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 present invention relates to an hyssop extract, a preparation method and an application thereof. Background Art

[0002] The skin is the largest organ of the human body and the first line of defense. Skin aging includes natural aging and exogenous aging. Natural aging refers to the skin aging phenomenon caused by irresistible factors such as gravity, decline of the body's physiological functions, age growth, and genetics. Exogenous aging includes aging caused by ultraviolet (UV) rays, smoking, wind and sunlight, exposure to harmful chemicals, and other factors. Since UV radiation is the main cause of skin aging, exogenous aging is also called skin photoaging. Research shows that 80% of facial aging is caused by photoaging, and the clinical manifestations are rough, loose, wrinkled, pigmented skin, and even induce the occurrence of skin cancer.

[0003] Honey frying is one of the frying methods, also known as honey processing, which is a frying method with honey as an adjuvant. It is a method of adding a certain amount of refined honey to stir-fry the selected or cut drugs. The purposes of honey frying are as follows: (1) enhancing the effect of moistening the lungs and relieving cough, such as Stemona japonica, Tussilago farfara, Aster tataricus, etc.; (2) enhancing the effect of tonifying the spleen and replenishing qi, such as Astragalus membranaceus, Glycyrrhiza uralensis, Codonopsis pilosula, etc.; (3) moderating the drug properties, such as Ephedra sinica, Cinnamomum cassia, Cimicifuga foetida, etc.; (4) correcting the taste and eliminating side effects, such as Aristolochia debilis, Stemona japonica, etc.

[0004] Hyssopus is a plant of the genus Hyssopus in the family Lamiaceae, with a total of 15 species, mostly perennial herbs or semi-shrubs, distributed from the Mediterranean to Central Asia. In China, there are two species, Hyssopus cuspidatus Boriss and Hyssopus latilabiatus C.Y.Wu et H.W.Li, which mostly grow in the northern part of Xinjiang, China (Altay region). They are commonly used medicinal materials in Uyghur medicine. The whole herb of Hyssopus cuspidatus Boriss is used as medicine and is called "Zupaqini" by the Uyghur people. The same genus of Hyssopus officinalis L. is also used as hyssopus for medicine. As a commonly used medicinal material in the folk of Xinjiang, China, Uyghur medicine traditionally believes that the property of hyssopus is second-level dry and hot, and its functions and indications are warming the lungs and relieving asthma, dispelling cold and relieving cough, drying dampness and resolving phlegm, sweating and detoxifying, anti-inflammatory and detumescence, etc. It is generally used to treat wet-cold and mucous-quality respiratory organ diseases. At present, the compounds isolated from plants of the genus Hyssopus mainly include terpenoids, flavonoids, phenolic acids, phenylpropanoids, steroids, etc.

[0005] Currently, the commonly used preparation methods of Hyssopus officinalis L. extract include solvent extraction method, resin adsorption and separation method, distillation method, etc. Hyssopus officinalis L. extract has anti-inflammatory and immunomodulatory effects and can be used to prepare anti-lung cancer drugs and anti-inflammatory drugs. Many scholars have studied the anti-inflammatory mechanism of Hyssopus officinalis L., but there is no research on the anti-photoaging effect of honey-roasted Hyssopus officinalis L. Summary of the Invention

[0006] In order to make up for the research gap in the anti-photoaging effect of Hyssopus officinalis L., the present invention provides an extract of Hyssopus officinalis L., a preparation method and an application. The extract of Hyssopus officinalis L. 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 preparation method of an extract of Hyssopus officinalis L., which comprises the following steps:

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

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

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

[0012] Among them, the moisture content of the Hyssopus officinalis L. is preferably 10-13%.

[0013] Among them, the Hyssopus officinalis L. is preferably cut into sections.

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

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

[0016] In the present invention, in step (1), in the mixture, 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 preparation method of the mixture preferably comprises the following steps: first mixing the honey and the solvent, and then mixing with the Hyssopus officinalis L.

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

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

[0020] In the present invention, in step (1), the moistening can be conventional in the art, generally by placing the material for a period of time. The moistening time is preferably 2 - 10 h, more preferably 3 - 5 h, such as 3 h, 5 h or 6 h.

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

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

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

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

[0025] Among them, generally a sieving step is also included after the pulverizing. The sieve mesh used for sieving is preferably 45 - 80 mesh, more preferably 45 - 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), such as 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 - 8 h, such as 5 h, 8 h, 9 h or 10 h.

[0030] In the present invention, in step (2), during the heating process, a stirring step generally may also be included. The stirring time is sufficient for the initial product and the polyol to be mixed evenly.

[0031] In the present invention, in step (2), the product obtained after heating is generally also called the leaching solution.

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

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

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

[0035] In the gauze filtration, the pore size of the gauze is preferably 100 - 400 mesh, such as 200 mesh.

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

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

[0038] In a preferred embodiment, the product after heating is filtered successively with a 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 product after heating is filtered successively with a 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 product after heating is filtered successively with a 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 product after heating is filtered successively with a 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 hyssop extract prepared by the preparation method of the foregoing hyssop extract.

[0043] The present invention also provides an application of the aforesaid hyssop extract in cosmetics.

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

[0045] In the present invention, preferably, the hyssop extract serves as an anti-photoaging active ingredient in cosmetics.

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

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

[0048] The positive and progressive effects of the present invention are as follows:

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

[0050] The hyssop 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-dermal cells, promote the expression of HSP27 in HaCaT cells, thereby helping to resist thermal aging, and down-regulate the expression of HMGB1 in HaCaT cells, and has good anti-photoaging effects. Specific Embodiments

[0051] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the examples described herein. For the experimental methods without specific conditions noted in the following examples, they are carried out 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 Hyssop Medical Technology Co., Ltd., and the moisture content is 10-13%.

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

[0054] Example 1

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

[0056] 2) Place the well-moistened hyssop obtained above in a preheated (100 - 150 °C) frying container and heat it with a gentle fire (120 - 250 °C) until the surface of the medicinal material turns slightly yellow and is not sticky to the touch (the specific frying time is 3 - 8 min). Take it out and let it cool;

[0057] 3) Pulverize the honey-fried hyssop obtained in step 2) and sieve it through a 45-mesh sieve to obtain hyssop powder. Mix the hyssop powder and butylene glycol in a mass ratio of 1:8 and stir for 3 - 5 min to obtain a mixed solution;

[0058] 4) Heat the mixed solution obtained in step 3) to 90 °C and heat for 5 h, then stir to obtain an extract;

[0059] 5) Filter the extract obtained in step 4) with a 200-mesh gauze, and filter the crude extract through a microfiltration membrane with a pore size of 10 μm and an ultrafiltration membrane with a pore size of 0.6 μm. Collect the filtrate to obtain hyssop extract.

[0060] Experimental Example 2

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

[0062] 2) Place the well-moistened hyssop obtained above in a preheated (100 - 150 °C) frying container and heat it with a gentle fire (120 - 250 °C) until the surface of the medicinal material turns slightly yellow and is not sticky to the touch (the specific frying time is 3 - 8 min). Take it out and let it cool;

[0063] 3) Pulverize the honey-fried hyssop obtained in step 2) and sieve it through a 60-mesh sieve to obtain hyssop powder. Mix the hyssop powder and butylene glycol in a mass ratio of 1:9 and stir for 3 - 5 min to obtain a mixed solution;

[0064] 4) Heat the mixed solution obtained in step 3) to 85 °C and heat for 8 h, then stir to obtain an extract;

[0065] 5) Filter the extract obtained in step 4) with a 100-mesh gauze, and filter the crude extract through a microfiltration membrane with a pore size of 8 μm and an ultrafiltration membrane with a pore size of 0.45 μm. Collect the filtrate to obtain hyssop extract.

[0066] Experimental Example 3

[0067] 1) Take 100g honey and add 30g boiling water to dilute it. Cut 1100g dried Hyssopus officinalis into sections and place them in the honey water, mix well, and let it soak 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) frying container, heat over a slow fire (120-250°C), stir-fry until the surface of the medicinal material is slightly yellow and not sticky (the specific stir-frying time is 3-8 minutes), take out and let cool;

[0069] 3) crushing the honey-roasted hyssop obtained in step 2) through a 60-mesh sieve to obtain hyssop powder, mixing the hyssop powder with a butylene glycol aqueous solution at 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) filtering the extract obtained in step 4) through 400-mesh gauze, filtering the crude extract through a microfiltration membrane with a pore size of 6 μm and an ultrafiltration membrane with a pore size of 0.6 μm, collecting the filtrate, and obtaining the Hyssopus officinalis extract.

[0072] Experimental Example 4

[0073] 1) Take 80g honey and add 35g boiling water to dilute. Cut 1000g dried Hyssopus officinalis into sections and place in the honey water, mix well, and let it soak 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) frying container, heat over a slow fire (120-250°C), stir-fry until the surface of the medicinal material is slightly yellow and not sticky (the specific stir-frying time is 3-8 minutes), take out and let cool;

[0075] 3) crushing the honey-roasted hyssop obtained in step 2) through a 45-mesh sieve to obtain hyssop powder, mixing the hyssop powder with a butylene glycol aqueous solution at 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) is filtered through 400-mesh gauze, and the crude extract is 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 is 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, and place it in 60 g of water for full soaking;

[0080] 2) Mix the soaked hyssop with the aqueous solution of butylene glycol at a mass ratio of 1:8, and stir for 3 - 5 min to obtain a mixed solution;

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

[0082] 4) Filter the extract obtained in step 3) with a 200 - mesh gauze, and filter the crude extract through a microfiltration membrane with a pore size of 10 μm and an ultrafiltration membrane with a pore size of 0.6 μm, and collect the filtrate to obtain the hyssop extract.

[0083] Comparative Example 2

[0084] Compared with Example 1, in Comparative Example 2, the heating time in step 4) is 3 h, and 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, which can regulate the expression of genetic genes with antioxidant functions such as HO - 1, NQO - 1, GST, etc., and is of great significance for the cell defense mechanism against oxidative damage. Promote antioxidant effects by activating the Nrf2 pathway, scavenge reactive oxygen species generated in the skin, and play a role in protecting 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]]> 1 mM Positive Control Group (PC) <![CDATA[H2O2+EGCG]]> 1 ppm Test Sample Group <![CDATA[H2O2 + Hyssop Extract]]> 0.5％、1％、2％

[0090] Preparation of the working solution:

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

[0092] The samples in the test sample group are mixtures of H2O2 and the hyssop extracts of each example and comparative example. The mixture corresponding to Example 1 is diluted with DMEM medium (purchased from Gibco, model number 10569-010) into solutions with hyssop extract concentrations of 0.5%, 1%, and 2%. The mixtures corresponding to other examples and comparative examples are diluted into solutions with a hyssop extract concentration of 2%. The % refers to the ratio of the hyssop extract to the mass of the mixture.

[0093] Operation steps:

[0094] The HaCaT cell line (purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) was treated with H2O2 solution, H2O2 solution + EGCG solution, and H2O2 solution + sample solution respectively. After culturing for 6 hours in an environment of 37°C and 5% CO2, proteins were extracted and separated by SDS-PAGE. After completion, the relative expression level of Nrf2 was detected by the antigen-antibody reaction in the Western blot method.

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

[0096] Table 2

[0097]

[0098] Among them, the relative expression level of Nrf2 refers to the ratio of the Nrf2 gene expression level to the expression level 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 levels of Nrf2 in the hyssop extracts obtained in Examples 1-4 are all higher than those in Comparative Examples 1-2, indicating that the hyssop extracts prepared in the examples have higher antioxidant effects than the comparative examples. When the concentration of the hyssop extract obtained in Example 1 is 2%, 1%, and 0.5%, the relative expression level of Nrf2 increases significantly, and the upregulation rates are 50.1%, 9.5%, and 17.2% respectively. The upregulation rate = (the relative expression level of Nrf2 in the test sample group - the relative expression level of Nrf2 in the negative control group) / the relative expression level of Nrf2 in the negative control group. This shows that the hyssop extract has antioxidant effects under this condition.

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

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

[0102] Studies have shown that ultraviolet light (UVA) induces skin wrinkles and photoaging by specifically decomposing the main matrix components in the dermis, such as Collagen I, III, V, and the main matrix components in the basement membrane, such as collagen IV, VII, proteoglycans, laminin, etc., through the regulation of the synthesis of various MMPs.

[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 the working solution:

[0107] Preparation of the EGCG solution: Dilute EGCG (purchased from sigma) to 0.5 μg / ml with DMEM medium (purchased from Gibco, model number 10567014).

[0108] Preparation of the sample solution: Dilute the hyssop extract of Example 1 with DMEM medium to sample solutions with concentrations of 0.5%, 1%, and 2%. Dilute the hyssop extracts of other examples and comparative examples to a sample solution with a concentration of 2%. The % refers to the mass ratio of the hyssop extract in the sample solution.

[0109] Operating procedure:

[0110] Inoculate human dermal fibroblasts HDF (Human dermal fibroblast) (purchased from Boxi Biology) in a 60 mm culture dish for culture. After washing with buffer, irradiate with UVA (intensity: 25 J / cm 2 ), replace with serum-free medium, add 2 ml of EGCG solution and 2 ml of sample solution, and then culture in a 37 °C, 5% CO2 microbial incubator. Use QIAzol TM lysis reagent to collect cells, and isolate RNA according to the manufacturer's method. After quantifying the isolated RNA, synthesize cDNA for Real-time PCR (Applied Biosystems kit, rapid real-time quantitative PCR instrument) to obtain the relative expression level of MMP-1.

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

[0112] Table 4

[0113]

[0114] Among them, the relative expression level of MMP-1 refers to the ratio of the expression level of the MMP-1 gene to the expression level of the internal reference gene β-Actin, with the unit of %.

[0115] It can be seen that when the concentration is 2%, the relative expression levels of MMP-1 in the hyssop extracts obtained in Examples 1-4 are all lower than those in Comparative Examples 1-2, indicating that the hyssop extracts prepared in Examples 1-4 have better anti-aging effects than those in Comparative Examples 1-2; when the concentration of the hyssop extract obtained in Example 1 is 2%, 1%, and 0.5%, the relative expression level of MMP-1 decreases significantly, and the down-regulation rates are 62.6%, 17.1%, and 16% respectively. The down-regulation rate = (the relative expression level of MMP-1 in the test sample group - the relative expression level of MMP-1 in the negative control group) / the relative expression level of MMP-1 in the negative control group. This shows that the hyssop extract has the effect of anti-wrinkle under this condition.

[0116] Effect Example 3, determination of elastin content

[0117] Fibroblasts are the main cell components of the skin, distributed in the dermis of the skin. They are the most common cells in loose connective tissue and 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] The content of elastin determines the firmness of the skin and thus affects the state of the skin. A decrease in the content of elastin will lead to skin aging. Therefore, the effect of the sample on the content of elastin can be tested by the enzyme-linked immunosorbent assay (ELISA) method to evaluate the firming effect of the sample.

[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 the working solution:

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

[0124] Preparation of the sample solution: Dilute the hyssop extract of Example 1 with DMEM medium (purchased from Gibco, product number 10567014) to sample solutions with concentrations of 0.5%, 1%, and 2%. Dilute the hyssop extracts of other examples and comparative examples to sample solutions with a concentration of 2%. % refers to the mass ratio of the hyssop extract to the sample solution.

[0125] Operating steps:

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

[0127] 2) Drug administration: After 24 h of cell culture, the supernatant was aspirated, and 200 μl of sample solution and 200 μl of TGF-β1 solution were added to the wells respectively, mixed evenly, and placed in the incubator for 24 h + 1 h.

[0128] 3) Cell viability detection: After 24 h, the supernatant was collected (for ELISA detection), 100 μl of CCK-8 working solution (purchased from Tongren Chemistry, model CK04) was added to each well, and placed in the incubator for 1 h to 4 h. The absorbance was measured at 450 nm, and the experimental data was saved in electronic format for subsequent analysis.

[0129] 4) ELISA detection: After collecting the cell culture supernatant, ELISA detection was performed.

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

[0131] Table 6

[0132]

[0133] It can be seen that when the concentration is 2%, the elastin content of the hyssop extracts obtained in Examples 1-4 is higher than that of Comparative Examples 1-2, indicating that the hyssop extracts prepared in Examples 1-4 have better firming effects than Comparative Examples 1-2. When the concentration of the hyssop extract obtained in Example 1 is 2%, 1%, and 0.5%, the elastin content increases significantly, and the upregulation rates are 29.4%, 22.7%, and 18.0% respectively. The upregulation rate = (elastin content of the test sample group - elastin content of the blank control group) / elastin content of the blank control group. It shows that the hyssop extract has a firming effect under this condition.

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

[0135] When cells are exposed to high temperatures, they will release proteins (heat shock proteins or stress proteins) to protect themselves. HSP (heat shock protein) absorbs heat itself to prevent cell death. It is secreted by cells exposed to stress and increases when exposed to various stimuli, such as electrical stimulation and chemicals. Promoting the release of HSP (heat shock protein) can improve skin aging.

[0136] The test plan 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 the working solution:

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

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

[0143] The operation steps are as follows:

[0144] 1) Grouping: Set up a blank control group (DMEM culture medium + HaCaT cells, BC), a negative control group (DMEM culture medium + HaCaT cells + 50 mJ UVB irradiation, NC), a positive control group (HaCaT cells + 50 mJ UVB irradiation + resveratrol solution, PC), and a test sample group (HaCaT cells + 50 mJ UVB irradiation + sample solution). The dosage for each group is 2 ml.

[0145] 2) Cell seeding: Take HaCaT cells in the logarithmic growth phase (purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences), discard the culture medium, wash twice with PBS (purchased from Procell), add 0.25% trypsin, place at 37 °C for 5 min, terminate digestion with the culture medium, centrifuge at 1000 rpm for 3 min, resuspend the cells with complete DMEM culture medium, count, and adjust the cell concentration to 1.5×10 5 / ml, and add 1 ml to each well of a 48-well plate. Culture in an environment of 37 °C and 5% CO2 for 24 hours.

[0146] 3) Administration: After 24 h, the cells adhered to the wall. Use a UV crosslinker for UVB irradiation with an intensity of 50 mJ. After irradiation, discard the culture medium, and add 2 ml of the test sample and 2 ml of 20 μM resveratrol to the corresponding wells respectively. Culture in an environment of 37 °C and 5% CO2 for 24 hours.

[0147] 4) RNA detection: After 24 h, RNA was extracted, reverse transcribed into cDNA, and the relative expression level of HSP27 mRNA was detected.

[0148] The test results are shown in Table 8.

[0149] Table 8

[0150]

[0151]

[0152] Among them, 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 %.

[0153] It can be seen that when the concentration is 2%, the relative expression levels of HSP27 in the hyssop extracts obtained in Examples 1-4 are all higher than those in Comparative Examples 1-2, indicating that the hyssop extracts prepared in Examples 1-4 have better anti-thermal aging effects than Comparative Examples 1-2; when the concentration of the hyssop extract obtained in Example 1 is 2%, 1%, and 0.5%, the relative expression level of HSP27 decreases significantly, and the up-regulation rates are 64.9%, 35.0%, and 21.8% respectively. The down-regulation rate = (the relative expression level of HSP27 in the test sample group - the relative expression level of HSP27 in the negative control group) / the relative expression level of HSP27 in the negative control group. It shows that the hyssop extract has an anti-photoaging effect under this condition.

[0154] Effect Example 5, determination of the content of high-mobility group protein HMGB1

[0155] HMGB1 is usually located in the cell nucleus, binds to chromatin, and participates in maintaining the stability of nucleosomes, etc. When cells are damaged by light, HMGB1 is released, can bind to RAGE and participate in mediating inflammatory reactions, further leading to skin aging. In this experimental protocol, human immortalized keratinocytes HaCaT were selected to determine the effect of the test substance on the content of HMGB1 in the cell culture supernatant after UVB stimulation, and the anti-photoaging efficacy can be evaluated.

[0156] The test protocol is shown in Table 9. The experiment was divided into a blank control group, a negative control group, a positive control group, and a test sample group.

[0157] Table 9

[0158]

[0159]

[0160] Preparation of the working solution:

[0161] Preparation of resveratrol solution: Resveratrol (purchased from sigma) was diluted with DMEM medium (purchased from Gibco, model number 10569 - 010) to 20 μM to obtain the resveratrol solution.

[0162] Preparation of sample solution: The hyssop extract of Example 1 was diluted with DMEM medium (purchased from Gibco, model number 10569 - 010) to sample solutions with concentrations of 0.5%, 1%, and 2%, and the hyssop extracts of other examples and comparative examples were diluted to sample solutions with a concentration of 2%. Here, "%" refers to the mass ratio of the hyssop extract in the sample solution.

[0163] The operation steps are as follows:

[0164] 1) Grouping: A blank control group (DMEM culture medium + HaCaT cells, BC), a negative control group (DMEM culture medium + HaCaT cells + 50 mJ UVB irradiation, NC), a positive control group (HaCaT cells + 50 mJ UVB irradiation + resveratrol solution, PC), and a test sample group (HaCaT cells + 50 mJ UVB irradiation + sample solution) were set up. The dosage for each group was 2 ml.

[0165] 2) Cell seeding: Cells were seeded into a 6 - well plate at a density of 2×10 5 / ml, 2 ml per well. The seeded cell culture plate was placed in an incubator and cultured for another 24 h (5% CO2, 37 °C).

[0166] 3) Administration: After 24 h, the cells adhered to the wall. UVB irradiation was performed using a UV cross - linker with an intensity of 50 mJ. 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 respectively. The cells were cultured for 24 hours in an environment of 37 °C and 5% CO2.

[0167] 4) ELISA detection: After 24 h, the cell culture supernatant was collected, and ELISA detection was performed according to the instructions of the ELISA kit.

[0168] The test results are shown in Table 10.

[0169] Table 10

[0170]

[0171]

[0172] It can be seen that when the concentration is 2%, the HMGB1 contents of the 2% hyssop extracts obtained in Examples 1-4 are all lower than those in Comparative Examples 1-2, indicating that the hyssop extracts prepared in Examples 1-4 have better anti-photoaging effects than those in Comparative Examples 1-2; when the hyssop extract obtained in Example 1 is at concentrations of 1% and 2%, the HMGB1 contents decrease significantly, and the down-regulation rates are 20.2% and 34.5% respectively. The down-regulation rate = (HMGB1 content in the test sample group - HMGB1 content in the negative control group) / HMGB1 content in the negative control group. This shows that the hyssop extract has an anti-photoaging effect under these conditions.

Claims

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

2. The preparation method of the hyssop extract according to claim 1, wherein, The mass ratio of the hyssop 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 preparation method of the hyssop extract according to claim 1, characterized in that, The solvent in the mixture is water; And / or, in the mixture, 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 mixture includes the following steps: first mix the honey and the solvent, and then mix with the hyssop.

4. The preparation method of the hyssop extract according to claim 1, wherein, The preparation method of the hyssop extract satisfies any one or more of the following conditions: ① The hyssop is dried hyssop, and the moisture content of the hyssop is preferably 10 - 13%; ② The primary product is in powder form, and the particle size of the primary product is preferably 250 - 350 μm; ③ After the stir-frying, a pulverizing step is further included; ④ The honey is refined honey, the refining temperature is preferably 100 - 120 °C, and the heat preservation time is preferably 5 - 15 min; and, ⑤ The honey is robinia honey.

5. The preparation method of the hyssop extract according to claim 1, characterized in that, The preparation method of the hyssop extract satisfies any one or more of the following conditions: ① The moistening time is 2 - 10 h, more preferably 3 - 5 h, such as 3 h, 5 h or 6 h; ② The stir-frying temperature is 120 - 250 °C; ③ The stir-frying time is 3 - 15 min, such as 3 - 8 min; and, ④ The stir-frying includes the following steps: placing the moistened material in a preheated stir-frying device for stir-frying; the preheating temperature is preferably 100 - 150 °C.

6. The preparation method of the hyssop extract according to claim 1, characterized in that, In step (2), the mass ratio of the primary product to the polyol is 1:(7 - 12), preferably 1:(8 - 10), such as 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 preparation method of the hyssop extract according to claim 1, characterized in that, The heating temperature is 85 - 100 °C, such as 85 °C, 90 °C or 100 °C; And / or, the heating time is 6 - 8 h; And / or, a stirring step is further included during the heating process.

8. The preparation method of the hyssop extract according to claim 1, characterized in that, In step (2), the heated product is filtered; preferably, the filtration includes one or more of gauze filtration, microfiltration membrane filtration and ultrafiltration membrane filtration; more preferably, the filtration sequentially includes gauze filtration, microfiltration membrane filtration and ultrafiltration membrane filtration; The aperture of the gauze in the gauze filtration is preferably 100 - 400 mesh; The aperture of the microfiltration membrane used in the microfiltration membrane filtration is preferably 2.5 - 10 μm, such as 2.5 μm, 6 μm, 8 μm or 10 μm; The aperture of the ultrafiltration membrane used in the ultrafiltration membrane filtration is preferably 0.45 - 0.8 μm, such as 0.45 μm or 0.6 μm.

9. A hyssop extract prepared by the preparation method of the hyssop extract according to any one of claims 1 to 8.

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

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