An enzyme for synthesizing anti-aging active substances, method and application thereof in the preparation of cosmetics

Abstract

The present invention discloses an enzyme for synthesizing anti-aging active substances, a method and an application thereof in the preparation of anti-aging cosmetics, and belongs to the field of biotechnology. The amino acid sequence of the enzyme is shown in SEQ ID NO.2. The enzyme is used in vitro to carry out an enzyme-catalyzed biosynthesis method to obtain a bioflavonoid compound with anti-aging activity, providing a new method for synthesizing a bioflavonoid compound with anti-aging activity, and can be used in the preparation of anti-aging cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Dendrobium fringed dendrobium methyltransferase and an application thereof in synthesizing anti-aging active flavonoid compounds, and further an application thereof in preparing cosmetics containing anti-aging active substances. Background Art

[0002] Bioflavonoids, often referred to as flavonoids, are a class of polyphenols widely found in plants. They are not only plant pigments that give flowers, fruits and leaves rich colors, but are also secondary metabolites of plants with a variety of biological activities, including antioxidant, anti-inflammatory, antiviral, anticancer, and antibacterial. According to their chemical structure, flavonoids can be divided into six major categories: flavones, flavonols, isoflavones, flavanones, flavanols and anthocyanidins, each of which contains many specific compounds. These flavonoids have important application value in drug development, nutrition and health care, food industry and cosmetics industry due to their wide range of health benefits.

[0003] As a natural flavonoid compound, isorhamnetin has powerful antioxidant, anti-inflammatory, anti-ultraviolet and anti-aging biological activities (https: / / doi.org / 10.1016 / j.biopha.2020.110301). It can help protect the skin from environmental damage and slow down the aging process. For example, isorhamnetin has been shown to protect human keratinocytes from ultraviolet B-induced cell damage (doi: 10.4062 / biomolther.2015.005), and isorhamnetin can reduce the risk of photoaging of human skin (doi: 10.2991 / icoh-17.2018.60). Therefore, it has broad application prospects in the field of cosmetics. However, isorhamnetin is mainly extracted from plants or chemically synthesized. Among them, the disadvantages of plant extraction are unstable supply of raw materials: the growth of plants is affected by factors such as season, climate and region, resulting in unstable supply of raw materials; low extraction purity: isorhamnetin extracted from plants is often accompanied by other impurities and needs further purification, which increases cost and complexity; environmental impact: large-scale extraction may burden the environment, such as over-exploitation of plant resources and the use of solvents. The disadvantages of chemical synthesis are: high cost: chemical synthesis involves multi-step reactions, harsh reaction conditions, and purification is required, which is costly. Environmental pollution: harmful by-products and waste may be produced in chemical reactions, which are potentially harmful to the environment. Safety issues: some chemical reagents are toxic or corrosive, and improper operation may lead to safety accidents. In comparison, biosynthesis can overcome the above determination and has the following advantages: environmental protection: the process is relatively environmentally friendly and does not produce harmful waste; high efficiency: efficient production can be achieved by optimizing the metabolic pathways of microorganisms or plant cells; sustainable: using biological resources, strong sustainability, and not dependent on the supply of plant raw materials. Therefore, it is of great significance to identify and develop functional enzymes and methods for the synthesis of methylated flavonoids with anti-aging activity.

[0004] Dendrobium is a plant of the genus Dendrobium in the family Orchidaceae. Dendrobium extract is rich in polyphenols and flavonoids and has strong antioxidant capacity. These ingredients can effectively neutralize free radicals and reduce the oxidative damage of free radicals to skin cells, thereby delaying skin aging and keeping the skin young. The functional identification of related enzymes in Dendrobium will help develop functional enzymes for the synthesis of methylated flavonoids with anti-aging activity. Summary of the invention

[0005] The present invention mainly aims at the above-mentioned technical problems and provides a methyltransferase gene that catalyzes the production of anti-aging active bioflavonoids, as well as a method for biosynthesizing anti-aging active bioflavonoid compounds, so as to solve the shortcomings of existing methods for obtaining anti-aging active bioflavonoid compounds.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In one aspect, the present invention provides a methyltransferase for synthesizing anti-aging active bioflavonoid compounds, wherein the amino acid sequence thereof comprises the sequence shown in SEQ ID NO.2.

[0008] On the other hand, the present invention provides a methyltransferase gene for synthesizing anti-aging active bioflavonoid compounds, the sequence of which is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2,

[0009] Or based on the principle of complementary pairing, the methyltransferase gene provided by the present invention can be a sequence that is fully complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0010] In a preferred embodiment, the nucleotide sequence of the methyltransferase gene comprises a nucleotide sequence as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0011] It is well known to those skilled in the art that a gene sequence may also contain introns, promoters and various regulatory elements, and thus the nucleotide sequence of the above-mentioned methyltransferase gene may also contain introns, promoters and various regulatory elements.

[0012] On the other hand, the present invention provides an application of a methyltransferase gene in the synthesis of anti-aging active bioflavonoid compounds, wherein the nucleic acid sequence of the methyltransferase gene is a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2, or a sequence that is fully complementary to a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0013] In a preferred embodiment, the methyltransferase gene sequence is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2, or a sequence that is completely complementary to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0014] In a preferred embodiment, the nucleotide sequence of the methyltransferase gene is as shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO. 1.

[0015] On the other hand, the present invention provides the use of the aforementioned methyltransferase in synthesizing bioflavonoid compounds with anti-aging activity.

[0016] In another aspect, the present invention provides a method for synthesizing anti-aging active bioflavonoid compounds, the method comprising the following steps:

[0017] 1) obtaining the aforementioned methyltransferase;

[0018] 2) Using the methyltransferase in step 1) to catalyze the synthesis of anti-aging active bioflavonoid compounds in an enzyme activity reaction system.

[0019] In a preferred embodiment, the methyltransferase is obtained by prokaryotic expression.

[0020] In a preferred embodiment, the methyltransferase is obtained by chemical synthesis.

[0021] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned methyltransferase, SAM, a substrate for synthesizing anti-aging active bioflavonoid compounds, and a buffer.

[0022] In a preferred embodiment, the enzyme activity reaction system in step 2) exists in the microorganism.

[0023] In another aspect, the present invention provides a method for synthesizing a bioflavonoid compound with anti-aging activity, characterized in that the method comprises the following steps:

[0024] 1) obtaining a methyltransferase comprising the amino acid sequence shown in SEQ ID NO.2;

[0025] 2) Using the above methyltransferase to catalyze the synthesis of anti-aging active bioflavonoid compounds in an enzyme activity reaction system.

[0026] In a preferred embodiment, the methyltransferase is obtained by prokaryotic expression.

[0027] In a preferred embodiment, the methyltransferase is obtained by chemical synthesis.

[0028] In a preferred embodiment, the enzyme activity reaction system contains the above-mentioned methyltransferase, SAM, a substrate for synthesizing anti-aging active flavonoid compounds, and a buffer.

[0029] In a preferred embodiment, the enzyme activity reaction system in step 2) exists in the microorganism.

[0030] In a preferred embodiment, the suitable reaction temperature of the enzyme activity reaction system is 10-70°C, preferably 50-60°C.

[0031] In a preferred embodiment, the pH of the enzyme activity reaction system is 7.0-10.0, preferably 8.0-9.0.

[0032] In a preferred embodiment, the substrate for synthesizing anti-aging active flavonoids is quercetin.

[0033] On the other hand, the present invention provides a method for preparing cosmetics rich in anti-aging active substances, characterized in that it comprises the following steps: 1) obtaining anti-aging active bioflavonoid compounds using the aforementioned method; 2) adding the anti-aging active bioflavonoid compounds described in step 1) into the cosmetics at a mass percentage of 0.001% to 10%.

[0034] In a preferred embodiment, the aforementioned method for preparing cosmetics is characterized in that the dosage form of the cosmetics is essence, lotion, emulsion, cream, gel, mask, foam or spray.

[0035] In a preferred embodiment, the aforementioned method for preparing cosmetics is characterized in that the mass percentage of the anti-aging active bioflavonoid compound in the cosmetics is 0.001% to 5%.

[0036] In a preferred embodiment, the aforementioned method for preparing cosmetics is characterized in that the mass percentage of the anti-aging active bioflavonoid compound in the cosmetics is 0.001% to 1%.

[0037] In a preferred embodiment, the aforementioned method for preparing cosmetics is characterized in that the mass percentage of the anti-aging active bioflavonoid compound in the cosmetics is 0.01% to 0.1%.

[0038] In a preferred embodiment, the aforementioned cosmetic preparation method is characterized in that the anti-aging active flavonoid compound is isorhamnetin.

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] 1) Compared with the prior art, the present invention utilizes an enzyme-catalyzed biosynthesis method to obtain anti-aging active bioflavonoid compounds, which has the advantages of simpler steps, less pollution, and more uniform products compared with chemical synthesis methods.

[0041] 2) The present invention provides a new methyltransferase.

[0042] 3) The methyltransferase of the present invention has a wide temperature range (10-70°C) suitable for reaction.

[0043] 4) The methyltransferase of the present invention has a wider suitable pH range. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0045] Figure 1Results of cloned gene fragments. A: Gene cloning, M is a 2000 bp DNA marker, and lane 1 is DfOMT1.

[0046] Figure 2 The SDS-PAGE gel electrophoresis diagram of pET-28a(+)-DfOMT1 fusion protein. M is a protein marker (25-180 kDa), a is empty load, b is crude enzyme, and c is pure enzyme.

[0047] Figure 3 It is the liquid phase diagram of the reaction between DfOMT1 and quercetin; a and b are the standard samples of quercetin and isorhamnetin, respectively, and c is the product detection result after the enzyme-catalyzed reaction.

[0048] Figure 4 The results of the test on the suitable conditions for DfOMT1 to catalyze the conversion of quercetin to isorhamnetin. A represents different pH conditions, B represents different temperature conditions, and C represents the catalytic results of different metal ions. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0051] Example 1

[0052] Cloning of target methyltransferase gene

[0053] 1. Total RNA extraction kit was used to extract total RNA from Dendrobium fimbriatum. The total RNA extracted above was taken and reverse transcribed to obtain cDNA. The full-length fragment of the DfOMT1 gene was amplified by polymerase chain reaction (PCR) using this as a template. The specific primers were designed according to the transcriptome data of this experiment. The cloning primer sequences were: DfOMT1-F: ATGTTATCTTGCCCTTACTAT, DfOMT1-R: TCATTCGATTCGCCTGCAAAT. The cDNA of Dendrobium fimbriatum extracted in the above experiment was used as a PCR template, and the target fragment was amplified using primers DfOMT1-F and DfOMT1-R. A 25 μL PCR reaction system was prepared according to the following system: 1 μL DNA template, 12.5 μL 2×Phanta Max Master Mix, 1 μL upstream primer, 1 μL downstream primer, 9.5 1 μL ddH2O.

[0054] Place the prepared PCR reaction system in a PCR instrument and amplify the target gene according to the PCR reaction program in the table below.

[0055] PCR reaction procedure for amplifying genes

[0056]

[0057] After the reaction, the product size was detected by 1% agarose gel electrophoresis. Figure 1 .

[0058] 2. Connect the amplified product to the T vector and transform it into E. coli DH5α. Select a single colony and culture it in LB medium and perform PCR verification. Send samples with correct PCR results for sequencing.

[0059] The nucleotide sequence obtained by sequencing is shown in SEQ ID NO.1. The gene sequence contains 681 nucleotides and encodes 226 amino acids (shown in SEQ ID NO.2).

[0060] >SEQ ID NO.1

[0061] ATGTTATCTTGCCCTTACTATTTTATGCAGTACATGTTGGATACCAGTGTATACCCTAGAGAGCATGAACAACTCAAGGC

[0062] TTTGAGAGAAGAAACAATGAAGAACAGTAGGTTTTATATGATTGTGCCTCCAGAAGAAGGCCAACTCCTCTCTATGCTGC

[0063] TCAAGTTGATGAATGCAAAGAAAACTATTGAAATTGGTGTCTTCACAGGCTACTCACTCCTCACTACAGCTCTTGCATTA

[0064] CCAGAAGATGGCAAAATAATGGCAATCGACATTGACAAATCGAACTACGAAATTGGGCGACCGTACATTCAGAAAGCTGG

[0065] CGTTGAGCACAAGATCCAGTCATTTCATTGAATCTCAGGCTCTTCCTGTTCTTGATAAATTGATTCAAGAGTTGAAGGAAGAAG

[0066] AATGTTTGATTTTGCATTTGTGGATGCTGATAAAGTTAACTACAGCAATTATCATAACAGGGTTATTAAGCTTGTCAGG

[0067] ATAGGTGGACTGATTGTGTATGATAACACACTTTGGGGAGGCAGCGTCGTTGGGCCAGTGAAACCTCAATTTCCAGGTCA

[0068] GTTCGAGGAAGCTCGAGATAGCATAATGCGTCTCAACAATGAATTAGCCTCGGATCATAGGATCGAAATGTCACAAGTTT

[0069] GTATAGGCGATGGCGTCACCATTTGCAGGCGAATCGAATGA

[0070] >SEQ ID NO.2

[0071] MLSCPYYFMQYMLDTSVYPREHEQLKALREETMKNSRFYMIVPPEEGQLLSMLLKLMNAKKTIEIGVFTGYSLLTTALAL

[0072] PEDGKIMAIDIDKSNYEIGRPYIQKAGVEHKIQFIESQALPVLDKLIQELKEEELFDFAFVDADKVNYSNYHNRVIKLVR

[0073] IGGLIVYDNTLWGGSVVGPVKPQFPGQFEEARDSIMRLNNELASDHRIEMSQVCIGDGVTICRRIE

[0074] Example 2 In vitro enzyme activity function identification

[0075] 1. Protein Purification

[0076] The bacterial culture containing the target DNA sequence was cultivated and enriched, and then the key recombinant plasmid pET-28a-DfOMT1 was isolated and extracted from the liquid. This plasmid was introduced into the sensitive cell strain of Escherichia coli BL21, and the single clones were screened and purified by the plate streaking method. After that, these clones were transferred to LB culture medium and cultured at 37°C and 200rpm for up to 16 hours to prepare the original bacterial liquid. Next, the original bacterial liquid was inoculated into LB medium containing 50μg / mL kanamycin at a ratio of 1:100, and continued to be cultured at 37°C until OD600 reached 0.6-0.8 (refer to LB medium without antibiotics). After reaching the specified density, 0.5mM IPTG was added to the culture medium to induce protein expression, and the temperature was lowered to 16°C and stirred for 20 hours. After the induction of expression was completed, the bacterial liquid was centrifuged and collected, and the bacteria were resuspended in 50mL Tris-HCl buffer pre-cooled to pH 9.0, and the culture medium residue was completely removed after two centrifugation and buffer exchange steps. Finally, a bacterial suspension was prepared by adding 15 ml of Tris-HCl buffer per gram of wet bacteria, and the suspension was placed in a -20°C refrigerator and frozen overnight. The next day, ultrasonic crushing technology was used under low-temperature conditions to lyse the bacteria. The power was set to 150 watts, and the crushing cycle was 3 seconds of operation and 3 seconds of pause until the suspension became clear. Subsequently, the supernatant was separated by centrifugation at 14000 rpm for 4 minutes at 4°C. The resulting supernatant was the required crude enzyme solution, which was suitable for storage at -20°C for later use. In order to further purify the target protein with a His-Tag tag, nickel ion affinity chromatography technology was used to elute the protein by gradiently increasing the imidazole concentration. Finally, the empty control, crude enzyme solution and purified products (marked as 1, 2, and 3) were analyzed by 10% SDS-PAGE gel electrophoresis. The results ( Figure 2 ) showed that the target protein band was clearly displayed at the position of about 26 kDa, which not only confirmed the effective expression of the target protein in the supernatant, but also proved the high quality of the purified protein, laying the foundation for subsequent experiments.

[0077] 2. In vitro enzyme activity detection

[0078] 1) The enzyme activity test system constructed contains the following components: 10 μg of pure enzyme preparation, 0.5 mM quercetin as substrate, 5 mM S-adenosylmethionine as methyl donor, and 14 mM β-mercaptoethanol, all of which are dissolved in 200 μL 50 mM Tris-HCl buffer with a pH of 9.0. The mixture is incubated at 45°C in a water bath for 45 minutes, and then an equal amount of pre-cooled methanol is added to terminate the reaction, and vortex mixing is used to ensure sufficient mixing. The mixture is then centrifuged at 14,000 rpm for 4 minutes at 4°C, after which the supernatant is extracted and analyzed by high-performance liquid chromatography.

[0079] 2) For high performance liquid chromatography analysis, the following gradient elution program was used: chromatographic acetonitrile (solution A) and ultrapure water containing 0.1% formic acid (solution B) were used as mobile phases, and gradient elution was used, with the elution gradient being 0-5 min, solution A 20%; 5-10 min, solution A 20%-27%; 10-15 min, solution A 27%; 15-25 min, solution A 27%-40%; 25-35 min, solution A 40%-60%; 35-40 min, solution A 60%-80%; 40-42 min, solution A 80%-100%; 42-45 min, solution A 100%-20%; 45-50 min, solution A 20%. The detection wavelength was 350 nm, the temperature was room temperature (25° C.), and the injection volume was 10 μL.

[0080] The experimental test results show (see Figure 3 ), when quercetin was used as a substrate, the DfOMT1 enzymatic reaction produced a significant chromatographic peak. By comparing the retention time of the peak with that of the standard, it was confirmed that the target product corresponding to the peak was isorhamnetin.

[0081] 3. Suitable temperature and pH detection

[0082] Using quercetin as the methyl acceptor and SAM as the methyl donor, the effects of pH, temperature and metal ions on the catalytic activity were investigated.

[0083] 1. Temperature: Quercetin was used as the methyl acceptor and SAM as the methyl donor to investigate the changes in enzyme activity at different reaction temperatures (10°C, 20°C, 30°C, 40°C, 50°C, 60°C and 70°C). Each 200μL reaction mixture contained 10μg of purified enzyme protein, 0.5mM substrate concentration, 5mM SAM and 14mMβ-mercaptoethanol. The system was adjusted to the specified volume using pH9.0, 50mM Tris-HCl buffer. The reaction at each temperature lasted for 45 minutes, and then the reaction process was quickly terminated with the help of an equal amount of pre-cooled methanol. The supernatant was obtained by centrifugation and analyzed by HPLC to evaluate the conversion rate. This process was performed three times per group to enhance data robustness.

[0084] 2. pH: Four different buffer systems were selected, covering the pH range of 5.0 to 10.0: Citric acid-sodiumcitrate (5.0-9.0), Na2HPO4-NaH2PO4 (5.0-9.0), Tris-HCl (7.0-10.0), and Na2CO3-NaHCO3 (7.0-10.0). The experiment was set up in a standardized 200μL reaction volume, with 14mM β-mercaptoethanol, 5mM SAM, 10μg protein and 0.5mM quercetin added uniformly, and the corresponding buffer was used to adjust the total volume to the required volume. All samples were reacted at a constant temperature of 45°C for 45 minutes, and the subsequent processing steps were the same as above to ensure the accuracy and repeatability of the data.

[0085] 3. Metal ions: In a total volume of 200 μl reaction mixture, 50 mM Tris-HCl buffer at pH 9.0 was added with 14 mM β-mercaptoethanol, 5 mM UDP-glucose, 0.5 mM specific substrate, and 10 μg of purified protein. In addition, 5 mM of selected divalent metal ions were added. The entire reaction was carried out at 37 °C for 1 hour. The specific effects of various divalent metal ions and EDTA·2Na on catalytic activity were evaluated.

[0086] The results show that ( Figure 4 ), DfOMT1 has the highest enzyme activity at 30℃, and has certain activity in the range of 10~70℃. The optimum pH of the reaction solution is 8.0~9.0, suitable for reaction in the pH range of 7.0~10.0, and the catalytic activity in NaCO3-NaHCO3 or Tric-HCl buffer is higher than that in other buffers. The enzyme is ion-dependent, and Mn and Mg ions can greatly improve its catalytic efficiency.

[0087] Example 3 Preparation of cosmetics rich in anti-aging active substances

[0088] The methyltransferase identified in Examples 1 and 2 is used in the preparation of cosmetics rich in anti-aging active substances, comprising the following steps: 1) using the aforementioned method to obtain an anti-aging active bioflavonoid compound, namely isorhamnetin; 2) adding the anti-aging active bioflavonoid compound described in step 1) into the cosmetics at a mass percentage of 0.001% to 10%.

[0089] In some products, the cosmetic is in the form of a solution, emulsion, suspension, cream, gel, foam or spray.

[0090] In some other products, the mass percentage of the anti-aging active bioflavonoid compounds in the cosmetics is 0.001% to 5%.

[0091] In some other products, the mass percentage of the anti-aging active bioflavonoid compounds in the cosmetics is 0.001% to 1%.

[0092] In some other products, the mass percentage of the anti-aging active bioflavonoid compound in the cosmetic is 0.01% to 0.1%.

[0093] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A methyltransferase for synthesizing anti-aging active bioflavonoid compounds, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. A methyltransferase gene for synthesizing anti-aging active bioflavonoid compounds, the sequence of which is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

3. Use of the methyltransferase according to claim 1 or the gene according to claim 2 in the synthesis of anti-aging active bioflavonoid compounds, wherein the anti-aging active bioflavonoid compounds are isorhamnetin synthesized using quercetin as a substrate and S-adenosylmethionine as a methyl donor.

4. Use of the methyltransferase according to claim 1 or the gene according to claim 2 in the preparation of cosmetics containing anti-aging active bioflavonoid compounds, wherein the anti-aging active bioflavonoid compound is isorhamnetin synthesized using quercetin as a substrate and S-adenosylmethionine as a methyl donor.

5. A method for synthesizing anti-aging active bioflavonoid compounds, the method comprising the following steps: 1) obtaining the methyltransferase according to claim 1; 2) Using the methyltransferase in step 1) to catalyze the synthesis of anti-aging active bioflavonoid compounds in an enzyme activity reaction system, the enzyme activity reaction system contains the methyltransferase in step 1), S-adenosylmethionine, quercetin and a buffer solution, and the flavonoid compound is isorhamnetin.

6. A method for synthesizing anti-aging active bioflavonoid compounds as claimed in claim 5, characterized in that: In step 1), the methyltransferase is obtained by prokaryotic expression or by chemical synthesis.

7. A method for preparing cosmetics containing anti-aging active substances, characterized in that: The method comprises the following steps: 1) obtaining an anti-aging active bioflavonoid compound by the method of claim 5; 2) adding the anti-aging active bioflavonoid compound of step 1) into cosmetics at a mass percentage of 0.01% to 10%, wherein the flavonoid compound is isorhamnetin.

8. The method for preparing cosmetics according to claim 7, characterized in that: The dosage form of the cosmetic is essence, lotion, emulsion, cream, gel, mask, foam or spray.

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