Natural eutectic solvent, perilla leaf extract and preparation method and application of perilla leaf extract

Abstract

The invention provides a natural eutectic solvent which is prepared by uniformly mixing D-fructose, glycerol, xylitol and deionized water, and the molar ratio of the D-fructose to the glycerol to the xylitol is (6-7): (4.5-5.5): 1; the invention also provides a preparation method of the perilla leaf extract, which comprises the following steps: mixing the natural eutectic solvent with perilla leaf powder, and then carrying out ultrasonic extraction to obtain an extracting solution; performing centrifugal treatment on the extracting solution, taking supernate, and performing sterilization and preservative treatment on the supernate. The invention also provides a perilla leaf extract extracted by the natural deep-eutectic solvent and application of the perilla leaf extract in preparation of cosmetics. The natural eutectic solvent is reasonable in preparation, simple to prepare, low in cost, low in toxicity, renewable and biodegradable; the natural eutectic solvent is used for extracting active ingredients in natural plants, can improve the extraction efficiency, save the cost and improve the preparation efficiency, is green and environment-friendly, and is particularly suitable for extracting the active ingredients of purple perilla.

Description

Technical Field

[0001] The present application belongs to the field of cosmetics, and specifically relates to a natural low eutectic solvent, a perilla leaf extract, and a preparation method and application thereof. Background Art

[0002] Due to the improvement of people's living standards, unreasonable diet and environmental pollution, inflammatory skin conditions are becoming more and more common. It is urgent to develop a product with good anti-inflammatory effect and no burden on the skin. Currently, there are many kinds of anti-inflammatory products on the market with different effects. Their formula components not only burden the skin, but also the amount of ingredients with real anti-inflammatory effect is very small. There are also products containing plant active ingredients with anti-inflammatory effect on the market, but many plant extracts are used as additives at a low ratio and cannot better exert the anti-inflammatory effect of plant active ingredients.

[0003] Perilla is a plant of the genus Perilla in the family Lamiaceae. It is one of the 60 Chinese medicines for both medicinal and edible use announced by the former Ministry of Health of my country. According to the Compendium of Materia Medica, perilla leaves can relieve coldness, promote qi circulation and soothe the stomach. Perilla leaves are rich in polyphenols, flavonoids and anthocyanins, mainly including catechins, folinic acid, apigenin, luteolin, rosmarinic acid, caffeic acid, anthocyanins, etc. The difference in their content is the main reason for the diversity of perilla colors, and is also the material basis for the significant antioxidant activity of perilla and the inhibition of inflammation and chronic diseases.

[0004] In the prior art, the active ingredients in Perilla are extracted by water extraction, organic solvents (such as methanol, ethanol, acetone, ethyl acetate, etc.), etc., which not only has high extraction production costs but also causes waste of resources and environmental pollution. Such organic solvents have certain toxicity and will remain in the extract after extraction. When used in the field of cosmetics, it will affect the safety of the product. The subsequent removal process is relatively cumbersome, the extraction efficiency is low, and the cost is high.

[0005] As a new type of low melting point mixture solvent, deep eutectic solvent (DES) has received extensive attention. Deep eutectic solvents are composed of hydrogen bond acceptors and hydrogen bond donors in a certain stoichiometric ratio, and have a significantly lower melting point than pure substances. Deep eutectic solvents have the advantages of simple preparation process, easy preparation, low cost, high safety, and biodegradability. Natural deep eutectic solvents (NaDES) have a relatively broad application prospect in the fields of cosmetics and food. However, the market still has a significant demand for more and better natural deep eutectic solvent formulas. Summary of the invention

[0006] A brief overview of the present application is provided below in order to provide a basic understanding of certain aspects of the present application. It should be understood that this overview is not an exhaustive overview of the present application. It is not intended to identify the key or important parts of the present application, nor is it intended to limit the scope of the present application. Its purpose is merely to provide certain concepts in a simplified form as a prelude to a more detailed description discussed later.

[0007] In view of the above-mentioned defects of the prior art, the purpose of the present application is to provide a natural low eutectic solvent, a perilla leaf extract and a preparation method and application thereof.

[0008] According to the first aspect of the present application, a natural low eutectic solvent is provided, which is made by uniformly mixing D-fructose, glycerol, xylitol and deionized water, wherein the molar ratio of D-fructose, glycerol and xylitol is 6-7:4.5-5.5:1 (for example, 6:4.5:1, 6.5:5:1, 6:5.5:1, 7:4.5:1, 7:5.5:1, etc.); preferably, it is 6.2-6.8:4.8-5.2:1.

[0009] Preferably, in the above-mentioned natural low eutectic solvent, the mass proportion of deionized water is 20% to 50% (for example, 25%, 30%, 35%, 40%, 45%, etc.), and more preferably 25% to 45% (for example, 28%, 32%, 36%, 38%, 42%, etc.).

[0010] Preferably, the preparation method of the above-mentioned natural low eutectic solvent comprises: mixing D-fructose, glycerol and xylitol uniformly to obtain a mixed solution; then mixing the mixed solution with deionized water, and then heating and dissolving it with a magnetic stirrer to form a transparent and uniform liquid without crystal precipitation at room temperature to obtain the natural low eutectic solvent; wherein the heating temperature is preferably 75-85°C, and the heating and stirring time is preferably 40-60min.

[0011] According to a second aspect of the present application, a method for preparing a perilla leaf extract is provided, comprising:

[0012] The natural deep eutectic solvent is mixed with perilla leaf powder, and then ultrasonically extracted to obtain an extract;

[0013] The extract is centrifuged to obtain the supernatant, and the supernatant is sterilized and preserved to obtain the perilla leaf extract.

[0014] Preferably, in the above-mentioned method for preparing the perilla leaf extract, the perilla leaf powder is obtained by drying the perilla leaves and crushing them through a 50-mesh sieve.

[0015] Preferably, in the preparation method of the above-mentioned perilla leaf extract, the dosage ratio (solid-liquid ratio, mass ratio) of perilla leaf powder and low eutectic solvent is 1g:40-60g (for example, 1g:42g, 1g:45g, 1g:50g, 1g:55g, 1g:58g, etc.), more preferably 1g:45-55g (for example, 1g:48g, 1g:52g, etc.).

[0016] Preferably, in the preparation method of the above-mentioned perilla leaf extract, the ultrasonic frequency is 30-45KHz (such as 32KHz, 35KHz, 38KHz, 40KHz, 42KHz, etc.), more preferably 35-45KHz; the ultrasonic power is 250-350W (such as 260W, 280W, 300W, 320W, 340W, etc.), more preferably 280-320W (such as 285W, 290W, 300W, 310W, 315W, etc.) ; The heating temperature during ultrasound is 50-80°C (for example, 55°C, 60°C, 65°C, 70°C, 75°C, 78°C, etc.), more preferably 55-70°C (for example, 58°C, 62°C, 68°C, etc.); The ultrasonic time is 60-120min (for example, 70min, 80min, 90min, 100min, 110min, etc.), more preferably 80-100min (for example, 85min, 88min, 92min, 96min, etc.).

[0017] Preferably, in the above-mentioned preparation method of perilla leaf extract, the rotation speed of the centrifugal treatment is 3000-9000r / min (such as 3500r / min, 4000r / min, 5000r / min, 6000r / min, 7000r / min, 8000r / min, 8500r / min, etc.), more preferably 4000-6000r / min (such as 4500r / min, 4800r / min, 5200r / min, 5500r / min, 5800r / min, etc.), and further preferably 5000r / min; the time of the centrifugal treatment is 10-40min (such as 15min, 20min, 25min, 30min, 35min, etc.), more preferably 20-40min, and further preferably 30min.

[0018] Preferably, in the preparation method of the above-mentioned perilla leaf extract, the sterilization temperature is 95-110°C (such as 98°C, 100°C, 105°C, 108°C, etc.), more preferably 95-100°C; the time is 20-40min (such as 25min, 30min, 35min, etc.), more preferably 25-35min.

[0019] Preferably, in the preparation method of the above-mentioned perilla leaf extract, the preservative treatment includes adding a preservative to the material obtained after sterilization and mixing, and the mixing temperature is 50-80°C (such as 52°C, 55°C, 60°C, 65°C, 70°C, 75°C, 78°C, etc.), more preferably 70-80°C; the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the material obtained after sterilization is 0.5%-2%, and the mass percentage of 1,2-hexanediol in the material obtained after sterilization is 0.5%-2%; preferably, the mass percentage of p-hydroxyacetophenone in the material obtained after sterilization is 0.5%, and the mass percentage of 1,2-hexanediol in the material obtained after sterilization is 0.5%.

[0020] According to the third aspect of the present application, a perilla leaf extract is provided, which is obtained by extraction with the above-mentioned natural low eutectic solvent; further, the extract is obtained by the above-mentioned preparation method of the perilla leaf extract.

[0021] According to the fourth aspect of the present application, there is also provided an application of the above-mentioned perilla leaf extract in the preparation of cosmetics; preferably, the perilla leaf extract accounts for 5% to 99% of the mass of the cosmetics (such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, etc.), more preferably 60% to 99% (such as 65%, 75%, 85%, 94%, etc.), and the cosmetics can be facial masks, essences, toners, lotions, etc.

[0022] Compared with the prior art, this application has the following beneficial effects:

[0023] 1. The present application first provides a natural low eutectic solvent, which has reasonable formulation, simple preparation, low cost, low toxicity, regeneration and biodegradability;

[0024] 2. The present application also uses the above-mentioned natural low eutectic solvent to extract active ingredients from natural plants, thereby achieving higher extraction efficiency, saving costs, improving preparation efficiency, not polluting the environment, being green and environmentally friendly, and being particularly suitable for extracting active ingredients from Perilla frutescens.

[0025] These and other advantages of the present application will become more apparent through the following detailed description of the best embodiments of the present application in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present application may be better understood by referring to the following description in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in and form a part of this specification and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application. Among them:

[0027] Figure 1 The DPPH free radical scavenging rate of the test solution prepared by the products obtained in each embodiment and comparative example in effect embodiment 1 is shown;

[0028] Figure 2 The flavonoid content of the samples to be tested prepared by the products obtained in each embodiment and comparative example in Effect Example 2 is shown;

[0029] Figure 3 The total phenol content of the test solution prepared by the products obtained in each embodiment and comparative example in effect embodiment 3 is shown;

[0030] Figure 4 The cell viability of the test solution prepared by the products obtained in each embodiment and comparative example in effect embodiment 4 is shown;

[0031] Figure 5 The expression level of inflammatory factor IL-6 in the test solution prepared by the products obtained in each example and comparative example in Effect Example 4 is shown. DETAILED DESCRIPTION

[0032] The extraction of natural products is still mainly based on traditional solvent extraction. The extraction solvents are mostly organic solvents (such as methanol, ethanol, acetone, etc.), which have unsafe factors such as flammability, explosiveness, toxicity, and strong oxidizing properties. In actual production and use, they are easy to cause harm to experimental results, the environment, and personal safety. There are also problems such as high solvent costs and serious pollution. After extraction, some residues will remain in the extracts and are difficult to remove. Using them in the cosmetics field will affect the safety of the products. Compared with traditional organic solvents, NaDES can generally achieve higher extraction efficiency. In addition to the principle of like dissolves like, there is also a strong interaction between NaDES and the extracts, such as ion exchange and hydrogen bonding forces.

[0033] The present application uses a natural low eutectic solvent prepared from D-fructose, glycerol, xylitol and deionized water to extract perilla, thereby reducing the loss of active ingredients in perilla. The prepared extract has a high total phenol content and has good antioxidant, anti-inflammatory and soothing effects, good safety, and good application prospects in the field of cosmetics.

[0034] The present application provides a natural low eutectic solvent, a perilla leaf extract, and a preparation method and application thereof. In a specific implementation manner, the following steps are included.

[0035] First, a natural deep eutectic solvent is prepared.

[0036] D-fructose, glycerol and xylitol are mixed to prepare a mixed solution, wherein 1 mol of D-fructose has a mass of 180 g, 1 mol of glycerol has a mass of 92 g, and 1 mol of xylitol has a mass of 152 g, and the molar ratio of the three is D-fructose: glycerol: xylitol = (6-7): (4.5-5.5): 1, preferably (6.2-6.8): (4.8-5.2): 1, for example 13: 10: 2; the mixed solution of D-fructose, glycerol and xylitol prepared according to the above molar ratio is mixed with deionized water according to a certain mass, and the proportion of deionized water is preferably 20% to 50%, and more preferably 25% to 45%. After mixing D-fructose, glycerol, xylitol and deionized water, a magnetic stirrer is used to heat and dissolve them, and the heating and dissolving temperature is preferably 75-85° C., and the heating and stirring time is preferably 40-60 min. The natural low eutectic solvent is obtained by heating and stirring to form a transparent and uniform liquid without crystal precipitation at room temperature.

[0037] Secondly, the perilla leaf extract is prepared.

[0038] The perilla leaves are dried and crushed to pass through a 50-mesh sieve to obtain perilla leaf powder; the prepared natural low eutectic solvent and the perilla leaf powder are mixed according to a certain solid-liquid ratio, and the solid-liquid ratio (g:g) is preferably 1: (40-60), and more preferably 1: (45-55); after mixing, ultrasonic extraction is performed, and the ultrasonic frequency is preferably 30-45KHz, and more preferably 35-45KHz; the ultrasonic power is preferably 250-350W, and more preferably 280-320W; the heating temperature during ultrasound is preferably 50-80°C, and more preferably 55-70°C; the ultrasonic time is preferably 60-120min, and more preferably 80-100min.

[0039] After the ultrasound is finished, the extract is centrifuged; the centrifugal speed is 3000-9000r / min, preferably 4000-6000r / min; more preferably 4800r / min; the centrifugal time after the sterilization is 10-40min, preferably 20-40min, more preferably 30min.

[0040] After centrifugation, the extract is sterilized at high temperature. The sterilization temperature is preferably 95-110° C., more preferably 95-100° C.; the sterilization time is preferably 20-40 min, more preferably 25-35 min.

[0041] After sterilization, if the material is to be stored for a long time, a preservative needs to be added to the material. During the mixing process with the preservative, the mixing temperature is 50-80°C, preferably 70-80°C; the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the material obtained after sterilization is 0.5%-2%, and the mass percentage of 1,2-hexanediol in the material obtained after sterilization is 0.5%-2%; preferably, the mass percentage of p-hydroxyacetophenone in the material obtained after sterilization is 0.5%, and the mass percentage of 1,2-hexanediol in the material obtained after sterilization is 0.5%.

[0042] The perilla leaf extract obtained in the present application can be used directly as a cosmetic, or added to a basic cosmetic formula for use.

[0043] A specific embodiment of the present application also provides a skin topical preparation, wherein the mass percentage of the perilla leaf extract in the skin topical preparation is preferably 5% to 99%, and more preferably 60% to 99%. The skin topical preparation can be a facial mask, essence, toner, lotion, etc.

[0044] Exemplary embodiments of the present application will be described hereinafter with reference to the accompanying drawings.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] The perilla leaves in the following examples were from Anguo City, Hebei Province.

[0048] Example 1

[0049] (1) 117 g of D-fructose, 46 g of glycerol and 15.2 g of xylitol were mixed to obtain 178.2 g of a mixed solution, i.e., the molar ratio of D-fructose, glycerol and xylitol was 6.5:5:1; 126 g of the mixed solution was mixed with 74 g of deionized water, and heated and stirred to dissolve with a magnetic stirrer at a heating temperature of 80° C. for 50 min to obtain a natural low eutectic solvent;

[0050] (2) 3.7 g of perilla leaf powder (obtained by drying perilla leaves and crushing them through a 50-mesh sieve) was mixed with 200 g of the natural low eutectic solvent prepared in step (1), and then subjected to ultrasonic extraction at a frequency of 40 kHz, a power of 300 W, a time of 90 min, and a heating temperature of 66° C. After the ultrasonic extraction was completed, the extract was centrifuged at a speed of 4800 r / min for 30 min. After centrifugation, the supernatant was taken and sterilized at 100° C. for 30 min to obtain a perilla leaf extract.

[0051] Example 2

[0052] The preparation method is the same as that in Example 1, except that the molar ratio of D-fructose, glycerol and xylitol is 6:5:1, and other conditions and parameters are the same as those in Example 1.

[0053] Example 3

[0054] The preparation method is the same as that in Example 1, except that the molar ratio of D-fructose, glycerol and xylitol is 6.5:4.5:1, and other conditions and parameters are the same as those in Example 1.

[0055] Example 4

[0056] The preparation method is the same as that in Example 1, except that the heating temperature during ultrasound is 75° C., and other conditions and parameters are the same as those in Example 1.

[0057] Example 5

[0058] The preparation method is the same as that in Example 1, except that the molar ratio of D-fructose, glycerol and xylitol is 6:4.5:1, and other conditions and parameters are the same as those in Example 1.

[0059] Comparative Example 1

[0060] The preparation method is the same as that in Example 1, except that 50% ethanol is used to replace the natural deep eutectic solvent for extraction, and other conditions and parameters are the same as those in Example 1.

[0061] Comparative Example 2

[0062] The preparation method is the same as that in Example 1, except that deionized water is used to replace the natural deep eutectic solvent for extraction, and other conditions and parameters are the same as those in Example 1.

[0063] Comparative Example 3

[0064] The preparation method is the same as that in Example 1, except that 117 g of D-fructose and 46 g of glycerol (i.e., the molar ratio of D-fructose to glycerol is 6.5:5) are mixed, and then 126 g of the mixture is mixed with 74 g of deionized water to prepare a natural deep eutectic solvent. Other conditions and parameters are the same as those in Example 1.

[0065] Comparative Example 4

[0066] The preparation method is the same as that in Example 1, except that 90 g of glucose and 92 g of glycerol (i.e., the molar ratio of glucose to glycerol is 1:2) are mixed, and then 126 g of the mixture is mixed with 74 g of deionized water to prepare a natural deep eutectic solvent. Other conditions and parameters are the same as those in Example 1.

[0067] Effect Example 1: DPPH free radical scavenging experiment

[0068] DPPH is an early synthesized organic free radical, which is often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, purple, and has a characteristic absorption peak at 517nm. When encountering free radical scavengers, the lone pair of electrons of DPPH is paired and it fades, that is, the absorbance at the maximum absorption wavelength becomes smaller. Therefore, the scavenging effect of the sample on DPPH free radicals can be evaluated by measuring the change in absorbance.

[0069] The steps for preparing the test solution are as follows: diluting the perilla extracts prepared in the above Examples 1 to 5 and Comparative Examples 1 to 4 by 80 times with deionized water to prepare the test solution.

[0070] The specific experimental steps of the DPPH free radical scavenging experiment are as follows:

[0071] (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 mol / L DPPH solution (A 1 Tube);

[0072] (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and 2×10 -4 mol / L DPPH solution (A 2 Tube);

[0073] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (A 3 Tube);

[0074] (4) After 30 minutes of reaction in dark, measure A at 517 nm. 1 Tube, A 2 Tube, A 3 The clearance rate is calculated as follows: Clearance rate = [(A 2 +A 3 )-A 1 ] / A 2 ×100%.

[0075] In this experiment, the DPPH free radical scavenging experiment was performed on the perilla extracts prepared in the examples and comparative examples. The results are shown in Tables 1 and Figure 1It can be seen that the DPPH free radical scavenging ability of the extracts obtained in Examples 1-5 of the present application is significantly better than the DPPH free radical scavenging ability of the extracts obtained in Comparative Examples 1-4.

[0076] Table 1 Free radical scavenging test results of the perilla leaf extract obtained in the examples and comparative examples

[0077] DPPH free radical scavenging rate (%) Example 1 88.23±3.00 Example 2 84.85±1.59 Example 3 84.69±2.05 Example 4 81.65±1.61 Example 5 83.47±1.77 Comparative Example 1 59.26±1.24 Comparative Example 2 52.90±2.67 Comparative Example 3 68.71±1.98 Comparative Example 4 65.23±2.30

[0078] Effect Example 2: Determination of total flavonoid content

[0079] The experimental drugs used in this experiment are as follows:

[0080] 1. Rutin standard. 2. 30% ethanol solution: 30mL anhydrous ethanol mixed with 70mL distilled water. 3. 5% NaNO 2 Solution: 5 g NaNO 2 Add water to 100g and dissolve. 4.10% Al(NO 3 ) 3 Solution: weigh 10g Al(NO 3 ) 3 Add water to 100g and dissolve. 5.1mol / L NaOH solution: weigh 40g NaOH and dissolve it in water, then dilute to 1L in a volumetric flask.

[0081] Experimental instruments: precision balance, spectrophotometer.

[0082] Experimental operation:

[0083] 1. Drawing of standard solution

[0084] Accurately weigh 21.6 mg of dry constant-weight rutin standard, place it in a 100-mL volumetric flask, add 60% ethanol to dissolve, dilute to the scale, accurately measure 25 mL, place it in a 50-mL volumetric flask, and dilute to the scale with distilled water to obtain a 0.108 mg / mL standard solution.

[0085] Accurately pipette 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of rutin standard solution into 6 10 mL stoppered colorimetric tubes, add 30% ethanol solution to each colorimetric tube to make it 5 mL, and then accurately add 5% NaNO 2 0.3 mL of solution was shaken and allowed to stand for 6 min before adding 10% Al(NO 3 ) 3 Add 0.3 mL of the solution, shake well and let stand for 6 minutes, then add 4 mL of 1 mol / L NaOH solution, add 0.4 mL of water, shake well and let stand for 15 minutes, use the first tube as the blank to measure the absorbance at a wavelength of 510 nm, plot the absorbance against the standard content, and draw a standard curve.

[0086] 2. Sample determination

[0087] Dilute the sample to be tested (i.e., the supernatant prepared in the examples and comparative examples) 5 times, then take 2 mL of the sample solution, add 30% ethanol solution to make the volume of the colorimetric tube reach 5 mL, and then accurately add 5% NaNO 2 0.3 mL of solution was shaken and allowed to stand for 6 min before adding 10% Al(NO 3 ) 3 0.3 mL of the solution was shaken and placed for 6 minutes, then 4 mL of 1 mol / L NaOH solution was added, 0.4 mL of water was added, and the solution was shaken and placed for 15 minutes. The absorbance was measured at a wavelength of 510 nm. The flavonoid content was calculated by comparing with the standard curve. The results are shown in Table 2 and Figure 2 , shows the total flavonoid content of the products obtained in the examples and comparative examples. It can be seen that the total flavonoid content of the extracts obtained in Examples 1-5 of the present application is significantly higher than the total flavonoid content of the extracts obtained in Comparative Examples 1-4. Table 2 Test results of total flavonoid content of the perilla leaf extracts obtained in the examples and comparative examples

[0088]

[0089]

[0090] Effect Example 3: Determination of total phenol content

[0091] In alkaline solution, polyphenols can reduce tungstomolybdic acid (W 6+ becomes W 3+ ) to generate a blue compound with maximum absorption at 760nm. The depth of the color is positively correlated with the polyphenol content. Gallic acid (or pyrogallic acid) is generally used as a reference standard, and the total polyphenol content in the extract is expressed as an amount equivalent to gallic acid.

[0092] Experimental drugs: 1. Pyrogallic acid standard; 2. Folin-phenol test solution; 3. 26.7% Na 2 CO 3 Solution: weigh 26.7g Na 2 CO 3 Solid powder, add distilled water to 100g, stir to dissolve.

[0093] Experimental instruments: precision balance, spectrophotometer;

[0094] Experimental operation:

[0095] 1. Drawing of standard curve

[0096] Accurately weigh 44.3 mg of pyrogallic acid standard that has been vacuum dried to constant weight, dissolve it in distilled water to 100 mL, take 2.0, 4.0, 8.0, 12.0, 16.0 and 20.0 mL in a 100 mL volumetric flask, and dilute to scale with distilled water. Then take 1 mL of the above solutions of different concentrations and add them to a 10 mL colorimetric tube, then add 1 mL of distilled water, 0.5 mL of 2-fold diluted Folin-phenol test solution,

[0097] 1.5mL 26.7% Na 2 CO 3 The solution was diluted to 10 mL with water, reacted at room temperature for 2 h, and the absorbance was measured at 760 nm. The absorbance was plotted against the content of the standard to obtain a typical standard curve.

[0098] 2. Sample determination

[0099] The product prepared in the embodiment or comparative example was diluted 10 times to obtain a test solution, and then 1 mL of the test solution was added to a 10 mL colorimetric tube, followed by 1 mL of distilled water, 0.5 mL of a 2-fold diluted Folin-phenol test solution, 1.5 mL of 26.7% Na 2 CO 3 The solution was diluted to 10 mL with water, reacted at room temperature for 2 h, and the absorbance was measured at 760 nm. The measured absorbance was substituted into the standard curve to calculate the polyphenol content in the test solution. The results are shown in Tables 3 and Figure 3 , shows the total phenol content of the products obtained in the examples and comparative examples. It can be seen that the polyphenol content of the extracts obtained in Examples 1-5 of the present application is significantly higher than the polyphenol content of the extracts obtained in Comparative Examples 1-4.

[0100] Table 3 Test results of total phenol content of perilla leaf extract obtained in Examples and Comparative Examples

[0101]

[0102]

[0103] Effect Example 4: In vitro after-sun repair

[0104] 1. Experimental steps:

[0105] The products obtained in the above-mentioned examples and comparative examples were respectively prepared with serum-free DMEM culture medium to prepare test solutions of the experimental group with a volume percentage of 0.5%.

[0106] HaCaT cells were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 The cells were grown in DMEM medium containing 100 U / L penicillin and 100 mg / L streptomycin. 2In a saturated humidity incubator, when the cell confluence reaches more than 85%, the cells in the logarithmic growth phase are digested with 0.05% trypsin, and the digestion reaction is terminated with serum-containing DMEM. The cell count plate is used to count and the cell suspension concentration is adjusted to 7×10 4 The cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well and incubated at 37°C and 5% CO 2 After incubation, the old culture medium was removed, the cells were washed twice with phosphate buffer, and then 100 μL of PBS solution was added. The model group and the experimental group were irradiated with UVB at a dose of 15 mJ / cm 2 The negative control group was not irradiated; PBS was discarded, serum-free DMEM medium was added to the model group and the negative control group, 100 μL of the above-prepared filtered and sterilized experimental group test solution was added to each well of the experimental group, and 3 replicates were made for each test solution; the blank control group had no cells and 100 μL of PBS was added. Then, the cells were incubated at 37°C and 5% CO 2 Incubate in an incubator for 24 h. Then add 10 μL of CCK-8 solution to each well and incubate for another 3 h. Measure the absorbance at 450 nm to calculate the cell survival rate of each group.

[0107] The calculation formula of cell survival rate is as follows: cell survival rate (%) = (A experimental group or model group-A blank control group) / (A negative control group-A blank control group)×100%.

[0108] The experimental results are shown in Table 4 and Figure 4 It can be seen that the cell survival rate of the products prepared in Examples 1-5 of the present application is significantly higher than the cell survival rate of the products prepared in Comparative Examples 1-4. Figure 4 In the table, *p<0.05 indicates that there is a statistical difference compared with the model group; **p<0.01 indicates that there is a significant statistical difference compared with the blank group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the model group, which is significantly increased; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the blank group, and a very significant decrease.

[0109] Table 4 In vitro after-sun repair test results

[0110] Cell survival rate (%) Blank Group 100±2.87 Model Group 56.5±1.01 Example 1 69.74±2.32 Example 2 72.41±5.13 Example 3 68.26±2.4 Example 4 71.15±4.44 Example 5 72.46±1.41 Comparative Example 1 61.93±2.84 Comparative Example 2 63.49±1 Comparative Example 3 64.44±1.66 Comparative Example 4 65.56±2.67

[0111] Effect Example 5: Determination of inflammatory factor (IL-6) content

[0112] HaCaT cells in logarithmic phase were taken and the cell suspension was inoculated into a 6-well cell culture plate at a density of 250,000 cells / mL. 2 mL of cell suspension was added to each well and cultured for 12 h. The experimental group and the model group were treated with 40 mJ / cm 2After UVB irradiation of cells, the supernatant was discarded. 2 mL of DMEM solution was added to the blank group and the model group, and 2 mL of DMEM solution containing samples (sample volume percentage was 0.5%) was added to the experimental group to treat the cells for 24 h. After the treatment, the supernatant was discarded, and the cells were washed 2 to 3 times with PBS, and then the cells were treated with cell lysis solution, centrifuged at 10000r / min and 4°C for 10 minutes, and the supernatant was obtained to obtain the cell lysis supernatant. 20 μL of cell lysate was taken to detect the total protein content in the sample using the BCA kit; the determination of inflammatory factors was carried out according to the instructions of the ELISA kit, and the OD values ​​were measured at 450nm.

[0113] BCA kit operation steps:

[0114] 1. Sample pretreatment: According to the usage amount, prepare lysis solution at a ratio of RIPA:PMSF of 100:1, remove the culture medium, wash one side with PBS, add lysis solution at a ratio of 200 μL lysis solution to each well of the 6-well plate, and pipette several times to make the lysis solution fully contact with the cells.

[0115] 2. Post-processing: Centrifuge the lysed sample at 10,000 r for 10 min, take the supernatant, and then proceed to the subsequent protein concentration determination.

[0116] 3. Prepare BCA working solution at a ratio of 50:1 of reagent (A) to reagent (B).

[0117] 4. Add 20 μL of the protein sample to be tested into a 96-well plate.

[0118] 5. Add 200 μL of the prepared BCA working solution to each well and incubate at 37°C for 60 min.

[0119] 6. Detect absorbance at 562 nm.

[0120] 7. Calculate the protein concentration of the sample based on the standard curve and the dilution factor.

[0121] ELISA kit detection steps:

[0122] 1. Reagent preparation:

[0123] 1) Reagent warming: First, warm the reagent kit 30 minutes before the experiment. Place the sample to be tested at room temperature. If crystals appear in the concentrated washing solution, place it in a 37°C water bath until all the crystals are dissolved.

[0124] 2) Prepare the washing solution: Calculate the volume of the diluted washing solution in advance, then dilute the 20-fold concentrated washing solution into 1-fold application solution with deionized water. Store the unused concentrated washing solution at 4°C.

[0125] 3) Sample gradient dilution: Add 1 mL of standard / sample diluent (SR1) to the lyophilized standard, let stand for 15 minutes, and gently mix after it is completely dissolved (concentration 1000 pg / mL). Then add 500 μL of standard / sample diluent (SR1) to each of the remaining 6 tubes and dilute according to the following concentrations: 500, 250, 125, 62.5, 31.25, 15.62, 0 pg / mL. 1000 pg / mL is the highest point concentration of the standard curve, and the standard / sample diluent (SR1) is used as the zero point of the standard curve. The unused reconstituted standard stock solution (concentration 1000 pg / mL) should be discarded or divided into a single use amount as needed and stored in a -80°C refrigerator.

[0126] 4) Biotinylated antibody working solution: Calculate the required amount for the test in advance, dilute the 100-fold antibody concentrate into 1-fold working solution with the detection diluent (SR2) (mix thoroughly before dilution), and add to the reaction well within 30 minutes.

[0127] 5) Enzyme conjugate working solution: Prepare the required amount for each test, dilute the 40-fold concentrated enzyme conjugate into 1-fold working solution with enzyme conjugate diluent (SR3) (centrifuge before dilution), and use within 30 minutes.

[0128] 6) Washing method: shake off all the liquid in the wells of the ELISA plate, pat dry on absorbent paper, add 300 μL / well of washing solution using a washing bottle, let it stand for 30 seconds, shake off all the liquid in the wells of the ELISA plate, and pat dry on absorbent paper.

[0129] 2. Testing steps:

[0130] 1) 30 minutes before the experiment, take out the test kit and return to room temperature. Before adding the standard / sample, wash the plate three times and spin dry.

[0131] 2) Add 100 μL of standard / sample to the reaction wells, seal the plate, incubate at 37°C for 90 min, tap and wash the plate 4 times.

[0132] 3) Add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate, incubate at 37°C for 60 min, tap and wash the plate 4 times.

[0133] 4) Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate, incubate at 37°C for 30 min, tap and wash the plate 5 times.

[0134] 5) Add 50 μL of color development substrate to the reaction wells, seal the plate and incubate in a 37°C incubator in the dark for 15 min.

[0135] 6) Add 50 μL of stop solution and immediately measure the OD value at 450 nm using an ELISA reader (within 5 min).

[0136] According to the OD value obtained from the above test, the release level of IL-6 was calculated. The release amount of each inflammatory factor needs to be corrected by BCA content. The expression amount A of the inflammatory factor was calculated according to the standard curve, and corrected with the protein content B to obtain C=A / B. The results are shown in Table 5 and Figure 5 It can be seen that the anti-inflammatory ability of the products prepared in Examples 1 to 5 is significantly better than that of the products prepared in Comparative Examples 1 to 4, and the relative expression level of IL-6 in the products prepared in Examples 1 to 5 is significantly lower than that in Comparative Examples 1 to 4. Figure 5 In the table, *p<0.05, indicating that there is a statistical difference compared with the model group; **p<0.01, indicating that there is a significant statistical difference compared with the model group, and the level of leukemia is significantly reduced; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the blank group, and a very significant increase.

[0137] Table 5 Inflammatory factor (IL-6) content determination results

[0138] IL-6 content (pg / mg) Blank Group 1.58±0.41 Model Group 8.71±1.16 Example 1 3.25±0.51 Example 2 4.48±0.21 Example 3 4.34±0.33 Example 4 4.54±0.33 Example 5 4.56±0.34 Comparative Example 1 6.36±0.56 Comparative Example 2 6.69±0.38 Comparative Example 3 5.96±0.38 Comparative Example 4 5.81±0.3

[0139] Finally, it should be noted that, in this application, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the statement "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0140] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A natural deep eutectic solvent, characterized in that: The invention is prepared by uniformly mixing D-fructose, glycerol, xylitol and deionized water, wherein the molar ratio of D-fructose, glycerol and xylitol is 6-7:4.5-5.5:1, and preferably 6.2-6.8:4.8-5.2:

1.

2. The natural deep eutectic solvent according to claim 1, characterized in that The mass proportion of deionized water is 20% to 50%, more preferably 25% to 45%; Optionally, the preparation method of the natural low eutectic solvent comprises: mixing D-fructose, glycerol and xylitol uniformly to obtain a mixed solution; then mixing the mixed solution with deionized water, and then heating and dissolving it with a magnetic stirrer to form a transparent and uniform liquid without crystal precipitation at room temperature to obtain the natural low eutectic solvent; wherein the heating temperature is preferably 75-85°C, and the heating and stirring time is preferably 40-60min.

3. A method for preparing a perilla leaf extract, characterized in that: include: Mixing the natural deep eutectic solvent according to claim 1 or 2 with perilla leaf powder, and then performing ultrasonic extraction to obtain an extract; The extract is centrifuged to obtain the supernatant, and the supernatant is sterilized and preserved to obtain the perilla leaf extract.

4. The method for preparing the perilla leaf extract according to claim 3, characterized in that: The perilla leaf powder is obtained by drying the perilla leaves and crushing them through a 50-mesh sieve.

5. The method for preparing the perilla leaf extract according to claim 3 or 4, characterized in that: The mass ratio of the perilla leaf powder to the low eutectic solvent is 1:40-60, more preferably 1:45-55.

6. The method for preparing the perilla leaf extract according to any one of claims 3 to 5, characterized in that: In the ultrasonic extraction, the ultrasonic frequency is 30-45KHz, more preferably 35-45KHz; the ultrasonic power is 250-350W, more preferably 280-320W; the heating temperature during ultrasound is 50-80°C, more preferably 55-70°C; and the ultrasonic time is 60-120min, more preferably 80-100min.

7. The method for preparing the perilla leaf extract according to any one of claims 3 to 6, characterized in that: The rotation speed of the centrifugal treatment is 3000-9000 r / min, more preferably 4000-6000 r / min, and further preferably 5000 r / min; the time of the centrifugal treatment is 10-40 min, more preferably 20-40 min, and further preferably 30 min; Optionally, the sterilization treatment temperature is 95-110°C, more preferably 95-100°C; the time is 20-40 min, more preferably 25-35 min.

8. The method for preparing the perilla leaf extract according to any one of claims 3 to 7, characterized in that: The antiseptic treatment comprises adding a preservative to the sterilized material and mixing the mixture, wherein the mixing temperature is 50 to 80° C., more preferably 70 to 80° C.; Optionally, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the material obtained after sterilization is 0.5% to 2%, and the mass percentage of 1,2-hexanediol in the material obtained after sterilization is 0.5% to 2%; preferably, the mass percentage of p-hydroxyacetophenone in the material obtained after sterilization is 0.5%, and the mass percentage of 1,2-hexanediol in the material obtained after sterilization is 0.5%.

9. A perilla leaf extract, characterized in that Prepared by extraction with the natural low eutectic solvent according to claim 1 or 2; further, prepared by any method of claims 1-8.

10. Use of the perilla leaf extract according to claim 9 in preparing cosmetics; Preferably, the perilla leaf extract accounts for 5% to 99% by weight of the cosmetic, and more preferably 60% to 99% by weight.

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