably, in step 2, the concentration of the solution is 1 g / L.

[0022] Preferably, in step 2, the specific steps of column chromatography separation are as follows: loading the sample at a speed of 2-5 mL / min, and after the adsorption is completed, standing for 2-4 hours.

[0023] Further preferably, in step 2, the specific steps of column chromatography separation are as follows: loading the sample at a rate of 2 mL / min, and after the adsorption is completed, standing for 3 hours.

[0024] Preferably, in step 2, the volume of water used for elution is 1-4 BV, and the volume of ethanol aqueous solution used for elution is 1-4 BV.

[0025] Further preferably, in step 2, the volume of water for elution is 1BV, and the volume of ethanol-water solution for elution is 2.5BV.

[0026] Preferably, in step 2, the concentration of the ethanol aqueous solution for elution is 60%-90%.

[0027] Further preferably, in step 2, the concentration of the ethanol aqueous solution for elution is 60%.

[0028] More preferably, the preparation process comprises the following steps:

[0029] Step 1, mixing hops and ethanol aqueous solution, performing heat reflux extraction for 3 times, combining the 3 extracts, filtering twice with a 0.45 μm filter plate to obtain a clear extract, concentrating the extract under reduced pressure, and freeze-drying the extract to obtain a crude hop extract;

[0030] Step 2, dissolving the crude hop extract in water to obtain a solution, performing column chromatography separation using AB-8 resin, eluting with water and ethanol aqueous solution, collecting the eluate, concentrating the eluate under reduced pressure, and then freeze-drying the eluate to obtain the hop extract.

[0031] In another aspect, the present invention provides a hop extract obtained by the above preparation process.

[0032] Finally, the present invention provides the use of the hop extract in preparing skin care products, cosmetics or medicines.

[0033] The beneficial effects of the present invention are:

[0034] (1) The present invention discloses a preparation process of a hop extract, in which only one column chromatography separation is used to achieve a high total flavonoid content.

[0035] (2) Under the optimal preparation process conditions, the present invention verifies the anti-inflammatory, antibacterial, soothing and skin barrier repairing effects of hop extract, indicating that it has antibacterial, anti-inflammatory, soothing and repairing effects and can be applied to skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The total flavonoids test results of the embodiments and comparative examples are shown in FIG.

[0037] Figure 2 This is the test result of the safe concentration of CCK-8 sample in Example 1.

[0038] Figure 3 This is the FLG immunofluorescence detection result of Example 1.

[0039] Figure 4 This is the TRPV4 immunofluorescence detection result of Example 1. DETAILED DESCRIPTION

[0040] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they should also fall within the scope of the present invention.

[0041] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0042] Hops are the immature flowering clusters of the hop plant of the Moraceae family. They are picked in summer and autumn when the clusters are green and slightly yellow, and then sun-dried or oven-dried. The drying temperature must not exceed 45°C.

[0043] Example 1

[0044] Weigh 15g of hops and crush them, add 750mL of 75% ethanol, put them in a heating mantle, extract them 3 times respectively, and reflux them for 210min, keeping them slightly boiling. After the extraction, combine the 3 extracts and filter them twice with a 0.45μm filter plate to obtain a clear extract. Rotary evaporate the ethanol, freeze-dry, and weigh to obtain product 1-1. Dissolve product 1-1 in water to obtain a solution (1g / L), fill the column with the pretreated AB-8 resin, load the sample at a rate of 2mL / min, and after the adsorption is completed, let it stand for 3h. Rinse the sample with water, volume 1BV, and then elute with 60% ethanol, the elution rate is 2mL / min, the elution volume is 2.5BV, collect the rinse solution and concentrate it, rotary evaporate it (40℃), and freeze-dry it to obtain the powder Example 1.

[0045] Example 2

[0046] Weigh 15g of hops, crush them, add 600mL of 60% ethanol, put them in a heating mantle, extract once, and reflux for 120min, keeping it slightly boiling. After the extraction, filter twice with a 0.45μm filter plate to obtain a clear extract. Rotary evaporate the ethanol, freeze-dry, weigh, and obtain product 2-1. Dissolve product 2-1 in water to obtain a solution (1g / L), fill the column with the pretreated AB-8 resin, load the sample at a rate of 5mL / min, and after the adsorption is completed, let it stand for 3h. Rinse the sample with water, volume 1BV, and then elute with 90% ethanol, the elution rate is 5mL / min, the elution volume is 3BV, collect the rinse solution and concentrate it, rotary evaporate and concentrate (40°C), and freeze-dry to obtain the powder Example 2.

[0047] Example 3

[0048] Weigh 15g of hops and crush them, add 900mL of 90% ethanol, put them in a heating mantle, extract them twice, heat reflux extraction for 180min, and keep them slightly boiling. After the extraction, combine the two extracts and filter them twice with a 0.45μm filter plate to obtain a clear extract. Rotary evaporate the ethanol, freeze dry, weigh, and obtain product 3-1. Dissolve product 3-1 in water to obtain a solution (1g / L), fill the pretreated AB-8 resin column, load the sample at a speed of 3mL / min, and after adsorption is completed, let it stand for 3h. Rinse the sample with water, volume 1B, and then elute with 75% ethanol, the elution rate is 3mL / min, the elution volume is 4BV, collect the rinse solution and concentrate it, rotary evaporate and concentrate (40℃), and freeze dry to obtain powder Example 3.

[0049] Comparative Example 1

[0050] Weigh 6g of hops, crush them, add 300mL of pure water, put them in a heating mantle, and heat reflux extract for 210min, keeping it slightly boiling. After reflux, cool to room temperature, filter twice with a 0.45μm filter plate to obtain a clear extract. After filtration, add 2% 1,2-hexanediol, 5% glycerol and 0.5% p-hydroxyacetophenone as a preservative. Finally dry to obtain powder comparative example 1.

[0051] Comparative Example 2

[0052] Weigh 10g of hops and crush them, add 300mL of 30% 1,3-butanediol aqueous solution, put it in a heating mantle, and heat reflux extract for 210min, keeping it slightly boiling. After reflux, cool it to room temperature and filter it twice with a 0.45μm filter plate to obtain a clear extract. After filtration, add 2% 1,2-hexanediol, 5% glycerol and 0.5% p-hydroxyacetophenone as a preservative. Finally dry it to obtain powder comparative example 2.

[0053] Comparative Example 3

[0054] Weigh 10g of hops and crush them, add 300mL of 30% 1,3-butanediol aqueous solution, put it in a heating mantle, and heat reflux extract for 210min, keeping it slightly boiling. After reflux, cool it to room temperature and filter it twice with a 0.45μm filter plate to obtain a clear extract. After filtration, add 2% 1,2-hexanediol, 5% glycerol, 0.5% p-hydroxyacetophenone and 10% Tween20 for preservation and precipitation treatment. Finally dry it to obtain powder comparative example 3.

[0055] Comparative Example 4

[0056] Weigh 6g of hops and crush them, add 300mL of 30% 1,3-butanediol aqueous solution, put it in a heating mantle, and heat reflux extract for 210min, keeping it slightly boiling. After reflux, cool it to room temperature and filter it twice with a 0.45μm filter plate to obtain a clear extract. After filtration, add 2% 1,2-hexanediol, 5% glycerol and 0.5% p-hydroxyacetophenone as a preservative. Finally dry it to obtain powder comparative example 4.

[0057] Comparative Example 5

[0058] Weigh 6g of hops and crush them, add 300mL of 30% 1,3-butanediol aqueous solution, put it in a heating mantle, and heat reflux extract for 210min, keeping it slightly boiling. After reflux, cool it to room temperature and filter it twice with a 0.45μm filter plate to obtain a clear extract. After filtration, add 2% 1,2-hexanediol, 5% glycerol, 0.5% p-hydroxyacetophenone and 10% Tween20 for preservation and precipitation treatment. Finally dry it to obtain powder comparative example 5.

[0059] Comparative Example 6

[0060] Weigh 16g of hops and crush them, add 800mL of pure water, put them in a heating mantle, and heat reflux extract for 210min, keeping them slightly boiling. After the extraction, cool the extract to room temperature and filter it twice with a 0.45μm filter plate to obtain a clear extract. Concentrate the filtrate to 600mL and dispense it for spray drying. Finally dry it to obtain powder comparative example 6.

[0061] Comparative Example 7

[0062] Weigh 1 g of hops and crush them, add 50 mL of 30% 1,3-butanediol aqueous solution, and flash extract for 90 seconds. Filter twice with a 0.45 μm filter plate to obtain a clear extract. Store the filtrate away from light and wait for testing. Finally dry to obtain powder Comparative Example 7.

[0063] Comparative Example 8

[0064] Weigh 5 g of hops, crush them, add 250 mL of 75% ethanol, and perform ultrasonic extraction for 30 min. After the extraction, filter twice with a 0.45 μm filter plate to obtain a clear extract. Ethanol is evaporated from the filtrate, freeze-dried, and weighed to obtain powder Comparative Example 8.

[0065] Comparative Example 9

[0066] Weigh 5g of hops, crush them, add 250mL of 95% ethanol, and perform ultrasonic extraction for 30min. After the extraction, filter twice with a 0.45μm filter plate to obtain a clear extract. Take out 50mL of the extract, rotary evaporate the ethanol, freeze-dry, and weigh to obtain a powder product 9-1. Take out 100mL of the remaining extract, concentrate to 20mL, add 80mL of water, precipitate at low temperature for 24h, take the supernatant, freeze-dry, and weigh to obtain a powder comparative example 9.

[0067] Comparative Example 10

[0068] Weigh 5g of hops, mash them, add 250mL of pure water, put them in a heating mantle, and extract them under hot reflux for 210min, keeping them slightly boiling. After the extraction, filter them twice with a 0.45μm filter plate to obtain a clear extract. Take out 50mL of the extract, freeze-dry it, and weigh it to obtain a powder product 10-1. Take out 100mL of the remaining extract, concentrate it to 20mL, add 80mL of anhydrous ethanol, precipitate it at low temperature for 24h, take the supernatant, freeze-dry it, and weigh it to obtain a powder comparative example 10.

[0069] Comparative Example 11

[0070] Weigh 15g of hops, mash them, add 750mL of pure water, put them in a heating mantle, extract them 3 times respectively, and reflux them for 210min, keeping them slightly boiling. After the extraction, combine the 3 extracts, filter them twice with a 0.45μm filter plate to obtain a clear extract. Rotary evaporate ethanol, freeze-dry, weigh, and obtain product 11-1. Dissolve product 11-1 in water to obtain a solution (1g / L), fill the column with the pretreated AB-8 resin, load the sample at a rate of 2mL / min, and after the adsorption is completed, let it stand for 3h. Rinse the sample with water, volume 1BV, and then elute with 60% ethanol, the elution rate is 2mL / min, the elution volume is 2.5BV, collect the rinse solution and concentrate it, rotary evaporate and concentrate (40°C), and freeze-dry to obtain powder comparative example 11.

[0071] Comparative Example 12 (preparation process refers to Chinese patent CN104586945B)

[0072] (1) 15 g of dried hops were extracted three times with 70% methanol under heating and reflux, with the weight volume ratio of the hops to the solvent being 1:10, and the extract was recovered under reduced pressure to obtain a crude extract;

[0073] (2) extracting the extract obtained in step (1) twice with petroleum ether, chloroform and ethyl acetate, with the volume ratio of the extraction solvent to the extract being 2:1, and concentrating the chloroform and ethyl acetate extracts under reduced pressure to obtain crude total flavonoids of hops;

[0074] (3) The crude total flavonoids of hops obtained in step (2) are eluted by polyamide column chromatography with the mobile phase being ethanol and water, the volume ratio of the mixed solvent being 1:9, 3:7, 6:4, 9:1, and the volume ratio of methanol / water or ethanol / water being 3:7, 6:4 to obtain the total flavonoids of hops.

[0075] Comparative Example 13 (preparation process refers to Chinese patent CN108017604B)

[0076] (1) 15 g of hops was extracted with 95% ethanol by heating and refluxing for 3 times (dosage: 0.15 L), and the extract was recovered under reduced pressure to obtain a crude extract;

[0077] (2) The ethanol extract obtained in step (1) was separated by silica gel column chromatography and eluted with petroleum ether:acetone 100:1, 100:3, 100:6, 100:9, 100:11, 100:15, 5:1, 1:1 in sequence;

[0078] (3) The petroleum ether:acetone 100:3 fraction obtained in the above step (2) was separated by ODS chromatography, and the total flavonoids of hops were obtained by gradient elution using acetonitrile / water 1:9, 2:8, 4:6, 1:1, 6:4 as the mobile phase.

[0079] Results

[0080] 1. Detection of total flavonoid content

[0081] (1) Standard curve determination

[0082] a. Accurately weigh 0.1 mg of rutin reference substance, dissolve it in 75% ethanol and dilute to 100 mL volumetric flask. The mass concentration of this solution is 1.0 mg / mL;

[0083] b. Accurately pipette 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mL into a 10 mL volumetric flask, dilute to the mark with 75% ethanol, and shake well to obtain reference solutions with mass concentrations of 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively;

[0084] c. Take 1 mL of rutin reference solution of different concentrations in a 10 mL volumetric flask, add 4 mL of deionized water, 0.3 mL of 5% NaNO 2 After 5 min, 0.3 mL of 10% Al(NO 3 ) 3 Aqueous solution, after reacting for 6 minutes, add 2 mL of 1 mol / L NaOH aqueous solution and 2.4 mL of deionized water in sequence, and measure the absorbance at 510 nm after 10 minutes;

[0085] d. Draw a standard curve with the absorbance value A as the ordinate and the concentration of the rutin reference substance as the abscissa. The standard curve equation is: y = ax-b (where x is the concentration and y is the absorbance value), and the correlation coefficient r 2 .

[0086] (2) Sample measurement

[0087] Take hop extract and dissolve it in pure water to obtain 50μg / mL and 100μg / mL. Take 1mL of each solution in a 10mL volumetric flask, add 4mL of deionized water, 0.3mL of 5% NaNO 2 After 5 min, 0.3 mL of 10% Al(NO 3 ) 3 aqueous solution, react for 6 minutes, add 2mL of 1 mol / L NaOH aqueous solution and 2.4mL of deionized water in sequence, and measure the absorbance at 510nm after 10 minutes (using pure water as the blank).

[0088] (3) Total flavonoids test results

[0089] The total flavonoids test results of the embodiments and comparative examples are as follows Figure 1 As shown, compared with the comparative example, the total flavonoids content of Example 1 is the highest.

[0090] 2. The DPPH free radical scavenging and COX-2 in vitro inhibition experiments were used to detect that Example 1 had antioxidant and anti-inflammatory effects.

[0091] 2.1. DPPH free radical scavenging antioxidant efficacy test

[0092] (1) Preparation of DPPH ethanol solution: Weigh 20 mg DPPH, add anhydrous ethanol to dissolve and dilute to a 250 mL volumetric flask. The DPPH concentration is 2 × 10 -4 mol / L; stored at 0-4℃ away from light, used immediately after preparation, valid within 4 hours (vitamin C is used as positive control: the same concentration is used);

[0093] (2) Preparation of the test solution: Prepare four concentrations, diluting 10 times, 20 times, 50 times, and 100 times respectively;

[0094] (3) Experimental procedures

[0095] Take 1.0mL of the test solution and 1.0mL of 2×10 -4 mol / L DPPH solution and mix well (tube A);

[0096] Take 1.0 mL of anhydrous ethanol and 1.0 mL of 2×10 -4 mol / L DPPH solution and mix well (tube B);

[0097] Take 1.0 mL of anhydrous ethanol and mix it with 1.0 mL of the test solution (tube C);

[0098] After reacting for 30 minutes, measure the absorbance of tubes A, B, and C at 517 nm.

[0099] Table 1 DPPH free radical scavenging rate detection reagent ratio table

[0100] serial number DPPH solution Anhydrous ethanol Test liquid Total volume A 1.0mL —— 1.0mL 2.0mL B 1.0mL 1.0mL —— 2.0mL C —— 1.0mL 1.0mL 2.0mL

[0101] (4) Data processing

[0102] The formula for calculating the clearance rate is: Clearance rate (%) = [(B + C) - A] / B

[0103] (5) DPPH free radical inhibition rate test results

[0104] Table 2 DPPH radical scavenging results of Example 1

[0105] Sample concentration Example 1 10 μg / mL 30.19±2.66 20 μg / mL 45.20±1.86 50 μg / mL 78.21±1.12 100 μg / mL 92.47±0.69

[0106] The DPPH free radical scavenging results of Example 1 are shown in Table 2. The above results indicate that Example 1 can significantly inhibit DPPH free radicals and has significant antioxidant effect.

[0107] 2.2. Anti-inflammatory efficacy test of COX-2 in vitro inhibition experiment

[0108] The inhibition rate of COX-2 of different samples was tested using the COX-2 kit. The specific operation was based on the instructions provided by the manufacturer:

[0109] (1) Sample preparation: Dilute the sample with DMSO to the concentration to be tested.

[0110] (2) Reagent preparation:

[0111] a. Dissolve all reagents except rhCOX-2 to room temperature, centrifuge slightly to allow the solution to settle to the bottom of the tube, and then mix well for later use. COX-2Probe, COX-2Cofactor (50X) and COX-2Substrate (50X) are prepared in DMSO and can be dissolved in a 37℃ water bath for 0.5-2min. Store at -20℃ away from light immediately after use.

[0112] b. Preparation of COX-2 Cofactor working solution: Prepare an appropriate amount of COX-2 Cofactor working solution according to the ratio of 5 μl of COX-2 Cofactor working solution required for each sample. Take an appropriate amount of COX-2 Cofactor (50X) and dilute it with COX-2 Assay Buffer at a ratio of 1:49. For example, 4 μl of COX-2 Cofactor (50X) is added to 196 μl of COX-2 Assay Buffer to prepare 200 μl of COX-2 Cofactor working solution. The prepared COX-2 Cofactor working solution can be stored at 4°C and can only be used on the same day.

[0113] c. Preparation of COX-2 working solution: Prepare an appropriate amount of COX-2 working solution according to the ratio of 5 μl of COX-2 working solution for each sample. Take an appropriate amount of rhCOX-2 (25X) and dilute it with COX-2Assay Buffer at a ratio of 1:24. For example, 8 μl of rhCOX-2 (25X) is added to 192 μl of COX-2Assay Buffer to prepare 200 μl of COX-2 working solution. The prepared COX-2 working solution can be temporarily stored in an ice bath, and the enzyme activity is basically stable within 1 hour. Note: All operations involving COX-2 should be performed on ice.

[0114] d. Preparation of COX-2 Substrate working solution: Prepare an appropriate amount of COX-2 Substrate working solution according to the ratio of 5 μL of COX-2 Substrate working solution for each sample. Take an appropriate amount of COX-2 Substrate (50X), add an equal volume of Substrate Buffer, vortex thoroughly, and dilute the mixture with Milli-Q grade pure water or deionized water at a ratio of 1:24, and vortex thoroughly. For example, add 20 μL of COX-2 Substrate (50X) to 20 μL of Substrate Buffer, vortex thoroughly, then add 960 μL of Milli-Q grade pure water or deionized water, and vortex thoroughly to finally obtain 1 ml of COX-2 Substrate working solution. The prepared COX-2 Substrate working solution can be temporarily stored in an ice bath and is relatively stable within 1 hour. Note: COX-2 Substrate working solution can also be prepared during the 10-minute incubation at 37°C during sample testing.

[0115] e. Preparation of positive inhibitor Celecoxib solution: The positive control inhibitor Celecoxib provided in this kit has a concentration of 100 μM and is prepared in DMSO. It can be diluted to the desired concentration or concentration gradient using the same solvent as the inhibitor to be tested. Usually, the IC50 of Celecoxib is about 10nM-100nM.

[0116] (3) Sample testing:

[0117] a. Refer to Table 3, use a 96-well black board to set up control wells and sample wells, and add samples and solutions in sequence according to the table below. After adding the sample to be tested, mix well and incubate at 37°C for 10 minutes.

[0118] Table 3 Reagent ratio table

[0119]

[0120] b. Add 5 μl of COX-2 Probe to each well.

[0121] c. Quickly add 5 μL of COX-2 Substrate working solution to each well and mix well. Note: The reaction will start immediately after adding COX-2 Substrate working solution. If there are many wells, you can operate at low temperature or use a discharge gun to reduce the error caused by the time difference of adding COX-2 Substrate working solution between wells. Mixing can also be done on a culture plate oscillator.

[0122] d. Incubate at 37℃ in the dark for 5 minutes and then measure the fluorescence. The excitation wavelength is 560nm and the emission wavelength is 590nm. If the fluorescence reading is low, the incubation time can be appropriately extended to 10-20 minutes.

[0123] (4) Calculation:

[0124] a. Calculate the average fluorescence value of each sample well and blank control well, which can be recorded as RFU blank control, RFU 100% enzyme activity control, RFU positive inhibitor control and RFU sample. RFU, Relative Fluorescence Unit.

[0125] b. Calculate the percent inhibition for each sample.

[0126] The calculation formula is as follows:

[0127] Inhibition rate (%) = (RFU 100% enzyme activity control - RFU sample) / (RFU 100% enzyme activity control - RFU blank control) × 100%

[0128] (5) COX-2 inhibition rate test results

[0129] Table 4 COX-2 in vitro inhibition results of Example 1

[0130]

[0131] The in vitro COX-2 inhibition results of Example 1 are shown in Table 4. The above results show that Example 1 has an inhibitory effect on COX-2 and has a significant anti-inflammatory effect.

[0132] 3. Investigate the inhibitory effect of Example 1 on fungi and bacteria, and detect the antibacterial efficacy of Example 1.

[0133] 3.1. Evaluation of the efficacy of Example 1 in inhibiting bacteria (Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Propionibacterium acnes)

[0134] (1) Culture of Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Propionibacterium acnes

[0135] Shake the culture medium with Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Propionibacterium acnes (until a sterile block is precipitated at the bottom). Use a sterile syringe to transfer 200 μL-800 μL of bacterial solution into the new culture medium and culture it in a biochemical incubator at 37°C. After 3-5 days of culture, subculture again.

[0136] (2) Microbroth method to test the antibacterial activity of samples

[0137] 1) Sample configuration

[0138] Prepare high-concentration stock solution according to the required concentration. Use DMSO for water-insoluble substances and PBS for water-soluble substances.

[0139] 2) Prepare fresh culture medium

[0140] Transfer the sterile BHI / LB / blister medium (in the anaerobic bottle) into a sterile 50 mL centrifuge tube. Centrifuge the sterile BHI / LB / blister medium for 10 min at 25°C, 3500 rpm. Take the supernatant and place it in another sterile 50 mL centrifuge tube for later use. For the samples, dilute each sample of the fresh medium to the concentration to be tested.

[0141] 3) Extraction of Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Propionibacterium acnes

[0142] Gently shake the culture medium with Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Propionibacterium acnes, and transfer to a sterile 50 mL centrifuge tube. Centrifuge at 25°C, 1000 rpm, for 30 seconds, transfer the supernatant to a sterile 50 mL centrifuge tube, and centrifuge at 25°C, 3500 rpm, for 10 minutes.

[0143] 4) Determination of bacterial liquid concentration

[0144] Turn on the microplate reader and set the wavelength to 600nm. Take 400μL of the bacterial solution and place it in a 1.5mL sterile centrifuge tube. Take 200μL of the bacterial solution from the 1.5mL centrifuge tube containing 400μL of the bacterial solution and place it in a regular 96-well plate. Measure the OD of the bacterial solution at 600nm 600 If OD 600 When the value is >0.5, dilute with sterile PBS. (You can explore from 2 times, 4 times, 6 times, 8 times) 600 When the value is equal to 0.5, the next experiment can be carried out.

[0145] 5) Dilution of bacterial solution

[0146] Calculate the volume of bacterial suspension required. 600 =0.5) was diluted 100 / 3000 times with fresh culture medium.

[0147] 6)Planning

[0148] In a sterile 96-well culture plate, 100 μL of diluted bacterial suspension was added to each test well, and then 100 μL of samples of different concentrations were added. 100 μL of samples of different concentrations + 100 μL BHI / LB / blister medium was used as a control group, 200 μL BHI / LB / blister medium without Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Propionibacterium acnes was used as a blank control, and 100 μL of diluted bacterial suspension + 100 μL BHI / LB / blister medium was used as a negative control, and 3 replicate wells were made for each.

[0149] (Tear open the anaerobic gas production bag and place it in a 2.5L anaerobic tank), then place the sterile 96-well culture plate in a 2.5L anaerobic tank / 37°C incubator. After culturing for 24 / 12 hours, measure the absorbance at 600nm.

[0150] 7) After laying the plate, place it in an anaerobic jar / 37°C in an incubator and culture it for 24 / 12 hours, then measure the OD value at 600nm.

[0151] 8) Inhibition rate of Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Propionibacterium acnes:

[0152]

[0153] 3.2. Evaluation of the efficacy of Example 1 in inhibiting fungi (Malassezia and Candida albicans)

[0154] (1) Cultivation of Malassezia and Candida albicans

[0155] Shake the culture medium with Malassezia and Candida albicans until a sterile block is precipitated at the bottom. Use a sterile syringe to transfer 200μL-800μL of bacterial solution into the new culture medium and culture it in a biochemical incubator at 30℃. After 1-3 days of culture, subculture again.

[0156] (2) Microbroth method to test the antibacterial activity of samples

[0157] 1) Sample configuration

[0158] Prepare high-concentration stock solution according to the required concentration. Use DMSO for water-insoluble substances and PBS for water-soluble substances.

[0159] 2) Prepare fresh culture medium

[0160] Transfer the sterile malt extract medium / sterile Sabouraud medium into a sterile 50 mL centrifuge tube. Centrifuge the sterile malt extract medium / sterile Sabouraud medium for 10 min at 25°C, 3500 rpm. Take the supernatant and place it in another sterile 50 mL centrifuge tube for later use. For the samples, dilute each sample of the fresh medium to the concentration to be tested.

[0161] 3) Extraction of Malassezia and Candida albicans

[0162] Gently shake the culture medium, Malassezia and Candida albicans, and transfer to a sterile 50 mL centrifuge tube. Centrifuge at 25°C, 1000 rpm for 30 seconds, transfer the supernatant to a sterile 50 mL centrifuge tube, and centrifuge at 25°C, 3500 rpm for 10 minutes.

[0163] 4) Determination of bacterial liquid concentration

[0164] Turn on the microplate reader and set the wavelength to 600nm. Take 400μL of the bacterial solution and place it in a 1.5mL sterile centrifuge tube. Add 1mL PBS and take 200μL of the bacterial solution from the 1.5mL centrifuge tube containing 400μL of bacterial solution and place it in a regular 96-well plate. Measure the OD of the bacterial solution at 600nm. 600 If OD 600 When the value is >1, dilute with sterile PBS. (You can explore from 2 times, 4 times, 6 times, 8 times) 600 When the value is equal to 1, the next step of the experiment can be carried out.

[0165] 5) Dilution of bacterial solution

[0166] Calculate the volume of bacterial suspension required. 600 =1) Dilute 100-fold with fresh culture medium.

[0167] 6)Planning

[0168] In a sterile 96-well culture plate, add 100 μL of diluted bacterial suspension to each test well, then add 100 μL of samples of different concentrations, 100 μL of samples of different concentrations + 100 μL of malt medium / Sabouraud medium as the control group, 200 μL of malt medium / Sabouraud medium without Malassezia and Candida albicans as the blank control, 100 μL of diluted bacterial suspension + 100 μL of malt medium / Sabouraud medium as the negative control, and make 3 duplicate wells for each. Place the sterile 96-well culture plate in a 30°C incubator. After 72 / 48 hours of culture, measure the absorbance at 600 nm.

[0169] 7) Test results of inhibition rate of Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Propionibacterium acnes, Malassezia, and Candida albicans

[0170] Table 5 Inhibition rate of Example 1 on different bacteria (%)

[0171]

[0172]

[0173] The inhibition rates of Example 1 on different bacteria are shown in Table 5. The above results show that when the concentration of Example 1 is above 100 μg / mL, it has an inhibitory effect on Candida albicans, and the inhibition rate is 53.15%; when the concentration of Example 1 is above 50 μg / mL, it has an inhibitory effect on Malassezia, and the inhibition rate is 52.19%; when the concentration of Example 1 is above 25 μg / mL, it has an inhibitory effect on Propionibacterium acnes, and the inhibition rate is 52.27%; when the concentration of Example 1 is above 12.5 μg / mL, it has an inhibitory effect on Escherichia coli, and the inhibition rate is 74.20%; When the concentration of Example 1 is above 25 μg / mL, it has an inhibitory effect on Staphylococcus aureus, and the inhibition rate is 59.75%; when the concentration of Example 1 is above 100 μg / mL, it has an inhibitory effect on Pseudomonas aeruginosa, and the inhibition rate is 51.63%; when the concentration of Example 1 is above 50 μg / mL, it has an inhibitory effect on Fusobacterium nucleatum, and the inhibition rate is 62.62%, and the MIC50 is 3.76%; when the concentration of Example 1 is above 12.5 μg / mL, it has an inhibitory effect on Streptococcus mutans, and the inhibition rate is 62.44%, and the MIC50 is 1.59%.

[0174] 4. Using TNF-a and PGE-2 as inflammation indicators, Elisa was used to detect the anti-inflammatory effect of Example 1.

[0175] 4.1. Inhibitory effect of Example 1 on inflammatory factors TNF-a and PGE-2

[0176] 4.1.1 Example 1 Cytotoxicity Assay

[0177] (1) Sample preparation

[0178] Example 1 is a water-soluble light yellow-green powder. The sample was dissolved in DMEM medium and diluted according to Table 6 to prepare the sample to be tested. Before loading the sample for cytotoxicity experiment, the sample was sterilized by filtration using a filter membrane.

[0179] Table 6 Sample information table

[0180] Sample name Sample No. Sample concentration Storage conditions Example 1 PJH 100μg / mL, 50μg / mL, 25μg / mL, 12.5μg / mL Store in a sealed container away from light

[0181] (2) Cell plating

[0182] 1) Preheat DMEM medium, PBS, and trypsin in a 37°C water bath.

[0183] 2) Rinse: Remove the cells from the incubator, discard the cell culture medium, and rinse twice with PBS, taking 3 mL of PBS each time (3 mL of PBS for large culture bottles and 1 mL of PBS for small culture bottles).

[0184] 3) Digestion: Add 3 mL of trypsin to digest the cells for 8 minutes and observe the digestion under a microscope. If the cells are digested by 80%-90%, add an equal volume of culture medium to terminate the digestion (3 mL of trypsin for large culture flasks and 1 mL of trypsin for small culture flasks).

[0185] 4) Transfer: Aspirate the cell solution in step (3) into an EP tube. Cells of the same generation can be placed in the same tube and centrifuged at 1000r for 4 minutes. After centrifugation, discard the supernatant, keep the precipitate, and add 1mL of culture medium to suspend the cells.

[0186] 5) Counting: Take 10 μL of the mixed cell solution and add it to a cell counting plate to detect the cell density. Calculate the required cell volume based on the cell density. Use the culture medium to prepare the cell solution required for plating.

[0187] 6) Laying planks: 1×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, and 100 μL was added to each well. The edge wells were filled with sterile PBS. The plate was placed in an incubator at 37°C and 5% CO. 2 After culturing in the environment, the sample is added to the wall.

[0188] (3) Cell loading

[0189] Place the 96-well plate in a 5% CO 2 After incubation at 37°C for 24 hours, the culture medium was aspirated and 100 μL of sample solution was added (three replicates for each concentration). The blank group was added with 100 μL of serum-free culture medium solution. The control group was dimethyl sulfoxide. Place in 5% CO 2 , incubate in a 37°C incubator for 24 h.

[0190] (4) CCK-8 assay

[0191] After 24 hours, the culture was terminated and 10 μL CCK-8 was added to each well of the 96-well plate (protected from light). 2 After incubation at 37℃ for 1 hour, the color of the culture medium changes from light pink to orange-yellow. At this time, the OD value is measured at a wavelength of 450nm using an enzyme marker. Cell activity is proportional to the OD value. When the cell survival rate is greater than 80%, it is the safe concentration of the sample.

[0192] Survival rate = (sample OD 450 / BlankOD 450 )*100%

[0193] (5) CCK-8 cytotoxicity test results

[0194] Table 7 CCK-8 sample safety concentration test results of Example 1

[0195] Concentration (μg / mL) 12.5 25 50 100 Cell survival rate / % 90.36％ 81.49％ 65.15％ 23.25％ SD 0.0378 0.0457 0.0452 0.0064

[0196] The results of the safe concentration test of CCK-8 samples in Example 1 are shown in Tables 7 and Figure 2 As shown, the above results show that Example 1 has no cytotoxicity at a concentration below 12.5 μg / mL. Example 1 will select a concentration of 12.5 μg / mL for subsequent experiments.

[0197] 4.1.2 Example 1 Determination of efficacy of inhibiting inflammatory factors TNF-a and PGE-2

[0198] (1) Hacat cell plating

[0199] 1) Digestion of cells:

[0200] UV irradiation for 20-30 minutes, spray paper towels with alcohol disinfection, wipe the clean bench. Take out the cell culture bottle, put it in the clean bench after disinfection, pour out the culture medium, wash twice with PBS, pour out PBS, add 1mL TE, pour out a small amount of TE after full contact, digest in the incubator for 8-9 minutes, and observe under a microscope.

[0201] 2) Stop digestion:

[0202] After the cells are completely digested from the wall of the culture flask, add 5 mL of complete culture medium to stop digestion and resuspend the cell solution with a disposable dropper.

[0203] 3) Cell counting:

[0204] Count the cell stock solution, take 10 μL to one side of the counting plate, and 10 μL to the other side, count 2-3 times, take the average value, calculate the cell density, and calculate and prepare the cell solution according to the cell density and volume required for plating.

[0205] 4)Planning:

[0206] Each well of a 24-well plate was inoculated with 0.5 mL of cell solution, and the cell density was 5.0 × 10 5 / mL, placed in an incubator at 37°C, 5% CO 2 Culture in the environment for 8-12 hours to allow the cells to adhere to the wall and grow.

[0207] (2) Cell loading

[0208] 1) Washing plate:

[0209] The 24-well plate was taken out, the culture medium was aspirated with a pipette, and then washed twice with 0.5 mL PBS.

[0210] 2) Loading:

[0211] A blank group, a UVB stimulation group, and a drug group were set up. The blank group was connected to 0.5 mL of serum-free culture medium; the UVB stimulation group was connected to 0.5 mL of serum-free culture medium; the drug group was connected to 0.5 mL of sample solutions of different concentrations, and cultured in the incubator for 6 hours. The UVB stimulation group and the drug group were irradiated with 0.15 J of UVB. After UVB irradiation, the blank group was connected to 0.5 mL of serum-free culture medium; the UVB stimulation group was connected to 0.5 mL of serum-free culture medium; the drug group was connected to 0.5 mL of sample solutions of different concentrations, and cultured for another 18 hours.

[0212] (3) TNF-α, PGE-2 ELISA kit detection

[0213] The collected cell culture supernatant was tested according to the instructions of the corresponding ELISA kit.

[0214] (4) Results of the measurement of the inhibition of inflammatory factors TNF-α and PGE-2 in Example 1

[0215] Table 8 TNF-α content detection experimental results of Example 1

[0216] blank UVB PJH-0.125 TNF-α content (pg / mL) 10.78±0.79 101.72±0.55 46.57±0.97

[0217] Table 9 PGE-2 content detection experimental results of Example 1

[0218] sample blank UVB PJH-0.25 PGE-2 content (pg / mL) 41.31±3.05 137.85±0.33 36.31±1.88

[0219] The results of the inhibition of inflammatory factors TNF-α and PGE-2 in Example 1 are shown in Tables 8 and 9. The above results show that compared with the blank control, the TNF-α and PGE-2 levels of Hacat cells were significantly increased after UVB stimulation, and after adding 12.5 μg / mL Example 1, the TNF-α and PGE-2 levels were decreased, indicating that it has a good anti-inflammatory effect.

[0220] 5. Through cell experiments (immunofluorescence) with FLG, Ca 2+ Channel protein (TRPV4) and itch factor (TSLP) were used as indicators to detect the soothing and repairing effects of Example 1.

[0221] 5.1. Immunofluorescence detection of FLG and TRPV4 in Example 1

[0222] (1) Sample preparation

[0223] Table 10 FLG, AQP-3 sample information table

[0224] Sample name Sample No. Sample concentration Storage conditions Example 1 PJH 12.5 μg / mL Store in refrigerator away from light

[0225] (2) Cell plating

[0226] 1) Preheat DMEM medium, PBS, and trypsin in a 37°C water bath.

[0227] 2) Rinse: Remove the cells from the incubator, discard the cell culture medium, and rinse twice with PBS, taking 3 mL of PBS each time (3 mL of PBS for large culture bottles and 1 mL of PBS for small culture bottles).

[0228] 3) Digestion: Add 3 mL of trypsin to digest the cells for 8 minutes and observe the digestion under a microscope. If the cells are digested by 80%-90%, add an equal volume of culture medium to terminate the digestion (3 mL of trypsin for large culture flasks and 1 mL of trypsin for small culture flasks).

[0229] 4) Transfer: Aspirate the cell solution in step (3) into an EP tube. Cells of the same generation can be placed in the same tube and centrifuged at 1000r for 4 minutes. After centrifugation, discard the supernatant, keep the precipitate, and add 1mL of culture medium to suspend the cells.

[0230] 5) Counting: Take 10 μL of the mixed cell solution and add it to a cell counting plate to detect the cell density. Calculate the required cell volume based on the cell density. Use the culture medium to prepare the cell solution required for plating.

[0231] 6) Laying planks: 1-2×10 5 The density of cells / mL was inoculated into a 48-well plate, and 0.5 mL was added to each well. The plate was placed in an incubator at 37°C and 5% CO 2 After the cells have been cultured in the environment for 12-24 hours and have adhered to the wall, samples are added.

[0232] (3) Cell loading

[0233] 1) Washing plate:

[0234] The 48-well plate was taken out, the culture medium was aspirated with a pipette, and then washed twice with 0.5 mL PBS.

[0235] 2) Loading:

[0236] A blank group, a UVB stimulation group, and a drug group were set up. The blank group was connected to 0.5 mL of serum-free culture medium; the UVB stimulation group was connected to 0.5 mL of serum-free culture medium; the drug group was connected to 0.5 mL of sample solutions of different concentrations, and cultured in the incubator for 6 hours. The UVB stimulation group and the drug group were irradiated with 0.15 J of UVB. After UVB irradiation, the blank group was connected to 0.5 mL of serum-free culture medium; the UVB stimulation group was connected to 0.5 mL of serum-free culture medium; the drug group was connected to 0.5 mL of sample solutions of different concentrations, and cultured for another 18 hours.

[0237] (4) Immunofluorescence detection

[0238] Preparation: PBS, 4% paraformaldehyde solution, 0.3% Tritonx-100, BSA

[0239] 1) Fixation: Wash twice with PBS, add 100 μL of 4% paraformaldehyde solution to each well to fix the cells, let stand at room temperature for 15 min, wash three times with PBS, let stand at room temperature for 5 min each time (prepare 0.3% Tritonx-100 and 5% BSA during the standing period).

[0240] Prepare 10 wells: Tritonx-100: 6μL Tritonx-100 + 2mL PBS

[0241] 2) Permeabilization: Add 100 μL of 0.3% Tritonx-100 (diluted with PBS) to each well for 10 min at room temperature, wash with PBS three times, and let stand at room temperature for 5 min each time.

[0242] Prepare blocking solution (5% BSA): 0.1g BSA + 1.9g PBS

[0243] 3) Blocking: Add 100 μL of 5% BSA (diluted with PBS) to each well and incubate at room temperature for 2 hours without washing.

[0244] Prepare the primary antibody in advance: 3 μL + 600 μL 5% BSA (1:200 dilution)

[0245] 4) Primary antibody binding: Add 100 μL of primary antibody diluted with antibody diluent (5% BSA in 1x PBS) to each well. Antibody ratio 1:200, incubate at 4°C for 12 hours, wash 3 times with PBS, 5 minutes each time. Prepare fluorescent secondary antibody in a dark place: 2 μL secondary antibody + 1 mL 5% BSA (1:500 dilution).

[0246] 5) Secondary antibody binding: Protect from light, add 100 μL of rabbit fluorescent secondary antibody diluted with antibody diluent (5% BSA in 1x PBS) to each well, the antibody ratio is 1:500, incubate at room temperature for 1.5 hours. Wash with PBS 3 times, 5 minutes each time.

[0247] 6) Staining cell nuclei: Protect from light, add 200 μL DAPI staining solution to each well, incubate at room temperature for 10 minutes, aspirate the staining solution, and wash with PBS three times, 5 minutes each time.

[0248] 7) Add fluorescence quencher to cover the bottom.

[0249] (5) FLG immunofluorescence detection results

[0250] Table 11 FLG immunofluorescence detection results of Example 1

[0251] sample blank UVB Example 1 (12.5 μg / mL) FLG relative fluorescence intensity 131.75 121.80 129.97 SD 0.5908 1.1681 0.8029

[0252] The results of FLG immunofluorescence detection are shown in Table 11 and Figure 3 As shown, the above results show that compared with the blank control, the fluorescence intensity of FLG expressed by Hacat cells after UVB stimulation was significantly downregulated. After adding 12.5 μg / mL Example 1, the fluorescence intensity of FLG increased by 6.71%, indicating that Example 1 can promote the expression of FLG after cell UVB damage and has a good barrier repair effect.

[0253] (6) TRPV4 immunofluorescence detection results

[0254] Table 12 TRPV4 immunofluorescence detection results of Example 1

[0255] sample blank UVB Example 1 (12.5 μg / mL) TRPV4 relative fluorescence intensity 144.09 152.34 136.55 SD 1.7232 0.7661 1.3173

[0256] The results of TRPV4 immunofluorescence detection are shown in Table 12 and Figure 4 As shown, the above results show that compared with the blank control, the fluorescence intensity of TRPV4 expressed by Hacat cells after UVB stimulation was significantly upregulated. After adding 12.5 μg / mL Example 1, the fluorescence intensity of TRPV4 decreased by 10.36%, indicating that 12.5 μg / mL Example 1 can inhibit the expression of TRPV4 after cell UVB damage, inhibit the influx of calcium ions, inhibit the release of ROS, and relieve inflammatory response.

[0257] 5.2. Example 1: Determination of the efficacy of inhibiting itch factor TSLP

[0258] (1) Hacat cell plating

[0259] 1) Digestion of cells:

[0260] UV irradiation for 20-30 minutes, spray paper towels with alcohol disinfection, wipe the clean bench. Take out the cell culture bottle, put it in the clean bench after disinfection, pour out the culture medium, wash twice with PBS, pour out PBS, add 1mL TE, pour out a small amount of TE after full contact, digest in the incubator for 8-9 minutes, and observe under a microscope.

[0261] 2) Stop digestion:

[0262] After the cells are completely digested from the wall of the culture flask, add 5 mL of complete culture medium to stop digestion and resuspend the cell solution with a disposable dropper.

[0263] 3) Cell counting:

[0264] Count the cell stock solution, take 10 μL to one side of the counting plate, and 10 μL to the other side, count 2-3 times, take the average value, calculate the cell density, and calculate and prepare the cell solution according to the cell density and volume required for plating.

[0265] 4)Planning:

[0266] Each well of a 24-well plate was inoculated with 0.5 mL of cell solution, and the cell density was 5.0 × 10 5 / mL, placed in an incubator at 37°C, 5% CO 2 Culture in the environment for 8-12 hours to allow the cells to adhere to the wall and grow.

[0267] (2) Cell loading

[0268] 1) Washing plate:

[0269] The 24-well plate was taken out, the culture medium was aspirated with a pipette, and then washed twice with 0.5 mL PBS.

[0270] 2) Loading:

[0271] A blank group, a UVB stimulation group, and a drug group were set up. The blank group was connected to 0.5 mL of serum-free culture medium; the UVB stimulation group was connected to 0.5 mL of serum-free culture medium; the drug group was connected to 0.5 mL of sample solutions of different concentrations, and cultured in the incubator for 6 hours. The UVB stimulation group and the drug group were irradiated with 0.15 J of UVB. After UVB irradiation, the blank group was connected to 0.5 mL of serum-free culture medium; the UVB stimulation group was connected to 0.5 mL of serum-free culture medium; the drug group was connected to 0.5 mL of sample solutions of different concentrations, and cultured for another 18 hours.

[0272] (3) TSLP ELISA kit detection

[0273] The collected cell culture supernatant was tested according to the instructions of the corresponding ELISA kit.

[0274] (4) Results of the measurement of TSLP, an inhibitory factor of itch, in Example 1

[0275] Table 13 TSLP content detection experimental results of Example 1

[0276] sample blank UVB Example 1 (12.5 μg / mL) TSLP content / pg / mL 6.56 22.59 4.69 SD 2.4001 3.3342 1.3294

[0277] The results of the TSLP content detection experiment are shown in Table 13. Compared with the blank control, the TSLP content of HaCaT cells was significantly increased after UVB stimulation. After adding 12.5 μg / mL of Example 1, the TSLP content was significantly decreased. Hops has an excellent soothing effect. The above results show that 12.5 μg / mL of Example 1 has significant antibacterial, anti-inflammatory, soothing and skin barrier repair effects, and can be developed and applied in functional cosmetic raw materials.

[0278] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A process for preparing a hop extract, characterized in that: The following steps are involved: Step 1, mixing hops and ethanol aqueous solution, performing heat reflux extraction for 1-3 times, concentrating the extract under reduced pressure and drying to obtain a crude hop extract; Step 2: Dissolve the crude hop extract in water to obtain a solution, perform column chromatography separation using AB-8 resin, elute with water and ethanol aqueous solution, collect the eluate, concentrate the eluate under reduced pressure, and then dry it to obtain the hop extract.

2. The preparation process according to claim 1, characterized in that: In step 1, the ratio of hops to ethanol aqueous solution is 15g:600-900mL.

3. The preparation process according to claim 1, characterized in that: In step 1, the concentration of the ethanol aqueous solution is 60%-90%.

4. The preparation process according to claim 1, characterized in that: In step 1, each thermal reflux extraction is performed for 120-210 min.

5. The preparation process according to claim 1, characterized in that: In step 2, the concentration of the solution is 0.5-5 g / L; the specific steps of the column chromatography separation are as follows: loading the sample at a speed of 2-5 mL / min, and after the adsorption is completed, standing for 2-4 hours.

6. The preparation process according to claim 1, characterized in that: In step 2, the volume of water used for elution is 1-4BV, and the volume of ethanol-water solution used for elution is 1-4BV.

7. The preparation process according to claim 1, characterized in that: In step 2, the concentration of the ethanol aqueous solution for elution is 60%-90%.

8. The preparation process according to claim 1, characterized in that: The preparation process comprises the following steps: Step 1, mixing hops and ethanol aqueous solution, performing heat reflux extraction for 3 times, combining the 3 extracts, filtering twice with a 0.45 μm filter plate to obtain a clear extract, concentrating the extract under reduced pressure, and freeze-drying the extract to obtain a crude hop extract; Step 2, dissolving the crude hop extract in water to obtain a solution, performing column chromatography separation using AB-8 resin, eluting with water and ethanol aqueous solution, collecting the eluate, concentrating the eluate under reduced pressure, and then freeze-drying the eluate to obtain the hop extract.

9. The hop extract obtained by the preparation process according to any one of claims 1 to 8.

10. Use of the hop extract according to claim 9 in the preparation of skin care products, cosmetics or medicines.

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