Hyssopus officinalis extract, preparation method and application

Abstract

The invention discloses a hyssopus officinalis extract and a preparation method and application thereof.The preparation method of the hyssopus officinalis extract comprises the following steps that a mixed solution of hyssopus officinalis and honey is covered, moistened and stir-fried, and a primary product is obtained; and heating a mixed solution of the head product and polyhydric alcohol at the temperature of 80-100 DEG C for 5-10 hours to prepare the hyssopus officinalis extract. The hyssopus officinalis extract prepared by the preparation method disclosed by the invention can activate endogenous antioxidation of a human body, inhibit expression of MMP-1, promote synthesis of elastin in human true epidermis cells, promote expression of HSP27 in HaCaT cells and down-regulate expression of HMGB1 in the HaCaT cells, and has a better anti-photoaging effect.

Description

Technical Field

[0001] The invention relates to a Hyssopus officinalis extract, a preparation method and application. Background Art

[0002] The skin is the largest organ in the human body and is also the body's first line of defense. Skin aging includes natural aging and exogenous aging. Natural aging refers to the phenomenon of skin aging caused by irresistible factors such as gravity, decreased physiological functions of the body, aging, and genetics. Exogenous aging includes aging caused by ultraviolet rays (UV), smoking, wind and sunlight, exposure to harmful chemicals, and other factors. Because UV radiation is the main cause of skin aging, exogenous aging is also called skin photoaging. Studies have shown that 80% of facial aging is caused by photoaging, with clinical manifestations such as rough skin, sagging, wrinkles, pigmentation spots, and even the induction of skin cancer.

[0003] Honey roasting is one of the roasting methods, also known as honey processing. It is a roasting method that uses honey as an auxiliary ingredient. It is a method of adding a certain amount of refined honey to the cleaned or cut medicinal materials and stir-frying them. The purpose of honey roasting is (1) to enhance the effect of moistening the lungs and relieving coughs: such as Stemona, Coltsfoot, Aster, etc. (2) to enhance the effect of tonifying the spleen and replenishing qi: such as Astragalus, Licorice, Codonopsis, etc. (3) to alleviate the medicinal properties: such as Ephedra, Cinnamon Twig, Cimicifuga, etc. (4) to correct the taste and eliminate side effects: such as Aristolochia, Stemona, etc.

[0004] Hyssopus officinalis, a genus of the Lamiaceae family, comprises 15 species, most of which are perennial herbs or semi-shrubs, distributed from the Mediterranean to Central Asia. Two species, Hyssopus cuspidatus Boriss and Hyssopus latilabiatus (CYWu et HWLi), are found in my country. These two species primarily grow in northern Xinjiang (Altai Prefecture), with the entire plant used as medicine. Hyssopus officinalis L., a member of the same genus, is also used as a medicinal herb. Hyssopus officinalis, a traditional folk medicinal herb in Xinjiang, China, has a secondary dry-heat property and is primarily used to warm the lungs and relieve asthma, dispel cold and relieve cough, dry dampness and resolve phlegm, induce sweating and detoxify, and reduce inflammation and swelling. It is generally used to treat respiratory diseases characterized by dampness, cold, and mucus. Currently, the main compounds isolated from Hyssopus plants include terpenes, flavonoids, phenolic acids, phenylpropanoids, and steroids.

[0005] Currently, common methods for preparing Hyssopus officinalis extract include solvent extraction, resin adsorption separation, and distillation. Hyssopus officinalis extract has anti-inflammatory and immunomodulatory effects and can be used to prepare anti-lung cancer and anti-inflammatory drugs. Numerous studies have investigated the anti-inflammatory mechanism of Hyssopus officinalis, but no research has examined its anti-photoaging properties. Summary of the Invention

[0006] The present invention provides a Hyssopus officinalis extract, a preparation method and an application thereof to fill the research gap of the anti-photoaging effect of Hyssopus officinalis. The Hyssopus officinalis extract extracted by the method of the present invention has good antioxidant and anti-photoaging properties as a skin care product.

[0007] The present invention solves the above technical problems through the following technical solutions:

[0008] The present invention provides a method for preparing a Hyssopus officinalis extract, which comprises the following steps:

[0009] (1) Stir-frying the mixture of Hyssopus officinalis and honey to obtain a primary product;

[0010] (2) heating the mixture of the primary product and the polyol to obtain the Hyssopus officinalis extract; the heating temperature is 80-100° C., and the heating time is 5-10 hours.

[0011] In the present invention, in step (1), the Hyssopus officinalis can be conventional in the art, preferably dried Hyssopus officinalis.

[0012] The moisture content of the Hyssopus officinalis is preferably 10-13%.

[0013] Wherein, the Hyssopus officinalis is preferably cut into sections.

[0014] In the present invention, in step (1), the mass ratio of the Hyssopus officinalis to the honey is preferably (5-20):1, more preferably (8-13):1, for example, 10:1, 11:1, 12:1 or 12.5:1.

[0015] In the present invention, in step (1), the solvent in the mixed solution is generally water.

[0016] In the present invention, in step (1), in the mixed solution, the mass ratio of the honey to the solvent is preferably (2-5):1, such as 2:1, 2.3:1, 2.5:1 or 3.3:1, more preferably (2-3:1).

[0017] In the present invention, in step (1), the method for preparing the mixed solution preferably comprises the following steps: firstly mixing the honey and a solvent, and then mixing the mixture with the Hyssopus officinalis.

[0018] In the present invention, in step (1), the honey is preferably refined honey. The refining process is conventional in the art. Preferably, in certain embodiments of the present invention, the refining temperature is preferably 100-120° C., and the holding time is preferably 5-15 minutes.

[0019] In the present invention, in step (1), the type of honey can be conventional in the art, preferably acacia honey.

[0020] In the present invention, in step (1), the humidification can be conventional in the art, generally leaving the material for a period of time. The humidification time is preferably 2 to 10 hours, more preferably 3 to 5 hours, for example 3 hours, 5 hours or 6 hours.

[0021] In the present invention, in step (1), the stir-frying can be conventional in the art. The stir-frying temperature is preferably 120-250° C. The stir-frying time is based on the surface of the medicinal material being slightly yellow and not sticky, preferably 3-15 minutes, for example 3-8 minutes.

[0022] In the present invention, in step (1), the frying step can be conventional in the art, and preferably includes the following steps: placing the moistened material in a preheated frying device for frying. The preheating temperature is preferably 100-150°C.

[0023] In the present invention, in step (1), the primary product is preferably in powder form and has a particle size of 250 to 350 μm.

[0024] In the present invention, in step (1), a crushing step can preferably be provided after the frying, so that the primary product is in powder form.

[0025] The crushing step generally includes a sieving step after the pulverization, wherein the sieve used for the sieving is preferably 45 to 80 mesh, more preferably 45 to 60 mesh.

[0026] In the present invention, in step (2), the mass ratio of the primary product to the polyol is preferably 1:(7-12), more preferably 1:(8-10), for example 1:8, 1:9 or 1:10.

[0027] In the present invention, in step (2), the polyol can be conventional in the art, such as one or more of butanediol, ethylene glycol, glycerol and 1,2-hexanediol.

[0028] In the present invention, in step (2), the heating temperature is preferably 85-100°C, such as 85°C, 90°C or 100°C.

[0029] In the present invention, in step (2), the heating time is preferably 6 to 8 hours, such as 5 hours, 8 hours, 9 hours or 10 hours.

[0030] In the present invention, in step (2), the heating process may generally include a stirring step. The stirring time is sufficient to uniformly mix the primary product and the polyol.

[0031] In the present invention, in step (2), the product obtained after heating is generally also referred to as an extract.

[0032] In the present invention, in step (2), preferably, the heated product is filtered.

[0033] The filtration may be conventional in the art, for example, including one or more of gauze filtration, microfiltration membrane filtration and ultrafiltration membrane filtration.

[0034] Wherein, the filtration preferably includes gauze filtration, microfiltration membrane filtration and ultrafiltration membrane filtration in sequence.

[0035] The pore size of the gauze in the gauze filtration is preferably 100-400 meshes, for example 200 meshes.

[0036] The pore size of the microfiltration membrane used in the microfiltration membrane filtration is preferably 2.5 to 10 μm, such as 2.5 μm, 6 μm, 8 μm or 10 μm.

[0037] The pore size of the ultrafiltration membrane used in the ultrafiltration membrane filtration is preferably 0.45 to 0.8 μm, such as 0.45 μm or 0.6 μm.

[0038] In a preferred embodiment, the heated product is filtered sequentially through 200-mesh gauze, a microfiltration membrane with a pore size of 10 μm, and an ultrafiltration membrane with a pore size of 0.6 μm.

[0039] In a preferred embodiment, the heated product is filtered sequentially through 100-mesh gauze, a microfiltration membrane with a pore size of 8 μm, and an ultrafiltration membrane with a pore size of 0.45 μm.

[0040] In a preferred embodiment, the heated product is filtered sequentially through 400-mesh gauze, a microfiltration membrane with a pore size of 6 μm, and an ultrafiltration membrane with a pore size of 0.6 μm.

[0041] In a preferred embodiment, the heated product is filtered sequentially through 400-mesh gauze, a microfiltration membrane with a pore size of 2.5 μm, and an ultrafiltration membrane with a pore size of 0.6 μm.

[0042] The present invention also provides a Hyssopus officinalis extract prepared by the preparation method of the Hyssopus officinalis extract.

[0043] The present invention also provides a use of the Hyssopus officinalis extract in cosmetics.

[0044] In the present invention, the concentration of the Hyssopus officinalis extract is preferably 0.4-2.5%, such as 0.5%, 1% or 2%, where % refers to the ratio of the mass of the Hyssopus officinalis extract to the sum of the mass of the Hyssopus officinalis extract and the culture medium.

[0045] In the present invention, preferably, the Hyssopus officinalis extract is used as an anti-photoaging active ingredient in cosmetics.

[0046] In the present invention, preferably, the Hyssopus officinalis extract is used in cosmetics as an Nrf2 expression promoter, an MMP-1 expression inhibitor, an elastin synthesis promoter in human epidermal cells, an HSP27 expression promoter in HaCaT cells, or an HMGB1 expression inhibitor in HaCaT cells.

[0047] In the present invention, the cosmetics can be aqueous solutions, emulsions, sprays, creams, facial masks, sunscreens, liquid foundations, etc.

[0048] The positive progress effect of the present invention is:

[0049] The Hyssopus officinalis extract of the present invention has a higher total flavonoid content;

[0050] The Hyssopus officinalis extract of the present invention can activate endogenous antioxidants in the human body, inhibit the expression of MMP-1, promote the synthesis of elastin in human epidermal cells, promote the expression of HSP27 in HaCaT cells, thereby helping to resist thermal aging, and downregulate the expression of HMGB1 in HaCaT cells, thereby having a good anti-photoaging effect. DETAILED DESCRIPTION

[0051] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0052] The Hyssopus officinalis L. used in the following examples is the dried whole herb purchased from Xinjiang Hyssopus officinalis Pharmaceutical Technology Co., Ltd., with a moisture content of 10-13%.

[0053] The honey in the following examples is refined honey, the refining process is heating at 100-120° C. for 5-15 minutes, and the type of honey is commercially available acacia honey.

[0054] Example 1

[0055] 1) Take 50g of honey and dilute it with 20g of boiling water. Cut 600g of dried Hyssopus officinalis into sections and place them in the honey water. Mix well and let it sit for 3 hours until the honey water is fully absorbed by the Hyssopus officinalis.

[0056] 2) Place the steamed Hyssopus officinalis in a preheated (100-150°C) stir-fry container and heat over a low heat (120-250°C). Stir-fry until the surface of the herb is slightly yellow and no longer sticky (the specific stir-frying time is 3-8 minutes). Remove and let cool.

[0057] 3) Grinding the honey-roasted hyssopus officinalis obtained in step 2) through a 45-mesh sieve to obtain hyssopus officinalis powder, mixing the hyssopus officinalis powder with butylene glycol in a mass ratio of 1:8, and stirring for 3 to 5 minutes to obtain a mixed solution;

[0058] 4) heating the mixed solution obtained in step 3) to 90° C. for 5 h, and stirring to obtain an extract;

[0059] 5) The extract obtained in step 4) was filtered through 200-mesh gauze, and the crude extract was filtered through a microfiltration membrane with a pore size of 10 μm and an ultrafiltration membrane with a pore size of 0.6 μm, and the filtrate was collected to obtain the Hyssopus officinalis extract.

[0060] Experimental Example 2

[0061] 1) Take 50g of honey and dilute it with 25g of boiling water. Cut 500g of dried Hyssopus officinalis into sections and place them in the honey water. Mix well and let it sit for 3 hours until the honey water is fully absorbed by the Hyssopus officinalis.

[0062] 2) Place the steamed Hyssopus officinalis in a preheated (100-150°C) stir-fry container and heat over a low heat (120-250°C). Stir-fry until the surface of the herb is slightly yellow and no longer sticky (the specific stir-frying time is 3-8 minutes). Remove and let cool.

[0063] 3) Grinding the honey-roasted hyssopus officinalis obtained in step 2) through a 60-mesh sieve to obtain hyssopus officinalis powder, mixing the hyssopus officinalis powder with butylene glycol in a mass ratio of 1:9, and stirring for 3 to 5 minutes to obtain a mixed solution;

[0064] 4) heating the mixed solution obtained in step 3) to 85° C. for 8 h, and stirring to obtain an extract;

[0065] 5) The extract obtained in step 4) was filtered through 100-mesh gauze, and the crude extract was filtered through a microfiltration membrane with a pore size of 8 μm and an ultrafiltration membrane with a pore size of 0.45 μm, and the filtrate was collected to obtain the Hyssopus officinalis extract.

[0066] Experimental Example 3

[0067] 1) Take 100g of honey and dilute it with 30g of boiling water. Cut 1100g of dried Hyssopus officinalis into sections and place them in the honey water. Mix well and let it sit for 5 hours until the honey water is fully absorbed by the Hyssopus officinalis.

[0068] 2) Place the steamed Hyssopus officinalis in a preheated (100-150°C) stir-fry container and heat over a low heat (120-250°C). Stir-fry until the surface of the herb is slightly yellow and no longer sticky (the specific stir-frying time is 3-8 minutes). Remove and let cool.

[0069] 3) Grinding the honey-roasted Hyssopus officinalis obtained in step 2) through a 60-mesh sieve to obtain Hyssopus officinalis powder, mixing the Hyssopus officinalis powder with a butylene glycol aqueous solution in a mass ratio of 1:10, and stirring for 3 to 5 minutes to obtain a mixed solution;

[0070] 4) heating the mixed solution obtained in step 3) to 90° C. for 10 h, and stirring to obtain an extract;

[0071] 5) The extract obtained in step 4) was filtered through 400-mesh gauze, and the crude extract was filtered through a microfiltration membrane with a pore size of 6 μm and an ultrafiltration membrane with a pore size of 0.6 μm, and the filtrate was collected to obtain the Hyssopus officinalis extract.

[0072] Experimental Example 4

[0073] 1) Take 80g of honey and dilute it with 35g of boiling water. Cut 1000g of dried Hyssopus officinalis into sections and place them in the honey water. Mix well and let it sit for 6 hours until the honey water is fully absorbed by the Hyssopus officinalis.

[0074] 2) Place the steamed Hyssopus officinalis in a preheated (100-150°C) stir-fry container and heat over a low heat (120-250°C). Stir-fry until the surface of the herb is slightly yellow and no longer sticky (the specific stir-frying time is 3-8 minutes). Remove and let cool.

[0075] 3) Grinding the honey-roasted hyssopus officinalis obtained in step 2) through a 45-mesh sieve to obtain hyssopus officinalis powder, mixing the hyssopus officinalis powder with a butylene glycol aqueous solution in a mass ratio of 1:9, and stirring for 3 to 5 minutes to obtain a mixed solution;

[0076] 4) heating the mixed solution obtained in step 3) to 100° C. for 9 hours, and stirring to obtain an extract;

[0077] 5) The extract obtained in step 4) was filtered through 400-mesh gauze, and the crude extract was filtered through a microfiltration membrane with a pore size of 2.5 μm and an ultrafiltration membrane with a pore size of 0.6 μm, and the filtrate was collected to obtain the Hyssopus officinalis extract.

[0078] Comparative Example 1

[0079] 1) Grind 600 g of dried Hyssopus officinalis through a 45-mesh sieve to obtain Hyssopus officinalis powder, place it in 60 g of water, and soak it thoroughly;

[0080] 2) Mixing the soaked Hyssopus officinalis with a butylene glycol aqueous solution in a mass ratio of 1:8 and stirring for 3-5 minutes to obtain a mixed solution;

[0081] 3) heating the mixed solution obtained in step 2) to 90° C. for 5 h, and stirring to obtain an extract;

[0082] 4) The extract obtained in step 3) was filtered through 200-mesh gauze, and the crude extract was filtered through a microfiltration membrane with a pore size of 10 μm and an ultrafiltration membrane with a pore size of 0.6 μm, and the filtrate was collected to obtain the Hyssopus officinalis extract.

[0083] Comparative Example 2

[0084] Comparative Example 2 is compared with Example 1, except that the heating time in step 4) is 3 h, and the other experimental methods are the same as those in Example 1.

[0085] Effect Example 1, Antioxidant Test

[0086] Nrf2 is a transcription factor present in the cytoplasm that regulates the expression of genes with antioxidant properties, such as HO-1, NQO-1, and GST. It is crucial for cellular defense against oxidative damage. By activating the Nrf2 pathway, it promotes antioxidant activity, scavenges reactive oxygen species generated within the skin, and protects cells.

[0087] The test scheme is shown in Table 1. The experiment is divided into a blank control group, a negative control group, a positive control group and a test sample group.

[0088] Table 1

[0089] Reagent name and volume Sample name Sample concentration Blank control group (BC) / / Negative control group (NC) <![CDATA[H2O2]]> 1mM Positive control group (PC) <![CDATA[H2O2+EGCG]]> 1ppm Sample group to be tested <![CDATA[H2O2 + Hyssop extract]]> 0.5％、1％、2％

[0090] Preparation of working fluid:

[0091] The sample in the positive control group was a mixture of H2O2 and EGCG. The mixture was diluted with DMEM medium (purchased from Gibco, model 10569-010) to a solution with an EGCG concentration of 1 ppm. PPM refers to the ratio of the mass of EGCG to the mixture. In the mixture, the H2O2 concentration was 1 mM. mM refers to the ratio of the molar amount of H2O2 to the volume of the mixture.

[0092] The samples in the sample group to be tested are mixtures of H2O2 and the Hyssopus officinalis extracts of various examples and comparative examples. The mixture corresponding to Example 1 is diluted with DMEM culture medium (purchased from Gibco, model number 10569-010) to solutions with Hyssopus officinalis extract concentrations of 0.5%, 1% and 2%, and the mixtures corresponding to the other examples and comparative examples are diluted to solutions with Hyssopus officinalis extract concentrations of 2%. % refers to the ratio of Hyssopus officinalis extract to the mass of the mixture.

[0093] Steps:

[0094] HaCaT cells (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) were treated with H2O2 solution, H2O2 solution + EGCG solution, and H2O2 solution + sample solution, respectively. After incubation at 37°C, 5% CO2 for 6 hours, proteins were extracted and separated by SDS-PAGE. The relative expression of Nrf2 was then determined by Western blotting using an antigen-antibody reaction.

[0095] The test results are shown in Table 2.

[0096] Table 2

[0097]

[0098] The relative expression of Nrf2 refers to the ratio of the expression of Nrf2 gene to the expression of the internal reference gene β-Actin, and the unit is %.

[0099] It can be seen that when the concentration is 2%, the relative expression of Nrf2 in the Hyssopus officinalis extracts obtained in Examples 1-4 is higher than that in Comparative Examples 1-2, indicating that the Hyssopus officinalis extracts obtained in the examples have a higher antioxidant effect than the comparative examples. When the Hyssopus officinalis extract obtained in Example 1 is at a concentration of 2%, 1%, and 0.5%, the relative expression of Nrf2 increases significantly, with an upregulation rate of 50.1%, 9.5%, and 17.2%, respectively. The upregulation rate = (relative expression of Nrf2 in the test sample group - relative expression of Nrf2 in the negative control group) / relative expression of Nrf2 in the negative control group. This indicates that the Hyssopus officinalis extract has an antioxidant effect under these conditions.

[0100] Effect Example 2, determination of matrix metalloproteinase MMP-1 content

[0101] MMP-1, also known as collagenase-1, is a marker for wrinkle formation.

[0102] Studies have shown that ultraviolet rays (UVA) regulate the synthesis of multiple MMPs such as collagen I, III, V, the main matrix components of the dermis, and collagen IV, VII, proteoglycans, laminin, the main matrix components of the basement membrane, to decompose the cells and other matrix components and elastic fibers of the dermis layer of the skin, thereby inducing skin wrinkles and photoaging.

[0103] The test scheme is shown in Table 3. The experiment is divided into a blank control group, a negative control group, a positive control group and a test sample group.

[0104] Table 3

[0105]

[0106] Preparation of working fluid:

[0107] Preparation of EGCG solution: EGCG (purchased from Sigma) was diluted to 0.5 μg / ml using DMEM culture medium (purchased from Gibco, model number 10567014).

[0108] Preparation of sample solution: The Hyssopus officinalis extract of Example 1 was diluted with DMEM culture medium to prepare sample solutions with concentrations of 0.5%, 1% and 2%, and the Hyssopus officinalis extracts of other examples and comparative examples were diluted to prepare sample solutions with a concentration of 2%, where % refers to the mass ratio of Hyssopus officinalis extract to the sample solution.

[0109] Steps:

[0110] Human dermal fibroblasts (HDF) (purchased from Boxi Biotechnology) were seeded in a 60 mm culture dish and cultured. After washing with buffer, the cells were irradiated with UVA (intensity of 25 J / cm 2 ), replaced with serum-free medium, added 2 ml EGCG solution and 2 ml sample solution, and cultured in a microbial incubator at 37°C and 5% CO2. TM Cells were collected with a lysis reagent and RNA was isolated according to the manufacturer's protocol. The isolated RNA was quantified and then synthesized into cDNA for real-time PCR (Applied Biosystems kit, fast real-time quantitative PCR instrument) to determine the relative expression level of MMP-1.

[0111] The test results are shown in Table 4.

[0112] Table 4

[0113]

[0114] The relative expression of MMP-1 refers to the ratio of the expression of MMP-1 gene to the expression of the internal reference gene β-Actin, and the unit is %.

[0115] It can be seen that at a concentration of 2%, the relative expression levels of MMP-1 in the Hyssopus officinalis extracts obtained in Examples 1-4 were all lower than those in Comparative Examples 1-2, indicating that the Hyssopus officinalis extracts obtained in Examples 1-4 have better anti-aging effects than Comparative Examples 1-2. The Hyssopus officinalis extract obtained in Example 1 significantly decreased at concentrations of 2%, 1%, and 0.5%, with downregulation rates of 62.6%, 17.1%, and 16%, respectively. Downregulation rate = (relative expression level of MMP-1 in the test sample group - relative expression level of MMP-1 in the negative control group) / relative expression level of MMP-1 in the negative control group. This indicates that the Hyssopus officinalis extract has anti-wrinkle efficacy under these conditions.

[0116] Effect Example 3, determination of elastin content

[0117] Fibroblasts are the main cellular components of the skin. They are distributed in the dermis of the skin and are the most common cells in loose connective tissue. They can produce a large amount of collagen and elastic fibers, playing an important role in maintaining the structural stability and elasticity of the skin.

[0118] Elastin content determines skin firmness and thus affects the condition of the skin. A decrease in elastin content can lead to skin aging. Therefore, the firming effect of a sample can be evaluated by testing its effect on elastin content using an enzyme-linked immunosorbent assay (ELISA).

[0119] The test scheme is shown in Table 5. The experiment is divided into a blank control group, a positive control group and a test sample group.

[0120] Table 5

[0121]

[0122] Preparation of working fluid:

[0123] TGF-β1 solution: 10 μg / mL TGF-β1 (purchased from GenScript, model Z03411) was diluted to 100 ng / mL using DMEM culture medium (purchased from Gibco, model 10567014) to obtain a TGF-β1 solution with a concentration of 100 ng / mL.

[0124] Preparation of sample solution: The Hyssopus officinalis extract of Example 1 was diluted with DMEM culture medium (purchased from Gibco, model number 10567014) to prepare sample solutions with concentrations of 0.5%, 1% and 2%, and the Hyssopus officinalis extracts of other examples and comparative examples were diluted to prepare sample solutions with a concentration of 2%, where % refers to the mass ratio of Hyssopus officinalis extract to the sample solution.

[0125] Steps:

[0126] 1) Cell plating: The cell density is 8×10 4 Human dermal fibroblasts HDF (purchased from Boxi Biotechnology) were seeded into a 96-well plate at a density of 100 μl / mL. The inoculated cell culture plate was placed in an incubator and cultured for 24 h (5% CO2, 37°C).

[0127] 2) Administration: After culturing the cells for 24 hours, remove the supernatant and add 200 μl of sample solution and 200 μl of TGF-β1 solution to each well, mix well, and place in an incubator for 24 hours. + 1h.

[0128] 3) Cell Viability Assay: After 24 hours, collect the supernatant (for ELISA analysis) and add 100 μl of CCK-8 working solution (purchased from Tongren Chemical, model CK04) to each well. Place the cells in an incubator for 1 to 4 hours. Measure absorbance at 450 nm and save the data electronically for subsequent analysis.

[0129] 4) ELISA assay: The cell culture supernatant was collected and then subjected to ELISA assay.

[0130] The test results are shown in Table 6.

[0131] Table 6

[0132]

[0133] It can be seen that at a concentration of 2%, the elastin content of the Hyssopus officinalis extracts obtained in Examples 1-4 was higher than that in Comparative Examples 1-2, indicating that the Hyssopus officinalis extracts obtained in Examples 1-4 had a better firming effect than Comparative Examples 1-2. The elastin content of the Hyssopus officinalis extract obtained in Example 1 increased significantly at concentrations of 2%, 1%, and 0.5%, with increase rates of 29.4%, 22.7%, and 18.0%, respectively. The increase rate = (elastin content of the test sample group - elastin content of the blank control group) / elastin content of the blank control group. This indicates that the Hyssopus officinalis extract has a firming effect under these conditions.

[0134] Effect Example 4, determination of the content of heat shock protein HSP27

[0135] When cells are exposed to high temperatures, they release proteins (heat shock proteins or stress proteins) to protect themselves. Heat shock proteins (HSPs) absorb heat to prevent cell death. They are secreted by cells exposed to stress and increase in levels in response to various stimuli, such as electrical stimulation and chemicals. Promoting the release of HSPs may improve skin aging.

[0136] The test scheme is shown in Table 7. The experiment is divided into a blank control group, a negative control group, a positive control group and a test sample group.

[0137] Table 7

[0138]

[0139] Preparation of working fluid:

[0140] Preparation of resveratrol solution: Resveratrol (purchased from Sigma) was diluted to 20 μM using DMEM culture medium (purchased from Gibco, model number 10569-010) to obtain a resveratrol solution.

[0141] Preparation of sample solution: The Hyssopus officinalis extract of Example 1 was diluted with DMEM culture medium (purchased from Gibco, model number 10569-010) to prepare sample solutions with concentrations of 0.5%, 1%, and 2%, and the Hyssopus officinalis extracts of other examples and comparative examples were diluted to prepare sample solutions with a concentration of 2%, where % refers to the mass ratio of Hyssopus officinalis extract to the sample solution.

[0142] The steps are as follows:

[0143] 1) Grouping: Set up a blank control group (DMEM culture medium + HaCaT cells, BC), a negative control group (DMEM culture medium + HaCaT cells + 50mJ UVB irradiation, NC), a positive control group (HaCaT cells + 50mJ UVB irradiation + resveratrol solution, PC), and a test sample group (HaCaT cells + 50mJ UVB irradiation + sample solution). The dosage of each group is 2ml.

[0144] 2) Cell plating: HaCaT cells in logarithmic phase growth (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were obtained, the culture medium was discarded, and the cells were washed twice with PBS (purchased from Pronose). 0.25% trypsin was added and the cells were incubated at 37°C for 5 min to terminate the digestion. The cells were centrifuged at 1000 rpm for 3 min, and resuspended in complete DMEM culture medium. The cells were counted and the cell concentration was adjusted to 1.5 × 10 5 1 ml was added to each well of a 48-well plate and cultured at 37°C in a 5% CO2 environment for 24 hours.

[0145] 3) Administration: After 24 hours, cells were allowed to adhere and irradiated with UVB at an intensity of 50 mJ using a UV crosslinker. After irradiation, the culture medium was discarded, and 2 ml of the test sample and 2 ml of 20 μM resveratrol were added to the corresponding wells. The cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours.

[0146] 4) RNA detection: After 24 hours, RNA was extracted and reverse transcribed into cDNA to detect the relative expression level of HSP27 mRNA.

[0147] The test results are shown in Table 8.

[0148] Table 8

[0149]

[0150]

[0151] The relative expression level of HSP27 refers to the ratio of the expression level of HSP27 to the expression level of the internal reference gene β-Actin, and the unit is %.

[0152] It can be seen that at a concentration of 2%, the relative expression levels of HSP27 in the Hyssopus officinalis extracts obtained in Examples 1-4 were all higher than those in Comparative Examples 1-2, indicating that the Hyssopus officinalis extracts obtained in Examples 1-4 have better anti-thermal aging effects than Comparative Examples 1-2. The relative expression levels of HSP27 in the Hyssopus officinalis extract obtained in Example 1 decreased significantly at concentrations of 2%, 1%, and 0.5%, with upregulation rates of 64.9%, 35.0%, and 21.8%, respectively. The downregulation rate = (relative expression level of HSP27 in the test sample group - relative expression level of HSP27 in the negative control group) / relative expression level of HSP27 in the negative control group. This indicates that the Hyssopus officinalis extract has an anti-photoaging effect under these conditions.

[0153] Effect Example 5, Determination of the content of high mobility group protein HMGB1

[0154] HMGB1 is normally located in the cell nucleus, bound to chromatin, and involved in maintaining nucleosome stability. When cells are damaged by light, HMGB1 is released and can bind to RAGE to mediate inflammatory responses, further contributing to skin aging. This experimental protocol uses immortalized human keratinocytes (HaCaT) to measure the effect of test substances on HMGB1 levels in the cell culture supernatant after UVB stimulation, thereby evaluating their anti-photoaging efficacy.

[0155] The test scheme is shown in Table 9. The experiment is divided into a blank control group, a negative control group, a positive control group and a test sample group.

[0156] Table 9

[0157]

[0158]

[0159] Preparation of working fluid:

[0160] Preparation of resveratrol solution: Resveratrol (purchased from Sigma) was diluted to 20 μM using DMEM culture medium (purchased from Gibco, model number 10569-010) to obtain a resveratrol solution.

[0161] Preparation of sample solution: The Hyssopus officinalis extract of Example 1 was diluted with DMEM culture medium (purchased from Gibco, model number 10569-010) to prepare sample solutions with concentrations of 0.5%, 1%, and 2%, and the Hyssopus officinalis extracts of other examples and comparative examples were diluted to prepare sample solutions with a concentration of 2%, where % refers to the mass ratio of Hyssopus officinalis extract to the sample solution.

[0162] The steps are as follows:

[0163] 1) Grouping: Set up a blank control group (DMEM culture medium + HaCaT cells, BC), a negative control group (DMEM culture medium + HaCaT cells + 50mJ UVB irradiation, NC), a positive control group (HaCaT cells + 50mJ UVB irradiation + resveratrol solution, PC), and a test sample group (HaCaT cells + 50mJ UVB irradiation + sample solution). The dosage of each group is 2ml.

[0164] 2) Cell plating: The cell concentration was 2×10 5 The cells were seeded into a 6-well plate at a density of 500 μg / ml, with 2 ml per well. The inoculated cell culture plate was placed in an incubator and cultured for 24 h (5% CO2, 37°C).

[0165] 3) Administration: After 24 hours, cells were allowed to adhere and irradiated with UVB at an intensity of 50 mJ using a UV crosslinker. After irradiation, the culture medium was discarded, and 2 ml of the test sample and 2 ml of 20 μM resveratrol were added to the corresponding wells. The cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours.

[0166] 4) ELISA assay: After 24 hours, the cell culture supernatant was collected and ELISA assay was performed according to the ELISA kit instructions.

[0167] The test results are shown in Table 10.

[0168] Table 10

[0169]

[0170]

[0171] As can be seen, at a concentration of 2%, the HMGB1 content of the 2% Hyssopus officinalis extracts obtained in Examples 1-4 was lower than that in Comparative Examples 1-2, indicating that the Hyssopus officinalis extracts obtained in Examples 1-4 had better anti-photoaging effects than those in Comparative Examples 1-2. The HMGB1 content of the Hyssopus officinalis extract obtained in Example 1 decreased significantly at concentrations of 1% and 2%, with downregulation rates of 20.2% and 34.5%, respectively. Downregulation rate = (HMGB1 content of the test sample group - HMGB1 content of the negative control group) / HMGB1 content of the negative control group. This indicates that the Hyssopus officinalis extract has anti-photoaging effects under these conditions.

Claims

1. A method for preparing Hyssopus officinalis extract, characterized in that: It includes the following steps: (1) Stir-frying the mixture of Hyssopus officinalis and honey to obtain a primary product; (2) heating the mixture of the primary product and the polyol to obtain the Hyssopus officinalis extract; the heating temperature is 80-100° C., and the heating time is 5-10 hours.

2. The method for preparing the Hyssopus officinalis extract according to claim 1, wherein The mass ratio of the hyssopus officinalis to the honey is (5-20):1, preferably (8-13):1, such as 10:1, 11:1, 12:1 or 12.5:

1.

3. The method for preparing the Hyssopus officinalis extract according to claim 1, wherein The solvent in the mixed liquid is water; And / or, in the mixed solution, the mass ratio of the honey to the solvent is (2-5):1, such as 2:1, 2.3:1, 2.5:1 or 3.3:1, more preferably (2-3):1; And / or, the preparation method of the mixed liquid comprises the following steps: firstly mixing the honey and the solvent, and then mixing with the Hyssopus officinalis.

4. The method for preparing the Hyssopus officinalis extract according to claim 1, wherein The preparation method of the Hyssopus officinalis extract satisfies any one or more of the following conditions: ① The Hyssopus officinalis is dried Hyssopus officinalis, and the moisture content of the Hyssopus officinalis is preferably 10-13%; ② The primary product is in powder form, and the particle size of the primary product is preferably 250 to 350 μm; ③ After the frying, the method further comprises the step of crushing; ④ The honey is refined honey, the refining temperature is preferably 100-120°C, and the insulation time is preferably 5-15 minutes; and, ⑤The honey is acacia honey.

5. The method for preparing the Hyssopus officinalis extract according to claim 1, wherein The preparation method of the Hyssopus officinalis extract satisfies any one or more of the following conditions: ① The moistening time is 2 to 10 hours, more preferably 3 to 5 hours, for example 3 hours, 5 hours or 6 hours; ② The frying temperature is 120-250° C.; ③ The frying time is 3 to 15 minutes, for example 3 to 8 minutes; and, ④ The frying comprises the following steps: placing the moistened material in a preheated frying device for frying; the preheating temperature is preferably 100-150°C.

6. The method for preparing the Hyssopus officinalis extract according to claim 1, wherein: In step (2), the mass ratio of the primary product to the polyol is 1:(7-12), preferably 1:(8-10), for example 1:8, 1:9 or 1:10; And / or, the polyol is one or more of butanediol, ethylene glycol, glycerol and 1,2-hexanediol.

7. The method for preparing the Hyssopus officinalis extract according to claim 1, wherein: The heating temperature is 85-100°C, for example 85°C, 90°C or 100°C; And / or, the heating time is 6 to 8 hours; And / or, the heating process further includes a stirring step.

8. The method for preparing the Hyssopus officinalis extract according to claim 1, wherein: In step (2), the heated product is filtered; preferably, the filtration comprises one or more of gauze filtration, microfiltration membrane filtration, and ultrafiltration membrane filtration; more preferably, the filtration comprises gauze filtration, microfiltration membrane filtration, and ultrafiltration membrane filtration in sequence; The pore size of the gauze in the gauze filtration is preferably 100 to 400 meshes; The pore size of the microfiltration membrane used in the microfiltration membrane filtration is preferably 2.5 to 10 μm, for example, 2.5 μm, 6 μm, 8 μm or 10 μm; The pore size of the ultrafiltration membrane used in the ultrafiltration membrane filtration is preferably 0.45 to 0.8 μm, such as 0.45 μm or 0.6 μm.

9. A Hyssopus officinalis extract prepared by the method for preparing the Hyssopus officinalis extract according to any one of claims 1 to 8.

10. Use of the Hyssopus officinalis extract according to claim 9 in cosmetics; Preferably, the concentration of the Hyssopus officinalis extract is 0.4-2.5%, for example 0.5%, 1% or 2%, where % refers to the ratio of the mass of the Hyssopus officinalis extract to the sum of the mass of the Hyssopus officinalis extract and the culture medium; Preferably, the Hyssopus officinalis extract is used as an anti-photoaging active ingredient in cosmetics; Preferably, the Hyssopus officinalis extract is used in cosmetics as an Nrf2 expression promoter, an MMP-1 expression inhibitor, an elastin synthesis promoter in human epidermal cells, an HSP27 expression promoter in HaCaT cells, or an HMGB1 expression inhibitor in HaCaT cells.

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