A mustard leaf extract and its application in preparing cosmetics

Abstract

The present invention discloses a mustard leaf extract and its application in the preparation of cosmetics, and belongs to the field of biomedicine. The extract of the present invention is obtained by extracting mustard leaves as raw materials, and can make full use of the mustard leaf parts that are usually discarded, so as to achieve the recycling of waste, save costs, reduce waste, and protect the environment; the mustard leaf extract of the present invention has flavonoids, polysaccharides, and phenol contents up to 48.45 mg / g, 654 mg / g, and 64.7 mg / g, respectively; the mustard leaf extract of the present invention also contains a variety of active ingredients. The mustard leaf extract of the present invention can be used to prepare cosmetics, and has the effects of moisturizing and soothing the skin.

Description

Technical Field

[0001] The invention relates to a mustard leaf extract and application thereof in preparing cosmetics, belonging to the field of biomedicine. Background Art

[0002] White mustard (scientific name: Sinapis alba L.) is a plant of the genus Sinapis, family Cruciferae. It is an annual herb, up to 75 (-100) cm tall; the stem is erect and branched. The lower leaves are pinnately lobed; the petiole is 1-1.5 cm long; the upper leaves are ovate or oblong-ovate. The raceme has many flowers, up to 30 cm long in fruiting period; the flowers are light yellow, about 1 cm in diameter; the pedicels are open or slightly folded outward; the petals are obovate, 8-10 mm long. The siliques are nearly cylindrical, 2-4 cm long, 3-4 mm wide, erect or curved; there are 1-4 seeds in each locule, spherical, about 2 mm in diameter, yellow-brown, with fine pits, and the flowering and fruiting period is June-August.

[0003] As a Chinese herbal medicine, white mustard has a long history of being discovered and studied. However, existing research reports on white mustard focus on its seeds, namely white mustard seeds. White mustard seeds are used as medicine, containing sinigrin, myrosinase, sinapines, 4-hydroxybenzoylcholine, 4-hydroxybenzylamine and other ingredients, which have the functions of warming the lungs, relieving diseases and promoting qi, dispersing knots and dredging collaterals and relieving pain. It is used for cold diseases, asthma and cough, chest and flank distension and pain, phlegm stagnation, numbness of meridians and joints, phlegm and dampness, and swelling and poison in the yin.

[0004] Using different parts of plants as medicine and studying their functions is a common method in the field of traditional Chinese medicine. The same plant grows in different environments, and the components of its parts are different, resulting in different medicinal effects. Minority medicine has its own unique characteristics due to geographical location, unique ethnic culture and other factors. Among them, Tibetan herbal medicine is an indispensable part of Chinese medicine, ranking first among the medicines of China's four major ethnic minorities - Tibetan, Mongolian, Uyghur and Dai. Compared with ordinary herbal plants, Tibetan herbal medicines have the characteristics of high activity, high nutritional content, low pollution and abundant medicinal power, and have outstanding effects in many aspects such as anti-oxidation, anti-aging, anti-corrosion and anti-inflammatory.

[0005] Plant polysaccharides are a relatively complex class of natural active ingredients. Some of them have very obvious functions in improving human immune function, some have obvious effects on anti-aging, and some have very strong anti-inflammatory and antibacterial properties. Most polysaccharides have good moisturizing properties. Among natural plant extracts, Chinese herbal flavonoids also have a wide range of biological activities. Chinese herbal flavonoids can directly capture and remove free radicals, and can also regulate related signal transduction pathways such as MAPK, NF-kB, PI3K / Akt, SIRT1, mTOR, AMPK, etc., significantly improve the activity of antioxidant enzymes, enhance the ability of cells to resist oxidative stress, and achieve good antioxidant effects. In addition, plant-derived phenolic substances are secondary metabolites with antioxidant, anti-inflammatory and antibacterial activities. They are widely present in the skin, roots, leaves, and fruits of plants. They can reduce the oxidative damage of lipids, proteins, enzymes, carbohydrates and DNA in living cells and tissues. Therefore, they can be used as natural antioxidants in different fields. At the same time, plant-derived phenolic substances also have good moisturizing properties. Current research on white mustard is centered around sinapinic acid and sinigrin, and no research has been conducted on flavonoids and phenolic substances.

[0006] It will be of great significance if more active ingredients or functional factors can be discovered from white mustard and on this basis, more valuable products can be developed by making full use of white mustard. Summary of the invention

[0007] In order to solve the above problems, the present invention selects Tibetan mustard leaves as raw materials, extracts and analyzes the usually discarded mustard leaf parts to explore their application value. The present invention prepares mustard leaf extracts by water extraction and 75% ethanol extraction, and finds that the extracts from Tibetan mustard leaves contain more polysaccharides, flavonoids, phenolic substances, and cordycepin, L-carnitine, L-tyrosine, eriodictyol, (+)-chambertone, α-cyperone and other substances; further, the present invention uses the extracts in cosmetics and conducts efficacy evaluation tests.

[0008] The first object of the present invention is to provide a mustard leaf extract, the preparation method of which comprises: taking mustard leaves, drying and grinding into powder, then using water or ethanol as an extraction solvent for extraction, and filtering to obtain an extract.

[0009] In one embodiment, the extraction solvent is deionized water or 75% ethanol.

[0010] In one embodiment, the extraction is performed by reflux extraction.

[0011] In one embodiment, the preparation method further comprises drying the extract.

[0012] In one embodiment, the solid-liquid ratio used in the reflux extraction is 1:10 to 1:30; and the reflux extraction time is 3 to 5 hours.

[0013] In one embodiment, the solid-liquid ratio used in the reflux extraction is 1:10, 1:20 or 1:30, and the time is 4 hours.

[0014] In one embodiment, the white mustard leaves are white mustard leaves from Tibet.

[0015] In one embodiment, the white mustard leaf extract contains flavonoids, polysaccharides, and phenols.

[0016] In one embodiment, the white mustard leaf extract contains cordycepin, L-carnitine, L-tyrosine, cytidine, L-phenylalanine, L-glutamic acid, nicotinic acid, guanine, guanosine, L-leucine, p-hydroxybenzaldehyde, stachydrine, isoguanosine, 5-hydroxymethylfurfural, nicotinamide, scopoletin, 4-hydroxy-3-methoxycinnamic aldehyde, 4-hydroxycinnamic acid methyl ester, phloretin, 7-hydroxycoumarin, 5,7-dihydroxy-4-methylcoumarin, mulberry yellow, quercetin, artemisinin, 7-methoxy-4-methylcoumarin, asarone, α-asarone, β-asarone, mace lignin, hordenine, L-tryptophan, 7,8-dihydroxy -6-methoxycoumarin, cyperoside, 4-methoxysalicylic acid, 3-hydroxy-4-methoxybenzoic acid, 2,4-dihydroxy-6-methylbenzoic acid, trilobatin, eriodictyol, genistein, chlorogenic acid, cryptochlorogenic acid, naringenin chalcone, abscisic acid, 3,5-dimethoxy-4-hydroxybenzaldehyde, 3-hydroxy-4-methoxycinnamic acid, isoeugenol acetate, naringenin, ethyl ferulate, propyl parahydroxybenzoate, parthenolide derivatives, curcumol, curdione, dihydroartemisinic acid, 6-shogaol, mace lignans, Wei Rui Kun Sen, (+)-Cyperidinone, α-Cyperidinone, Aristolochicone, Germacone, Abietic acid, α-linolenic acid.

[0017] The second object of the present invention is to provide a method for preparing a composition containing a plurality of active substances, wherein the active substances include flavonoids, polysaccharides, phenols, and cordycepin, L-carnitine, L-tyrosine, cytidine, L-phenylalanine, L-glutamic acid, nicotinic acid, guanine, guanosine, L-leucine, p-hydroxybenzaldehyde, stachydrine, isoguanosine, 5-hydroxymethylfurfural, nicotinamide, scopoletin, 4-hydroxy-3-methoxycinnamic aldehyde, methyl 4-hydroxycinnamate, phloretin, 7-hydroxycoumarin, 5,7-dihydroxy-4-methylcoumarin, mulberry yellow, quercetin, artemisinin, 7-methoxy-4-methylcoumarin, asarone, α-asarone, β-asarone, macetin, hordenine, L-tryptophan, 7,8-dihydroxy-6-methoxycoumarin, artemisinin, 4-methoxysalicylic acid , 3-hydroxy-4-methoxybenzoic acid, 2,4-dihydroxy-6-methylbenzoic acid, trilobatin, eriodictyol, genistein, chlorogenic acid, cryptochlorogenic acid, naringenin chalcone, abscisic acid, 3,5-dimethoxy-4-hydroxybenzaldehyde, 3-hydroxy-4-methoxycinnamic acid, isoeugenol acetate, naringenin, ethyl ferulate, propyl parahydroxybenzoate, parthenolide derivatives, curcumol, curdione, dihydroartemisinic acid, 6-shogaol, mace lignans, Weiruikunsen, (+)-chambertone, α-cyperone, aristolochic acid, germazine, abietic acid, α-linolenic acid; the method is to prepare an extract using white mustard leaves as raw materials; the preparation of the extract comprises: taking white mustard leaves, drying and grinding them into powder, then using water or ethanol as an extraction solvent for extraction, and filtering to obtain the extract

[0018] The third object of the present invention is to provide a product, wherein the product is mainly composed of the extract of white mustard leaves, the product can inhibit hyaluronidase, and / or improve the cell viability of keratinocytes, and the product contains a variety of active substances.

[0019] The fourth object of the present invention is the use of the white mustard leaf extract in the preparation of cosmetics, or the use of the method for preparing cosmetics containing multiple active substances in the preparation of cosmetics.

[0020] In one embodiment, the cosmetic is a cosmetic having skin moisturizing and / or soothing effects.

[0021] In one embodiment, the cosmetic is one of water, gel, emulsion and cream.

[0022] Advantages and effects of the present invention:

[0023] (1) The extract of the present invention is obtained by extracting the mustard leaves as raw materials, which can make full use of the mustard leaves that are usually discarded, thereby realizing the recycling of waste, saving costs, reducing waste, and protecting the environment.

[0024] (2) The contents of flavonoids, polysaccharides and phenols in the mustard leaf extract of the present invention can reach up to 48.45 mg / g, 654 mg / g and 64.7 mg / g, respectively.

[0025] (3) The white mustard leaf extract of the present invention also contains a variety of active ingredients, such as cordycepin, L-carnitine, L-tyrosine, cytidine, L-phenylalanine, L-glutamic acid, nicotinic acid, guanine, guanosine, L-leucine, p-hydroxybenzaldehyde, stachydrine, isoguanosine, 5-hydroxymethylfurfural, nicotinamide, scopoletin, 4-hydroxy-3-methoxycinnamic aldehyde, 4-hydroxycinnamic acid methyl ester, phloretin, 7-hydroxycoumarin, 5,7-dihydroxy-4-methylcoumarin, mulberry yellow, quercetin, artemisinin, 7-methoxy-4-methylcoumarin, asarone, α-asarone, β-asarone, mace lignin, hordenine, L-tryptophan, 7,8- Dihydroxy-6-methoxycoumarin, cyperoside, 4-methoxysalicylic acid, 3-hydroxy-4-methoxybenzoic acid, 2,4-dihydroxy-6-methylbenzoic acid, trilobatin, eriodictyol, genistein, chlorogenic acid, cryptochlorogenic acid, naringenin chalcone, abscisic acid, 3,5-dimethoxy-4-hydroxybenzaldehyde, 3-hydroxy-4-methoxycinnamic acid, isoeugenol acetate, naringenin, ethyl ferulate, propyl parahydroxybenzoate, parthenolide derivatives, curcumol, curdione, dihydroartemisinic acid, 6-shogaol, mace lignans, Wei Rui Kun Sen, (+)-Cyperidinone, α-Cyperidinone, Aristolochicone, Germacone, Abietic acid, α-linolenic acid, etc.

[0026] (4) The mustard leaf extract of the present invention can be used to prepare cosmetics, which have the effects of moisturizing and soothing the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The content of flavonoids, polysaccharides and phenols in the mustard leaf extract obtained by extraction with water or 75% ethanol.

[0028] Figure 2 This is the ingredient list of the extract.

[0029] Figure 3 This is a continuation of the extract ingredient table.

[0030] Figure 4 This is the material spectrum of the mustard leaf extract in Example 3.

[0031] Figure 5 Comparison of cell viability differences among different groups in Example 5, wherein Control is a blank control group (NC), M is a model group, PC is a positive control group, and TA is formulation 1 (0.20% (v / v), 0.10% (v / v), 0.05% (v / v) test concentrations). DETAILED DESCRIPTION

[0032] 1. Experimental Materials

[0033] Ethanol, sulfuric acid (95.0-98.0%), phenol (≥99.0%), anhydrous sodium sulfite (≥97.0%), aluminum nitrate nonahydrate (≥99.0%), sodium nitrite (≥99.0%), sodium hydroxide (≥96.0%), and Folin phenol reagent (1 mol / L) were all purchased from Sinopharm Group; glucose (≥97.0%) was purchased from Shanghai Haohong Biotechnology Co., Ltd.; gallic acid (99%) was purchased from Beijing Inokai Technology Co., Ltd.; vitamin C (99.0%) and dexamethasone (≥99%) were purchased from Yuanye Biotechnology; hyaluronidase, hyaluronic acid, and hexadecyltrimethylammonium bromide (CTAB) were all purchased from McLean; sodium acetate buffer solution was (Guangzhou Chemical Reagent Factory).

[0034] The white mustard leaves in Tibetan areas use the white mustard leaves from Medog County.

[0035] 2. Determination of total flavonoids, polysaccharides and phenol content

[0036] The total flavonoids content of white mustard leaf extract was determined with reference to GB_T 20574-2006; the polysaccharide content was determined with reference to DB45 / T2143-2020; and the total phenol content was determined with reference to T / GZCX 020-2022.

[0037] (1) Determination of total flavonoid content

[0038] The flavonoid content was determined by sodium nitrite-aluminum nitrate colorimetric method. Accurately weigh 10.0 mg of rutin standard, dissolve it in 60% ethanol, transfer it to a 100 mL volumetric flask, dilute to volume and shake well to obtain a 0.1 mg / mL rutin standard solution.

[0039] Accurately pipette 0, 1, 2, 4, 5, and 10 mL of 0.1 mg / mL rutin mother solution into a 50 mL volumetric flask, and add 1.5 mL of 5% NaNO2 to each volumetric flask. Shake well and let stand for 6 minutes, then add 1.5 mL of 10% Al(NO3)3, shake well and let stand for 6 minutes. Add 20 mL of freshly prepared 1 mol / L NaOH solution, dilute to the scale line of the volumetric flask with 60% ethanol, shake well and let stand for 15 minutes, measure the absorbance of the solution at a wavelength of 510 nm, and draw a rutin standard curve using the absorbance-rutin concentration plot; perform linear regression between rutin concentration (x) and absorbance (y), and the regression equation is y=0.016x+0.03998, with a correlation coefficient R 2 =0.9976, the results show that the linear relationship is good within this range.

[0040] The dried extract samples were prepared into a solution with a concentration of 0.1 mg / mL with 60% ethanol, 1.00 mL was accurately drawn from it and placed in a 10 mL volumetric flask, 5% NaNO2 0.30 mL was added, 10% Al(NO3) 3 0.3 mL was added after shaking and standing for 6 min, 1 mol / L sodium hydroxide solution 4.00 mL was added, 60% ethanol was added to make the volume to 10 mL mark, and after shaking and standing for 15 min, the absorbance value was measured at a wavelength of 510 nm by ultraviolet spectrophotometry, and the average value of 3 times was taken to calculate the total flavonoid content in the sample. The calculation method is as follows:

[0041]

[0042] Where: A-absorbance of sample solution; X-intercept of standard curve; B-intercept of standard curve

[0043]

[0044] Where: m-mass of sample solution after concentration; C0-concentration of sample solution after constant volume, mg / mL; V0-volume of sample solution after constant volume, mL.

[0045] (2) Determination of total phenol content

[0046] Accurately weigh 8.20 mg of gallic acid reference substance, place it in a 50 mL volumetric flask, add an appropriate amount of 60% ethanol, ultrasonicate until completely dissolved, add 60% ethanol to the scale, and obtain 0.164 mg / mL gallic acid reference substance solution. Accurately pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5 mL of the reference substance solution into a 10 mL volumetric flask, add 3 mL of deionized water, add 2.5 mL of 10% Folin phenol reagent, add 2 mL of 7.5% sodium carbonate solution, add deionized water to the 10 mL scale, shake well, place in the dark for 30 minutes, and measure the absorbance value at a wavelength of 742 nm by ultraviolet spectrophotometry. Draw a gallic acid standard curve by plotting absorbance-gallic acid concentration; perform linear regression on gallic acid concentration (x) and absorbance (y), and the regression equation is y=0.1114x+0.0549, with a correlation coefficient R 2 =0.99673, the results show that the linear relationship is good within this range.

[0047] Prepare a 0.1 mg / mL solution of each extract sample with deionized water after drying, and accurately draw 0.10 mL from each sample and place it in a 10 mL volumetric flask. Add 3 mL of deionized water, 2.5 mL of 10% Folin phenol reagent, 2 mL of 7.5% sodium carbonate solution, and deionized water to 10 mL mark, shake well, place in a dark place for 30 minutes, and measure the absorbance at a wavelength of 742 nm by ultraviolet spectrophotometry. Take the average of 3 times and calculate the total phenol content in the sample. The calculation method is as follows:

[0048]

[0049] Wherein: C-total phenol concentration in the extract sample obtained after substituting into the standard curve, mg / mL; A-absorbance of the test solution after color development.

[0050]

[0051] P-total polyphenol content of the sample to be tested (calculated as gallic acid), mg / g; C-total phenol concentration in the extract sample obtained after substituting into the standard curve, mg / mL; V-volume of the extract sample, mL; N=1 / 1000; M-mass of the sample to be tested, g.

[0052] (3) Determination of polysaccharide content

[0053] The polysaccharide content was determined by phenol-sulfuric acid colorimetric method. Accurately weigh 100 mg of glucose standard, dissolve it in deionized water, transfer it to a 100 mL volumetric flask, dilute to volume and shake well, accurately pipette 0, 2, 4, 6, 8, and 10 mL of 1 mg / mL glucose mother solution into a 100 mL volumetric flask, dilute to volume and shake well to obtain the glucose standard solution.

[0054] Accurately pipette 1 mg / mL glucose standard solution into 10 mL volumetric flasks, add 1 mL of 4% phenol solution to each volumetric flask, quickly add 7 mL of concentrated sulfuric acid, shake for 4 min, and then let stand for 30 min. Measure the absorbance of the solution at 490 nm wavelength, and draw a glucose standard curve by plotting absorbance-glucose concentration; perform linear regression on glucose concentration (x) and absorbance (y), and the regression equation is y=6.165x+0.0297, with a correlation coefficient R 2 =0.995, the results show that the linear relationship is good within this range.

[0055] The dried extract samples were prepared into a solution with a concentration of 0.1 mg / mL with deionized water. 1.00 mL was accurately drawn from each sample and placed in a 10 mL volumetric flask. 1 mL of 4% phenol was added, and 7 mL of concentrated sulfuric acid was quickly added. The solution was shaken for 4 min. After color development for 30 min, the absorbance was measured at a wavelength of 490 nm by ultraviolet spectrophotometry to calculate the total polysaccharide content in the sample.

[0056]

[0057] Where A is the absorbance of the test solution.

[0058]

[0059] Where C0 is the concentration of the sample solution after volume adjustment, mg / mL; C1 is the total polysaccharide concentration of the sample, mg / mL.

[0060] 3. CALT / TM / SOP270-01 Standard operating procedures for keratinocyte anti-irritation test:

[0061] Cell line: human keratinocytes (HaCaT), from Shanghai Jingkang Bioengineering Co., Ltd., generation: 32; complete culture medium: MEM medium containing 10% fetal bovine serum (FBS) (FBS, Lot: 2500251P); maintenance culture medium: MEM medium containing 1% fetal bovine serum (FBS, Lot: 2500251P); test conditions: incubator temperature 37±1°C, humidity 90±5%, CO25±1%; MTT solution: MTT was prepared into a MTT stock solution with PBS at a concentration of 5 mg / mL, filtered and sterilized using a 0.22 μm syringe filter, and stored at -20°C (Lot: 20230209).

[0062] (1) Cytotoxicity screening: The cultured cells were inoculated into a 96-well plate and placed in an incubator for 18-24 hours until the cells were 80% confluent. The original culture medium was removed, and a certain amount of sample dilutions of different concentrations were added to each well, and the plates were exposed in an incubator for 72±1 hours. The culture plates were removed, the original liquid in the wells was discarded, and 100 μL of MEM medium containing 1% FBS was added to each well. 20 μL of MTT solution was added to each well and incubated at 37°C for 3±0.5 hours. Then, the MTT solution was removed, 100 μL of DMSO was added to each well, and the absorbance was measured at a wavelength of 570 nm after oscillation in the dark for 10-15 minutes.

[0063] (2) Keratinocyte anti-SDS stimulation test: Prepare a cell suspension of a certain density, inoculate the cell suspension into a 96-well cell culture plate, and culture for 18-24 hours. Remove the culture plate, discard the original culture medium in the well, add a certain amount of maintenance culture medium containing SDS to each well except the blank control group, and add a certain amount of maintenance culture medium to the blank control group, and return to the incubator for incubation for 30 minutes. Remove the culture plate, discard the original culture medium in the well, add a certain amount of PBS to each well for washing once, then add a certain amount of maintenance culture medium to each well of the blank control group and model group, and add maintenance culture medium containing samples or positive controls to the other groups, and return to the incubator for incubation for 24±1 hours. Then add a certain amount of maintenance culture medium containing MTT to each well, and incubate in the incubator for 3-4 hours. Remove the liquid in the well, add 100μLDMSO to each well, place it on an oscillator for 10-15 minutes, and measure the absorbance at a wavelength of 570nm on an ELISA reader.

[0064] Example 1: Extract of Sinapis alba leaves using deionized water

[0065] The white mustard leaves from the Tibetan area were dried and ground into powder, deionized water was used as the extraction solvent, and reflux extraction was performed for 4 hours at a solid-liquid ratio of 1:10, 1:20, and 1:30. After filtering, they were dried in an oven at 45°C to obtain the white mustard leaf extract.

[0066] Example 2: Extract of Sinapis alba leaves using 75% ethanol

[0067] The white mustard leaves from Tibetan areas were dried and ground into powder, and 75% ethanol was used as the extraction solvent. Reflux extraction was performed for 4 hours at a solid-liquid ratio of 1:10, 1:20, and 1:30. After filtering, the white mustard leaf extract was dried in an oven at 45°C to obtain the white mustard leaf extract.

[0068] Example 3: Analysis of components of mustard leaf extract

[0069] 0.1 g of the mustard leaf extract prepared in Examples 1-2 was dissolved in 100 mL of deionized water, ultrasonicated for 60 min, and then centrifuged at 10000 r / min for 10 min at room temperature. The supernatant was injected into the HPLC-MS system.

[0070] The HPLC conditions are as follows: gradient elution using ultrapure water and methanol as mobile phases: 0-1.5 min 5% methanol; 1.5-2.5 min methanol increased from 5% to 10%; 2.5-14 min methanol increased from 10% to 40%; 14-30 min methanol increased from 40% to 95%; 30-33 min methanol maintained at 95%; 33-38 min methanol decreased from 95% to 5%; 38-41 min methanol maintained at 5%.

[0071] The instrument model is: Ultra High Performance Liquid Chromatography-Quadrupole Electrostatic Orbital Hydrazine Mass Spectrometry (Thermo, UHPLC-MS-QExactive) and the chromatographic column is: Rapid Resolution HD 2.1*100mm 1.8μm.

[0072] The extract of white mustard leaves was analyzed for the total amount of flavonoids, polysaccharides, and phenolic substances. Figure 1 And as shown in Table 1.

[0073] Table 1 Content of ingredients in mustard leaf extract

[0074]

[0075] like Figure 2 and Figure 3As shown in the figure, the water extract of white mustard leaves obtained with a solid-liquid ratio of 1:30 was used to detect the specific substances obtained by UHPLC-MS-QExactive, including cordycepin, L-carnitine, L-tyrosine, cytidine, L-phenylalanine, L-glutamic acid, nicotinic acid, guanine, guanosine, L-leucine, p-hydroxybenzaldehyde, stachydrine, isoguanosine, 5-hydroxymethylfurfural, nicotinamide, scopoletin, 4-hydroxy-3-methoxycinnamic aldehyde, methyl 4-hydroxycinnamate, phloretin, 7-hydroxycoumarin, 5,7-dihydroxy-4-methylcoumarin, mulberry yellow, quercetin, artemisinin, 7-methoxy-4-methylcoumarin, asarone, α-asarone, β-asarone, mace lignin, and scutellaria baicalensis. Malt alkaloids, L-tryptophan, 7,8-dihydroxy-6-methoxycoumarin, cypermethrin, 4-methoxysalicylic acid, 3-hydroxy-4-methoxybenzoic acid, 2,4-dihydroxy-6-methylbenzoic acid, trilobatin, eriodictyol, genistein, chlorogenic acid, cryptochlorogenic acid, naringenin chalcone, abscisic acid, 3,5-dimethoxy-4-hydroxybenzaldehyde, 3-hydroxy-4-methoxycinnamic acid, isoeugenol acetate, galangin, ethyl ferulate, propyl parahydroxybenzoate, parthenolide derivatives, curcumol, curdione, dihydroartemisinic acid, 6-shogaol, mace lignans, Wei Rui Kun Sen, (+)-Cyperidinone, α-Cyperidinone, Aristolochicone, Germacone, Abietic acid, α-linolenic acid.

[0076] Example 4: Application of mustard leaf extract in inhibiting hyaluronidase and moisturizing the skin

[0077] One of the key ingredients involved in skin moisturizing is hyaluronic acid, which acts together with natural moisturizing factor (NMF) and the natural lipid bilayer of skin to promote and maintain the moisturizing of epidermis and bottom layer. When skin disorder is damaged, hyaluronidase will be produced, and hyaluronic acid will be further decomposed. The present embodiment analyzes the inhibitory effect of mustard leaf extract on hyaluronidase. Complexation reaction can occur between hyaluronic acid and CTAB cationic surfactant to produce turbidity, and then the inhibition result of hyaluronidase can be evaluated by colorimetric detection sample.

[0078] 500 μL of sample (the extract of white mustard leaves obtained directly after reflux filtration and before drying obtained in Example 1 according to a solid-liquid ratio of 1:30) was taken, 500 μL of ultrapure water was added, mixed, and a sample solution with a concentration of 50% (v / v) was prepared, followed by 1:1 dilution, mixed, and a total of five concentrations. 0.1000 g of vitamin C (control group) was weighed, 1 mL of sodium acetate buffer solution was added, and a concentration of 100 mg / mL was prepared, followed by 1:1 stepwise dilution, and a total of 5 concentrations.

[0079] The moisturizing efficacy was evaluated using the CALT / TM / SOP282-01 in vitro hyaluronidase inhibition test standard operating procedure.

[0080] Blank group: 50 μL of hyaluronic acid and 300 μL of sodium acetate buffer solution were added to a 1.5 mL EP tube;

[0081] Reaction group: 50 μL of hyaluronic acid, 150 μL of sodium acetate buffer solution, and 200 μL of hyaluronidase were added to a 1.5 mL EP tube in sequence;

[0082] Positive control group: 50 μL of hyaluronic acid, 50 μL of sodium acetate buffer solution, 200 μL of hyaluronidase, and 100 μL of vitamin C were added to a 1.5 mL EP tube in sequence;

[0083] Sample group: 50 μL of hyaluronic acid, 50 μL of sodium acetate buffer solution, 200 μL of hyaluronidase, and 100 μL of test sample were added to a 1.5 mL EP tube in sequence;

[0084] Each group was reacted at 37±1°C for 150 min. After the reaction, 500 μL of CTAB solution was added and incubated at 37±1°C for 3 min. 200 μL was taken to a 96-well plate and the absorbance value was measured at a wavelength of 400 nm using an ELISA reader (CALT / 2017E004).

[0085] The inhibition rate was calculated according to the following formula:

[0086]

[0087] Use GraphPrism to fit and calculate IC 50 , that is, the concentration of the test sample when the inhibition rate is 50%.

[0088] The results are shown in Table 2. When the concentration of white mustard leaf extract is 50% (v / v), the inhibition rate of hyaluronidase is as high as about 76%. As the concentration decreases, the inhibition effect decreases. In addition, the positive control group IC 50 is 54.44 mg / mL (95% Cl: 51.96-57.04 mg / mL), while the white mustard leaf extract IC 50 The IC of the tested substance cannot be calculated (extremely low). 50 The smaller the value, the stronger the ability of the test substance to inhibit hyaluronidase. These results all indicate that the water extract of white mustard leaves has an inhibitory effect on hyaluronidase, which further indicates that it has a certain moisturizing effect.

[0089] Table 2 Hyaluronidase inhibition rate of water extract of white mustard leaves

[0090]

[0091] Example 5: Application of mustard leaf extract in improving cell viability of keratinocytes and soothing skin

[0092] The present invention firstly uses SDS to stimulate HaCaT keratinocytes, and then the mustard leaf extract (1:30 extract) obtained in Example 1 acts on the stimulated keratinocytes, and the soothing effect is evaluated by detecting the change in cell viability of the keratinocytes.

[0093] 0.5 mL FBS (fetal bovine serum) was added to 49.5 mL MEM culture medium to prepare a MEM culture medium containing 1% FBS, which was used as a blank group (NC). 0.200 mL of the sample (the extract of white mustard leaves obtained directly after reflux filtration and before drying obtained in Example 1 according to a solid-liquid ratio of 1:30) was weighed, and 0.800 mL of 1% FBSMEM culture medium was added to prepare a 20% (v / v) reserve solution, which was then diluted to 0.20% (v / v), 0.10% (v / v), and 0.05% (v / v) concentrations in sequence, for a total of 3 concentrations. A certain amount of dexamethasone (positive control group PC) was weighed, dissolved in methanol, and prepared into a dexamethasone reserve solution, which was then diluted to a working concentration using maintenance culture medium, as shown in Table 3.

[0094] Table 3. Experimental groups

[0095]

[0096] The in vitro soothing efficacy was evaluated using the CALT / TM / SOP270-01 keratinocyte anti-irritation test standard operating procedure.

[0097] The data were analyzed using SPSS and presented as mean ± SD. The differences were considered statistically significant if p < 0.05.

[0098] The cell activity of the blank control group (Control) was taken as 100%, and the relative cell activity (Viability) of each group was calculated using the following formula:

[0099]

[0100] The results are shown in Table 4 and Figure 5As shown, under the method selected in this embodiment, the cell viability of the model group (M) was significantly reduced compared with the blank control group (NC) (p<0.05); the cell viability of the positive control group was significantly increased compared with the model group (p<0.05), indicating that the model was successfully established. Compared with the model group, the cell viability of the water extract of white mustard leaves (1:30 solid-liquid ratio) at the test concentrations of 0.20% (v / v), 0.10% (v / v), and 0.05% (v / v) was significantly increased by 19.51%, 15.89%, and 6.05%, respectively.

[0101] Table 4. Comparison of cell viability in different groups (Mean±SD)

[0102]

[0103] Note: # indicates that the difference is statistically significant compared with the blank control group (p<0.05); * indicates that the difference is statistically significant compared with the model group (p<0.05), the same below.

Claims

1. A use of a mustard leaf extract from Tibet in the preparation of cosmetics that moisturize by inhibiting hyaluronidase and soothe by increasing the activity of keratinocytes, characterized in that: The preparation method of the mustard leaf extract comprises: taking mustard leaves, drying and grinding them into powder, then using water as an extraction solvent for extraction, filtering to obtain the extract, wherein the extraction is performed by reflux extraction, the solid-liquid ratio used in the reflux extraction is 1:30, and the reflux extraction time is 4 hours.

2. The use according to claim 1, characterized in that: The mustard leaf extract contains flavonoids, polysaccharides and phenols.

3. The use according to claim 1, characterized in that: The cosmetic is one of water, gel, emulsion and cream.

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