Mulberry leaf extract as well as preparation method and application thereof in cosmetics

Abstract

The invention provides a mulberry leaf extract as well as a preparation method and application thereof in cosmetics, and relates to the technical field of fermentation and extraction of mulberry leaves. The preparation method of the mulberry leaf extract comprises the following steps: S1, inoculating a culture medium with compound bacteria to obtain a culture solution; s2, adding a mulberry leaf sample into the culture solution for fermentation, and feeding supplementary materials in the fermentation process to obtain fermentation liquor; s3, sterilizing, filtering, centrifuging and concentrating the fermentation liquid to obtain a crude extract; and S4, carrying out aqueous two-phase extraction, extraction, ultrafiltration and drying on the crude extract to obtain the mulberry leaf extract. The mulberry leaf extract prepared by the method has high contents of flavone, chlorogenic acid and peptide; the effects of inhibiting tyrosinase and removing DPPH free radicals are good.

Description

Technical Field

[0001] The invention belongs to the technical field of mulberry leaf fermentation extraction, and in particular is a mulberry leaf extract and a preparation method thereof and application in cosmetics. Background Art

[0002] Mulberry leaf is a widely recognized medicinal plant rich in bioactive ingredients such as flavonoids, proteins, amino acids and chlorogenic acid. These ingredients have shown significant therapeutic effects in terms of anti-oxidation, anti-inflammatory and hypoglycemic effects. Although traditional methods for preparing mulberry leaf extracts, such as physical methods such as hot water extraction and ultrasonic extraction, and chemical methods such as acid hydrolysis and alkaline hydrolysis, can extract some active ingredients, these methods have low extraction efficiency and are difficult to effectively extract proteins.

[0003] In recent years, microbial fermentation can significantly increase the content of active ingredients in plant materials, and convert macromolecules into more easily absorbed small molecules through microbial metabolism. However, the application of fermentation technology to the preparation of mulberry leaf extract still faces technical challenges in controlling fermentation conditions and extracting fermentation products. Summary of the invention

[0004] The present invention aims to solve the above problems and proposes a mulberry leaf extract and a preparation method thereof and application in cosmetics. The method utilizes composite bacteria fermentation technology to significantly increase the content of flavonoids in mulberry leaves. At the same time, the effective ingredients in the fermentation broth are efficiently extracted by two-phase aqueous extraction technology. In addition, the present invention also optimizes the fermentation conditions and extraction process to ensure the high purity and high activity of the extract.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The present invention provides a method for preparing a mulberry leaf extract, comprising the following steps:

[0007] S1. inoculating the composite bacteria into a culture medium to obtain a culture solution;

[0008] S2. adding the mulberry leaf sample to the culture medium for fermentation, and feeding the fermentation process to obtain a fermentation liquid;

[0009] S3. The fermentation broth is sterilized, filtered, centrifuged, and concentrated to obtain a crude extract;

[0010] S4. The crude extract is subjected to aqueous two-phase extraction, extraction, ultrafiltration and drying to obtain mulberry leaf extract.

[0011] In S1, the composite bacteria includes the following raw materials in parts by weight: 10 parts of Bacillus subtilis, 8 to 12 parts of Lactobacillus plantarum and 3 to 5 parts of Candida utilis.

[0012] In the present invention, the composite bacteria can significantly increase the flavonoid content in mulberry leaves, and can degrade macromolecular proteins into peptides, soluble proteins and free amino acids. Among them, Bacillus subtilis can secrete esterase and hydrolase during the fermentation process, and these enzymes can decompose the cell wall structure in mulberry leaves, thereby increasing the solubility and extractability of flavonoid compounds. Lactobacillus plantarum can enhance the antioxidant capacity of flavonoid components, because Lactobacillus plantarum can secrete enzymes to decompose cellulose and other complex polysaccharides in mulberry leaves during the fermentation process, thereby making active ingredients such as flavonoids easier to be absorbed by the human body. Candida utilis is mainly responsible for the degradation and conversion of proteins during the fermentation process. It decomposes the proteins in mulberry leaves into smaller peptides and amino acids by secreting proteases, thereby improving the utilization rate and digestion and absorption rate of proteins.

[0013] In S1, the inoculation was 4×10 7 ~7×10 7 10 mL of bacterial solution with a CFU / mL was inoculated into 1 L of culture medium.

[0014] In S1, the culture medium is MRS culture medium.

[0015] In S2, the mulberry leaf sample is obtained by washing, drying, sterilizing and crushing the mulberry leaves.

[0016] In S2, 80 to 130 g of the mulberry leaf sample is added to every 1 L of the culture solution.

[0017] In S2, the fermentation temperature is 25-30°C, preferably 26-28°C.

[0018] In S2, the fermentation time is 10 to 25 hours.

[0019] In S2, the stirring speed of the fermentation is 200-500 rpm.

[0020] In S2, the feed includes 17-25 g / L glucose, 9-15 g / L trehalose, 8-12 g / L peptone, 2-6 g / L yeast extract powder, 3-7 g / L sodium acetate, 0.1-0.9 g / L magnesium sulfate, and 0.1-0.3 g / L potassium dihydrogen phosphate.

[0021] In S2, the volume ratio of the feed to the culture medium is 10:2-4, preferably 10:2.5-3.3.

[0022] In S2, the flow rate of the feed is 30 to 50 mL / min.

[0023] In S3, the concentration method is reduced pressure concentration at a temperature of 50-60°C.

[0024] In S3, the concentration is performed to a solid content of 50-65%.

[0025] In S4, the first phase of the aqueous two-phase system is an organic solvent and water.

[0026] In S4, the second phase of the aqueous two-phase is salt and water.

[0027] In S4, the temperature of the aqueous two-phase extraction is 15-20°C.

[0028] In S4, the aqueous two-phase extraction may be performed by ultrasound-assisted extraction, and the ultrasound-assisted extraction time is 5 to 20 minutes.

[0029] In S4, the aqueous two-phase extraction retains the first phase.

[0030] Further in S4, the organic solvent is ethylene glycol.

[0031] Further in S4, the content of the organic solvent in the first phase is 18-35 wt %, preferably 20-30 wt %.

[0032] Further in S4, the salt is dipotassium hydrogen phosphate, disodium hydrogen phosphate or sodium citrate.

[0033] Further in S4, the content of the salt in the second phase is 6 to 20 wt%, preferably 9 to 15 wt%.

[0034] In S4, the extraction is to extract the first phase with ethyl acetate, and retain the ethyl acetate layer.

[0035] In S4, the molecular weight cut-off of the ultrafiltration is 5 KDa.

[0036] In S4, the drying method is vacuum drying, and the drying temperature is 45-55°C.

[0037] The present invention also provides a mulberry leaf extract prepared by the aforementioned preparation method, comprising 119.72-123.58 mg / g flavonoids, 36.78-46.54 mg / g polypeptides and 3.84-6.28 mg / g chlorogenic acid.

[0038] In some preferred embodiments, the mulberry leaf extract contains 121.47-123.58 mg / g flavonoids, 43.42-46.54 mg / g peptides and 5.94-6.28 mg / g chlorogenic acid.

[0039] In the present invention, the flavonoids and chlorogenic acid in the extract can both remove DPPH free radicals and inhibit the ability of tyrosinase; the peptide also has a good scavenging effect on DPPH free radicals.

[0040] In the present invention, the molecular weight of the peptide is ≤5 kDa.

[0041] In the present invention, the mulberry leaves are selected from small-leaved mulberry, Husang 197 or Nongsang No. 14.

[0042] In the present invention, the mulberry leaves are selected from mulberry leaves grown for 25 to 35 days after germination.

[0043] The present invention provides application of the mulberry leaf extract in cosmetics, wherein the cosmetics have at least one of the functions of anti-oxidation, anti-aging, promoting cell repair and regeneration, enhancing skin barrier and whitening.

[0044] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The present invention significantly increases the content of flavonoids in mulberry leaves through composite bacteria fermentation technology. At the same time, the technology can also degrade macromolecular proteins into peptides, soluble proteins and free amino acids. These changes not only improve the bioavailability of beneficial ingredients, but also improve their extraction efficiency. In order to ensure the high efficiency of the fermentation process, the present invention strictly controls the fermentation conditions to make the fermentation more efficient.

[0047] 2. The aqueous two-phase extraction system used in the present invention is particularly suitable for extracting various effective ingredients from the fermentation broth. The high extraction rate of the system ensures the efficient extraction of components such as flavonoids, peptides and chlorogenic acid.

[0048] 3. The fermented extract prepared by the present invention is rich in effective ingredients such as flavonoids, peptides and chlorogenic acid, which not only improves the extraction efficiency, but also enhances the medicinal value of mulberry leaf extract. At a concentration of 0.05 mg / mL, these extracts show significant antioxidant capacity, and the DPPH free radical scavenging rate can reach 66.5-82.4%, especially in some preferred embodiments, the scavenging rate can be further increased to 79.2-82.4%. In addition, these extracts also show good tyrosinase inhibition effect, with an inhibition rate between 43.2% and 66.5%, and the inhibition rate in the preferred embodiment can reach 58.7-66.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the HPLC chromatogram of the total flavonoids in the mulberry leaf extract in Example 1.

[0050] Figure 2 This is the high performance liquid chromatogram of the determination of chlorogenic acid in mulberry leaf extract in Example 1. DETAILED DESCRIPTION

[0051] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and carefully in combination with preferred embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0052] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0053] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of a particular range. The range defined in this way can be inclusive or exclusive of end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60-120 and 80-110 is listed for a specific parameter, it is understood that the range of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present invention, unless otherwise specified, the numerical range "ab" represents an abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" represents that all real numbers between "0-5" have been fully listed herein, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0054] If not otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution.

[0055] Unless otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0056] If not otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0057] If there is no special explanation, the "include" and "comprising" mentioned in the present invention represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may represent that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0058] If not specifically stated, in the present invention, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0059] The Bacillus subtilis used in the following examples and comparative examples was purchased from Jiangsu Greentech Biotechnology Co., Ltd., with a live bacterial content of 2×10 10 CFU / g; Lactobacillus plantarum was purchased from Jiangsu Greentech Biotechnology, with a live bacteria content of 1.0×10 10 CFU / g; Candida utilis was purchased from Chengdu Jiaye Biological, with a live bacterial content of 1×10 8 CFU / g.

[0060] Example 1

[0061] The preparation method of the mulberry leaf extract of this embodiment comprises the following steps:

[0062] The mulberry leaves are washed, dried, sterilized and crushed to obtain mulberry leaf samples; the mulberry leaves are selected from small-leaf mulberry leaves grown for 30 days after germination; the drying process makes the water content of the mulberry leaves 65%.

[0063] S1. inoculating the composite bacteria into a culture medium to obtain a culture solution;

[0064] S2. adding the mulberry leaf sample to the culture medium for fermentation, and feeding the fermentation process to obtain a fermentation liquid;

[0065] S3. The fermentation broth is sterilized, filtered, centrifuged, and concentrated to obtain a crude extract;

[0066] S4. The crude extract is subjected to aqueous two-phase extraction, extraction, ultrafiltration and drying to obtain mulberry leaf extract.

[0067] In this embodiment S1, the composite bacteria includes the following raw materials in parts by weight: 10 parts of Bacillus subtilis, 10 parts of Lactobacillus plantarum and 5 parts of Candida utilis.

[0068] In this embodiment S1, the inoculation is to inject 5×10 ^7 100 mL of bacterial solution with a CFU / mL was inoculated into 10 L of culture medium.

[0069] In this embodiment S1, the culture medium used is 10 L MRS culture medium.

[0070] In this embodiment S2, 100 g of the mulberry leaf sample is added to 1 L of the culture solution.

[0071] In Example S2, the fermentation temperature is 28°C.

[0072] In this embodiment S2, the fermentation time is 18 hours.

[0073] In this embodiment S2, the stirring speed of the fermentation is 300 rpm.

[0074] In this embodiment S2, the feed includes 20 g / L glucose, 9 g / L trehalose, 10 g / L peptone, 6 g / L yeast extract powder, 4 g / L sodium acetate, 0.7 g / L magnesium sulfate, and 0.3 g / L potassium dihydrogen phosphate.

[0075] In this embodiment S2, the volume ratio of the feed to the culture medium is 10:3.

[0076] In this embodiment S2, the flow rate of the feed is 30 mL / min.

[0077] In this embodiment S3, the concentration method is reduced pressure concentration at a temperature of 50°C.

[0078] In this embodiment S3, the concentration is performed to a solid content of 60%.

[0079] In the present embodiment S4, the first phase of the aqueous double phase is an organic solvent and water; wherein the organic solvent is ethylene glycol, and the content of the organic solvent in the first phase is 25 wt %.

[0080] In this embodiment S4, the second phase of the aqueous double phase is salt and water; wherein the salt is dipotassium hydrogen phosphate, and the content of the salt in the second phase is 12 wt %.

[0081] In this embodiment S4, the temperature of the two-phase aqueous extraction is 20°C.

[0082] In this embodiment S4, the two-phase aqueous extraction is also assisted by ultrasound, and the ultrasonic dispersion is carried out for 10 minutes.

[0083] In this embodiment S4, the two-phase aqueous extraction retains the first phase and discards the second phase.

[0084] In this embodiment S4, the extraction is to extract the first phase with ethyl acetate, and after three extractions, the ethyl acetate layer is retained and the water layer is discarded.

[0085] In this embodiment S4, the aforementioned water layer is subjected to ultrafiltration, and the filtrate is collected; the pore size of the ultrafiltration is 5KDa.

[0086] In this embodiment S4, the drying method is vacuum drying, the drying temperature is 50° C., and the drying is performed until constant weight.

[0087] The HPLC chromatogram of total flavonoids in mulberry leaf extract in this example is shown in Figure 1 , the HPLC chromatogram of chlorogenic acid is shown in Figure 2 Among them, total flavonoids include rutin, isoquercetin, astragalus glycoside, quercetin, etc.

[0088] Example 2

[0089] The difference between this embodiment and embodiment 1 is that:

[0090] The mulberry leaves of the present embodiment are selected from the mulberry leaves of Husang 197 grown for 25 days after germination.

[0091] In this embodiment S1, the composite bacteria includes the following raw materials in parts by weight: 10 parts of Bacillus subtilis, 8 parts of Lactobacillus plantarum and 3 parts of Candida utilis.

[0092] In this embodiment S1, the inoculation is to inject 4×10 ^7 10 mL of bacterial solution with a CFU / mL was inoculated into 10 L of culture medium.

[0093] Example 3

[0094] The difference between this embodiment and embodiment 1 is that:

[0095] In this embodiment S2, the fermentation temperature is 25°C.

[0096] In this embodiment S2, the fermentation time is 24 hours.

[0097] In this embodiment S2, the stirring speed of the fermentation is 500 rpm.

[0098] In this embodiment S2, the feed includes 25 g / L glucose, 12 g / L trehalose, 12 g / L peptone, 5 g / L yeast extract powder, 3 g / L sodium acetate, 0.4 g / L magnesium sulfate, and 0.3 g / L potassium dihydrogen phosphate.

[0099] In this embodiment S2, the volume ratio of the feed to the culture medium is 10:2.8.

[0100] Example 4

[0101] The difference between this embodiment and embodiment 1 is that:

[0102] In the present embodiment S4, the first phase of the aqueous double phase is an organic solvent and water; wherein the organic solvent is ethylene glycol, and the content of the organic solvent in the first phase is 20 wt %.

[0103] In this embodiment S4, the second phase of the aqueous double phase is salt and water; wherein the salt is disodium hydrogen phosphate, and the content of the salt in the second phase is 15 wt %.

[0104] Example 5

[0105] The difference between this embodiment and embodiment 1 is that:

[0106] In the present embodiment S4, the first phase of the aqueous double phase is an organic solvent and water; wherein the organic solvent is ethylene glycol, and the content of the organic solvent in the first phase is 23 wt %.

[0107] In this embodiment S4, the second phase of the aqueous two-phase system is salt and water; wherein the salt is sodium citrate, and the content of the salt in the second phase is 14 wt %.

[0108] Comparative Example 1

[0109] The difference between this comparative example and Example 1 is:

[0110] The mulberry leaves used in this comparative example are 40-day-old green mulberry leaves.

[0111] Comparative Example 2

[0112] The difference between this comparative example and Example 1 is:

[0113] In this comparative example S1, the composite bacteria includes the following raw materials in parts by weight: 10 parts of Bacillus subtilis and 10 parts of Lactobacillus plantarum.

[0114] Comparative Example 3

[0115] The difference between this comparative example and Example 1 is:

[0116] In this comparative example S2, no feed was added.

[0117] Comparative Example 4

[0118] The difference between this comparative example and Example 1 is:

[0119] In comparative example S4, the first phase of the aqueous biphase is an organic solvent and water; wherein the organic solvent is ethylene glycol, and the content of the organic solvent in the first phase is 15 wt %.

[0120] Comparative Example 5

[0121] The difference between this comparative example and Example 1 is:

[0122] In this comparative example S4, the second phase of the aqueous two-phase is salt and water; wherein the salt is ammonium sulfate.

[0123] Effect Example

[0124] The test results of the above embodiments and comparative examples are shown in Table 1.

[0125] The total flavonoid content was determined by high performance liquid chromatography (HPLC);

[0126] Test conditions: Octadecylsilane bonded silica gel was used as filler; methanol A and 0.5% phosphoric acid solution B were used as mobile phases for gradient elution;

[0127] Gradient elution program:

[0128] 0~5min, 10~30%A;

[0129] 5-15min, 30-60% A;

[0130] 15-20min, 60% A;

[0131] 20-30min, 60-90% A;

[0132] The detection wavelength is 330 nm; the theoretical plate number calculated based on the quercetin peak should be no less than 2000.

[0133] The peptide content was determined using high performance liquid chromatography (HPLC).

[0134] The determination of chlorogenic acid content refers to the determination standard of chlorogenic acid content in honeysuckle in the Chinese Pharmacopoeia.

[0135] The method for determining tyrosinase inhibition rate was based on T / SHRH 015-2018.

[0136] The DPPH free radical scavenging rate was determined using Solarbio's DPPH free radical scavenging ability test kit (specification: BC4750), and the concentration of the extract was 0.05 mg / mL.

[0137] Table 1

[0138]

[0139] In the above comparative examples, at the beginning of the study, when different types of mulberry leaves and mulberry leaves at different growth stages were selected, it was found that the contents of various substances were significantly different. Comparative Example 1 selected 40-day-old mulberry leaves, in which the contents of various substances were relatively low, resulting in poor extraction effect.

[0140] In comparative example 2, when exploring the effect of the composite bacteria species on the fermentation process, it was found that when Candida utilis was not added, less peptides were produced during the fermentation process.

[0141] In comparative example 3, no supplementary feed was added, and the fermentation degree was affected. The addition of supplementary feed can improve the fermentation efficiency.

[0142] When the amount of the organic solvent in the first phase of Comparative Example 4 is relatively small, the extraction efficiency will be reduced, thereby affecting the extraction of the target product.

[0143] In the second phase of the aqueous two-phase extraction of Comparative Example 5, when ammonium sulfate is selected as the salt, ammonium sulfate and ethylene glycol will compete for water molecules in the system. This competition leads to an increase in the mass fraction of ethylene glycol in the first phase, which is not conducive to the dissolution of target products such as flavonoids and peptides. Therefore, the selection of appropriate salts is key to improving the extraction efficiency and the extraction of target products.

[0144] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a mulberry leaf extract, characterized in that: The following steps are involved: S1. inoculating the composite bacteria into a culture medium to obtain a culture solution; S2. adding the mulberry leaf sample to the culture medium for fermentation, and feeding the fermentation process to obtain a fermentation liquid; S3. The fermentation broth is sterilized, filtered, centrifuged, and concentrated to obtain a crude extract; S4. The crude extract is subjected to aqueous two-phase extraction, extraction, ultrafiltration and drying to obtain mulberry leaf extract.

2. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: The S1 satisfies at least one of the following conditions (1) to (3): (1) The composite bacteria comprises the following raw materials in parts by weight: 10 parts of Bacillus subtilis, 8-12 parts of Lactobacillus plantarum and 3-5 parts of Candida utilis; (2) The inoculation is 4×10 7 ~7×10 7 CFU / mL 10mL of bacterial solution was inoculated into 1L of culture medium; (3) The culture medium is MRS culture medium.

3. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: The S2 satisfies at least one of the following conditions (1) to (7): (1) adding 80-130 g of the mulberry leaf sample to every 1 L of the culture solution; (2) The fermentation temperature is 25 to 30° C.; (3) The fermentation time is 10 to 25 hours; (4) The fermentation stirring speed is 200 to 500 rpm; (5) The feed includes 17-25 g / L glucose, 9-15 g / L trehalose, 8-12 g / L peptone, 2-6 g / L yeast extract, 3-7 g / L sodium acetate, 0.1-0.9 g / L magnesium sulfate, and 0.1-0.3 g / L potassium dihydrogen phosphate; (6) The volume ratio of the feed to the culture medium is 10:2-4; (7) The flow rate of the feed is 30 to 50 mL / min.

4. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: The aqueous two-phase extraction satisfies at least one of the following conditions (1) to (5): (1) The first phase of the aqueous biphase is an organic solvent and water; (2) The second phase of the aqueous double phase is salt and water; (3) The temperature of the aqueous two-phase extraction is 15 to 20° C.; (4) The aqueous two-phase extraction can be ultrasonic-assisted extraction; (5) The aqueous two-phase extraction retains the first phase.

5. The method for preparing the mulberry leaf extract according to claim 4, characterized in that: The aqueous two-phase extraction satisfies at least one of the following conditions (1) to (4): (1) The organic solvent is ethylene glycol; (2) the content of the organic solvent in the first phase is 18 to 35 wt %; (3) The salt is dipotassium hydrogen phosphate, disodium hydrogen phosphate or sodium citrate; (4) The content of the salt in the second phase is 6 to 20 wt%.

6. The method for preparing the mulberry leaf extract according to claim 1, characterized in that: The S4 satisfies at least one of the following conditions (1) to (3): (1) The extraction is to extract the first phase with ethyl acetate, and retain the ethyl acetate layer; (2) The ultrafiltration molecular weight cut-off is 5 KDa; (3) The drying method is vacuum drying.

7. The mulberry leaf extract prepared by the method for preparing the mulberry leaf extract according to any one of claims 1 to 6, characterized in that: Contains 119.72-123.58 mg / g flavonoids, 36.78-46.54 mg / g peptides and 3.84-6.28 mg / g chlorogenic acid.

8. The mulberry leaf extract according to claim 7, characterized in that The molecular weight of the peptide is ≤5 kDa.

9. The mulberry leaf extract according to claim 7, characterized in that Satisfy at least one of the following conditions (1) to (2): (1) The mulberry leaves are selected from Microphylla mulberry, Husang 197 or Nongsang 14; (2) The mulberry leaves are selected from mulberry leaves grown for 25 to 35 days after germination.

10. The use of the mulberry leaf extract in cosmetics according to any one of claims 7 to 9, characterized in that: The cosmetics have at least one of the functions of anti-oxidation, anti-aging, promoting cell repair and regeneration, enhancing skin barrier and whitening.

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