Preparation method of decapeptide-4, composition for preventing hair loss prepared by compounding the prepared decapeptide-4 with a plant extract, and hair care product

Abstract

The present invention discloses a preparation method of decapeptide-4, a composition for preventing hair loss prepared by compounding the prepared decapeptide-4 with plant extracts, and a hair care product. When preparing decapeptide-4, first, a polypeptide resin is synthesized by the Fmoc solid-phase method and cleaved to obtain a crude polypeptide. After dissolving the crude polypeptide, a polypeptide powder is obtained by freeze-drying. Finally, after dissolving the polypeptide powder, decapeptide-4 is obtained through oxidation, purification, and freeze-drying. The composition for preventing hair loss includes decapeptide-4, Poria cocos sclerotium extract, and Ginkgo biloba extract; the hair care product can be formulated in a water-soluble or liposome form. The preparation method of the present invention can effectively improve the purity and yield of decapeptide-4; and the prepared decapeptide-4 and plant extracts are compounded to form a composition, thereby effectively preventing hair loss.

Description

Technical Field

[0001] The present invention belongs to the application field of decapeptide-4, and particularly relates to a preparation method of decapeptide-4, a composition for preventing hair loss prepared by compounding the prepared decapeptide-4 with a plant extract, and a hair care product. Background Art

[0002] Decapeptide-4 is a synthetic peptide composed of arginine, aspartic acid, cysteine, glutamic acid, leucine, methionine and tyrosine. It can penetrate the dermis and increase collagen, reverse the aging process through internal and external reconstruction, stimulate the proliferation of collagen, elastic fibers and hyaluronic acid, increase the water content and lock water, increase the skin thickness and reduce fine lines. Decapeptide-4 can penetrate the dermis, stimulate the synthesis of collagen and elastin, and reverse the skin aging process through internal and external reconstruction; it can also stimulate the proliferation of collagen, elastic fibers and hyaluronic acid, improve the skin water content and moisturizing power, and increase the skin antibodies to keep sensitive skin insensitive. At the same time, studies have shown that small molecule decapeptide-4 can penetrate into the scalp to play a role in preventing hair loss and promoting hair growth. However, when directly formulating decapeptide-4 into a hair care product for application, due to its poor water solubility, it is difficult to exert its activity to the greatest extent, and at the same time, the products prepared by the existing preparation methods of decapeptide-4 have low purity and low yield, which will further limit its application in anti-hair loss products.

[0003] Poria cocos is the sclerotium of the fungus Poria cocos Wolf of the family Polyporaceae. So far, the chemical components found in Poria cocos mainly include polysaccharides, triterpenoids, cyclic alcohols, amino acids, fatty acids, etc., and the reported bioactive compounds mainly focus on triterpenoids and polysaccharide compounds. At present, 84 triterpenoid components and more than 60 polysaccharide components have been isolated and identified. Modern research shows that Poria cocos has biological activities such as anti-tumor, immunomodulatory, anti-inflammatory, antioxidant, anti-aging, improving memory, regulating the urinary system, lowering blood sugar, lowering blood lipid, sedative, hypnotic, liver protecting, etc., and its main active components are triterpenoids and polysaccharides.

[0004] Ginkgo biloba extract is a class of products rich in active ingredients extracted from dried Ginkgo biloba leaves. It mainly contains flavonoids, terpene lactones, polysaccharides, phenols, organic acids, alkaloids, amino acids, steroid compounds and trace elements, etc. The most important medicinal value components are flavonoids and terpene lactones, such as total flavonoids of Ginkgo biloba and ginkgolides. And these components have strong antioxidant and free radical scavenging abilities. Antioxidation and free radical scavenging are based on the fact that the flavonoids and terpene lactones in Ginkgo biloba extract can effectively scavenge free radicals in the body and protect cells from oxidative damage. Summary of the Invention

[0005] Objective of the Invention: The present invention provides a novel method for preparing decapeptide-4, which can effectively improve the purity and yield of decapeptide-4; and compound it with plant extracts to form a composition, thereby effectively preventing hair loss.

[0006] Technical Solution: The method for preparing decapeptide-4 of the present invention includes the following steps:

[0007] (1) Synthesize polypeptide resin by Fmoc solid-phase method and cleave it to obtain crude polypeptide.

[0008] (2) Dissolve the crude polypeptide and obtain polypeptide powder by freeze-drying.

[0009] (3) Dissolve the polypeptide powder, and obtain decapeptide-4 after oxidation, purification and freeze-drying.

[0010] When preparing decapeptide-4 of the present invention, the process steps of synthesis-cleavage-freeze-drying-oxidation-purification-freeze-drying are adopted. That is, compared with the existing method, after freeze-drying the crude polypeptide after cleavage and then dissolving it for oxidation before cleavage and oxidation, the state of the crude polypeptide can be made more fluffy, further increasing the solubility of the crude polypeptide, promoting the completion of the oxidation process, and effectively improving the purity of the crude product after oxidation.

[0011] Furthermore, in the method for preparing decapeptide-4 of the present invention, the synthesis of polypeptide resin by Fmoc solid-phase method includes the following steps:

[0012] (1) Immerse dichloro resin in dichloromethane, wash and drain it, add Fmoc-L-Cys(Trt)-OH, and cap it with methanol; then add piperidine to react to remove the protecting group.

[0013] (2) Add Fmoc-L-Tyr(tbu)-OH and HOBT to step (1) for condensation reaction; then add piperidine to react to remove the protecting group.

[0014] (3) Sequentially couple the subsequent Fmoc-L-Met-OH, Fmoc-L-Glu(otbu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Asp(otbu)-OH, Fmoc-L-Cys(Trt)-OH amino acids according to the steps, remove the Fmoc protecting group, wash and drain to obtain polypeptide resin.

[0015] The anti - hair - loss composition of the present invention, the anti - hair - loss composition comprises decapeptide - 4, Poria cocos sclerotium extract and Ginkgo biloba extract prepared by the above - mentioned preparation method, and the mass ratio of the three is 1:(15 - 20):(20 - 25).

[0016] The present invention combines Poria cocos sclerotium extract and Ginkgo biloba extract with decapeptide - 4 to prepare an anti - hair - loss composition. This composition can not only promote the proliferation of dermal papilla cells and regulate key growth factors, but also reduce the adverse effects brought by androgens and improve androgenetic alopecia. In addition, it can also resist oxidation and aging, improve blood circulation and energy metabolism, and promote hair follicle revival.

[0017] The hair care product of the present invention comprises 0.5 - 5% of the above - mentioned anti - hair - loss composition, 80 - 88% of an alcohol solution and the balance of water.

[0018] Furthermore, the hair care product of the present invention may also comprise 0.005 - 0.1% of an extended peptide, and the extended peptide comprises at least one of myristoyl pentapeptide - 4, biotin tripeptide - 1, acetyl tetrapeptide - 3, octapeptide - 2, decapeptide - 18, oligopeptide - 54, oligopeptide - 71, decapeptide - 10, myristoyl dipeptide - 13, myristoyl pentapeptide - 17, myristoyl pentapeptide - 16 or copper peptide.

[0019] Furthermore, the hair care product of the present invention may also comprise 0.3 - 1.0% of a solubilizer, and the solubilizer comprises LRI, Tween 20, Tween 40, Tween 60, Span 20 or PEG - 40. Or based on the extended peptide, it may also comprise 0.3 - 1.0% of a solubilizer, and the solubilizer comprises LRI, Tween 20, Tween 40, Tween 60, Span 20 or PEG - 40.

[0020] Furthermore, the hair care product of the present invention may also comprise 0.01 - 0.5% of an antioxidant, and the antioxidant comprises sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutylhydroxytoluene, tocopherol, butylated hydroxyanisole or tert - butylhydroquinone.

[0021] Furthermore, the hair care product of the present invention comprises 1 - 5% of the above - mentioned anti - hair - loss composition, 1 - 2% of soy lecithin, 0.4 - 0.6% of cholesterol, 15 - 18% of ethanol, 0.2 - 0.5% of polysorbate - 80, 0.5 - 1.5% of a preservative and the balance of water.

[0022] The hair care product of the present invention is prepared by the following steps:

[0023] (1) Put the Poria cocos sclerotium extract into an alcohol solution, heat and stir at 40 - 50 °C until the Poria cocos sclerotium extract is completely dissolved to obtain a mixed solution;

[0024] (2) Add water to the mixed solution in step (1) to form a transparent and clear solution;

[0025] (3) Add the ginkgo biloba leaf extract and decapeptide-4 to the solution in step (2), and stir to dissolve to obtain the anti-hair loss composition.

[0026] When preparing the water-soluble hair product of the present invention, the extract of Poria cocos sclerotium, the ginkgo biloba leaf extract and decapeptide-4 are compounded. First, an alcohol solution is added, that is, the extract of Poria cocos sclerotium is added to the alcohol solution. Based on the action of the alcohol solution, the alcoholic hydroxyl group on the alcoholic hydroxyl group or carboxyl group of the extract of Poria cocos sclerotium (mainly Poria triterpenoids) forms a hydrogen bond with the hydroxyl group of the alcohol solution. At the same time, the hydroxyl group of decapeptide-4 interacts with the alcohol solution, and the long alkane chain of decapeptide-4 interacts with the alkyl chain or cycloalkyl group of Poria triterpenoids, further improving the compatibility of the two in the alcohol solution to fully exert the activities of the extract of Poria cocos sclerotium and decapeptide-4.

[0027] Furthermore, the hair product of the present invention is prepared by the following steps:

[0028] (1) Add soy lecithin and cholesterol to ethanol, heat up to 50-55 °C, and stir until completely dissolved to obtain mixture I;

[0029] (2) Dissolve polysorbate-80 and a part of water to obtain mixture II, slowly drop mixture I into mixture II, and stir for 30-50 min to obtain a milky white suspension;

[0030] (3) Perform ethanol evaporation treatment on the above milky white suspension. After the system temperature drops to room temperature, add the anti-hair loss composition, preservative and the remaining water, stir to make the system uniform, and finally perform homogenization treatment to obtain the liposomal hair product.

[0031] Beneficial effects: Compared with the prior art, the remarkable advantages of the present invention are as follows: The method for preparing decapeptide-4 of the present invention can effectively improve its purity and yield; and compounding the decapeptide-4 prepared by the present invention with the extract of Poria cocos sclerotium and the ginkgo biloba leaf extract can not only promote the proliferation of dermal papilla cells and regulate key growth factors, but also reduce the adverse effects brought by androgens and improve male pattern baldness; at the same time, it can also resist oxidation and aging, improve blood circulation and energy metabolism, and promote hair follicle revival, and finally effectively achieve the effect of preventing hair loss and protecting hair. Description of the Drawings

[0032] Figure 1 1H-NMR spectrum of the decapeptide-4 prepared in Example 1-1; 1 1H-NMR spectrum;

[0033] Figure 2 Effect diagram of the hair product of Example 1-2 on cell viability in the range of 10-640 μg / mL; among them, compared with the blank group, *p<0.05, **p<0.01;

[0034] Figure 3 Inhibitory rate graph of 5α-reductase for the hair care products of Examples 1-2 in the range of 1-16 mg / mL;

[0035] Figure 4 Content graph of IL-1α for the hair care products of Examples 1-2 in the range of 10-160 μg / mL; wherein, compared with the blank group, p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001;

[0036] Figure 5 Content graph of IL-6 for the hair care products of Examples 1-2 in the range of 10-160 μg / mL; wherein, compared with the blank group, p<0.001; compared with the model group, **p<0.01, ***p<0.001;

[0037] Figure 6 Relative expression level graph of SRD5A2 for the hair care products of Examples 1-2 in the range of 20-160 μg / mL; wherein, compared with the blank group, p<0.001; compared with the model group, *p<0.05, **p<0.01;

[0038] Figure 7 Relative expression level graph of the signal transduction pathway P53 gene for the hair care products of Examples 1-2 in the range of 20-160 μg / mL; wherein, compared with the blank group, p<0.001; compared with the model group, ***p<0.001;

[0039] Figure 8 Histogram of relative fluorescence intensity of ROS for the hair care products of Examples 1-2 in the range of 20-160 μg / mL; wherein, compared with the model group, p<0.01; compared with the blank group, ***p<0.001;

[0040] Figure 9 Relative fluorescence intensity graph of ROS for the hair care products of Examples 1-2 in the range of 20-160 μg / mL;

[0041] Figure 10 Histogram of relative area of β-galactosidase for the hair care products of Examples 1-2 in the range of 20-80 μg / mL; wherein, compared with the model group, ***p<0.001;

[0042] Figure 11 Staining graph of β-galactosidase for the hair care products of Examples 1-2 in the range of 20-80 μg / mL;

[0043] Figure 12VEGF content graph of the hair products of Examples 1-2 in the range of 10-320 μg / mL; among them, compared with the blank group, ***p<0.001;

[0044] Figure 13 Graphs of different states of the hair products of Examples 1-2 under the condition of -18°C; among them, I is the state graph of the freshly prepared aqueous solution, and II is the state graph after standing for 22 days;

[0045] Figure 14 Graphs of different states of the hair products of Examples 1-2 under the condition of -4°C; among them, I is the state graph of the freshly prepared aqueous solution, and II is the state graph after standing for 22 days;

[0046] Figure 15 Graphs of different states of the hair products of Examples 1-2 at room temperature; among them, I is the state graph of the freshly prepared aqueous solution, and II is the state graph after standing for 22 days;

[0047] Figure 16 Graphs of different states of the hair products of Examples 1-2 under light conditions; among them, I is the state graph of the freshly prepared aqueous solution, and II is the state graph after standing for 22 days;

[0048] Figure 17 Graphs of different states of the hair products of Examples 1-2 under the condition of 40°C; among them, I is the state graph of the freshly prepared aqueous solution, and II is the state graph after standing for 22 days;

[0049] Figure 18 Graphs of different states of the hair products of Examples 1-2 under the condition of 45°C; among them, I is the state graph of the freshly prepared aqueous solution, and II is the state graph after standing for 22 days. Detailed implementation manners

[0050] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0051] It should be noted that the alcohol solution used in the present invention can be butylene glycol, glycerol, 1,3-butylene glycol, 1,3-propylene glycol, propylene glycol, pentylene glycol, 1,2-hexanediol, isopentylene glycol or ethanol.

[0052] The poria cocos sclerotium extract of the present invention can be a commercially available product directly purchased. For example, directly adopt the poria cocos sclerotium extract (powder) sold by Nanjing Specco Biotechnology Co., Ltd.

[0053] The ginkgo leaf extract of the present invention can be obtained by using existing well-known technologies for extraction. For example, the product extracted by the extraction process disclosed in the invention application No. 202110322467.8, titled "Preparation Method of a Water-Soluble Ginkgo Leaf Extract Free of Ginkgolic Acid". Or it can directly use the (water-soluble) ginkgo (GINKGO BILOBA) extract already sold by Nanjing Specco Biotechnology Co., Ltd.

[0054] In the attached drawings of the present invention, the blank group is the experimental group without adding the composition; the model group is the corresponding experimental group for the detection, and the stimuli added for the detection can be the well-known stimuli in the art according to each detection.

[0055] The preparation method of the decapeptide-4 of the present invention includes the steps:

[0056] (1) Soak the dichloro resin with dichloromethane. After washing and drying by suction, add Fmoc-L-Cys(Trt)-OH with a mass ratio of (1-3):1 to the dichloro resin, 0.07-0.13 L of DIEA, 25-50 L of DMF, and 4-6 L of DCM, and react for 6 h, then cap it with methanol; subsequently, add piperidine with a volume ratio of 1:4 to DMF to react and remove the protecting group;

[0057] (2) Add 0.8-1.2 kg of Fmoc-L-Tyr(tbu)-OH, 80-120 g of HOBT, 0.4-0.6 L of DIC, and 25-50 L of DMF to step (1) for condensation reaction; subsequently, add piperidine with a volume ratio of 1:4 to DMF to react and remove the protecting group;

[0058] (3) Sequentially couple the subsequent Fmoc-L-Met-OH, Fmoc-L-Glu(otbu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Asp(otbu)-OH, Fmoc-L-Cys(Trt)-OH amino acids in sequence, then remove the Fmoc protecting group, wash and dry by suction to obtain the crude polypeptide resin; wherein, the molar ratio of Fmoc-L-Met-OH, Fmoc-L-Glu(otbu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Asp(otbu)-OH, Fmoc-L-Cys(Trt)-OH amino acids to the dichloro resin is 3:1;

[0059] (4) Dissolve the crude polypeptide and then freeze-dry it at 15 - 25 °C to obtain polypeptide powder;

[0060] (5) Stir the crude polypeptide evenly with an acetonitrile solution with a concentration of 15 - 25%, adjust the pH to 8 - 9 with ammonia water, and obtain the decapeptide-4 after oxidation, purification, and freeze-drying.

[0061] Example 1-1: Preparation of decapeptide-4

[0062] The method for preparing decapeptide-4 in this Example 1-1 includes the following steps:

[0063] (1) Weigh 1 kg of dichloro resin (substitution degree 1.56 mmol / g) into a 50 L reaction kettle, soak it in dichloromethane (DCM) for 15 min; wash and drain it with DMF with a volume three times that of the resin, and repeat this four times;

[0064] (2) Weigh 1.5 kg of Fmoc-L-Cys(Trt)-OH and add it to the reaction kettle, add 0.1 L of DIEA, 30 L of DMF, and 5 L of DCM, react for 6 h, then seal the head with methanol (methanol:DIEA = 1:1) for half an hour, and then wash it three times with DMF with a volume three times that of the resin and drain it for use;

[0065] (3) Add 20% piperidine (piperidine / DMF = 1:4) to the reaction kettle, react for 20 min, then wash it twice with DMF, methanol, and DMF respectively, and then drain it to detect whether the protection is removed;

[0066] (4) Weigh 1 kg of Fmoc-L-Tyr(tbu)-OH and 100 g of HOBT and add them to the reaction kettle, add 0.5 L of DIC and 30 L of DMF, react for 1 hour; take a small amount of resin and detect it by the ninhydrin method (if the resin is colorless, it means the reaction is complete; if the resin has a color, it means the condensation is incomplete and continue the reaction);

[0067] (5) After the reaction is complete, wash it three times with DMF, then drain it, add 20% piperidine (piperidine / DMF = 1:4) to the reaction kettle, react for 20 min, then wash it twice with DMF, methanol, and DMF, and then drain it to detect whether the protection is removed;

[0068] (6) Subsequently, Fmoc-L-Met-OH, Fmoc-L-Glu(otbu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Asp(otbu)-OH, and Fmoc-L-Cys(Trt)-OH amino acids were successively coupled step by step. For each step of amino acid coupling, HOBT and DIC were added for reaction; among them, the molar ratio of Fmoc-L-Met-OH, Fmoc-L-Glu(otbu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Leu-OH, Fmoc-L-Asp(otbu)-OH, and Fmoc-L-Cys(Trt)-OH amino acids to dichlororesin was all 3:1;

[0069] (7) After the last Fmoc-L-Cys(Trt)-OH was coupled and the Fmoc protecting group was removed, it was washed twice with DMF and methanol, and the resin was dried by suction to obtain the polypeptide resin; after the Fmoc protecting group was removed, it was washed three times with DMF, then washed four times with methanol, the resin was dried by suction, and then dried at 40 °C with a blower until the polypeptide resin reached a constant weight;

[0070] (8) The polypeptide was cleaved from the resin with a cleavage solution to obtain a crude polypeptide product;

[0071] (9) The crude polypeptide product was stirred evenly with an acetonitrile solution with a concentration of 20%, and then freeze-dried with a freeze dryer at 20 °C to obtain a fluffy polypeptide powder;

[0072] (10) Weigh 100 g of the freeze-dried polypeptide powder, dissolve it with an aqueous acetonitrile solution with a concentration of 20%, adjust the pH to 8 with ammonia water, and stir overnight to oxidize the two cys in the polypeptide to obtain a crude liquid of oxidized decapeptide-4.

[0073] Performance Test 1: Structural Characterization

[0074] Take a portion of the crude decapeptide-4 liquid, filter it, and purify it to obtain a finished polypeptide liquid. The purity was detected by a high-performance liquid chromatograph (Jiangsu Hanbang DAC100 preparative chromatograph equipped with an ultraviolet detector), and the molecular weight was detected by a mass spectrometer (waters zq-2000 mass spectrometer). The qualified liquid of the purified finished polypeptide was freeze-dried with a vacuum freeze dryer to obtain a product powder. The purity of this product reached 99%, and the yield reached 40%.

[0075] The decapeptide-4 prepared in Example 1-1 was subjected to finished product testing, and the obtained results are as Figure 1As shown. Through this detection, it can be known that the molecular weight of the decapeptide-4 prepared by the present invention is consistent with that of the target product, and the structure of the compound is consistent with that of the target product.

[0076] Comparative Example: Preparation of decapeptide-4

[0077] The basic steps of this comparative example are the same as those of Example 1-1, except that its preparation process is: synthesis-cleavage-oxidation-purification-concentration-lyophilization. That is, compared with Example 1-1, the step of prior lyophilization is not adopted between cleavage and oxidation. The purity of the decapeptide-4 prepared in this comparative example was detected, and the purity of this product was 98%, and the yield was only 25%.

[0078] Example 1-2: Hair care product

[0079] The hair care product (water-soluble) of this Example 1-2 includes the raw materials shown in Table 1.

[0080] Table 1 Raw material composition of the hair care product of Example 1-2

[0081] Serial number Raw material Content / % 1 Poria cocos sclerotium extract 0.35 2 Ginkgo biloba leaf extract 0.5 3 Decapeptide-4 0.02 4 1,3-Butanediol 84 5 Water To 100

[0082] This hair care product is prepared by the following steps:

[0083] (1) Add the Poria cocos sclerotium extract to 1,3-butanediol, and heat and stir at 45°C until the Poria cocos sclerotium extract is completely dissolved to obtain a mixed solution;

[0084] (2) Add water to the mixed solution in step (1) to obtain a mixed solution;

[0085] (3) Add the Ginkgo biloba extract and decapeptide-4 to the solution in step (2), and stir and dissolve to obtain this hair care product.

[0086] Performance test 2: Cell proliferation

[0087] The hair care product prepared in Example 1-2 of the present invention was subjected to a cell proliferation test with single or two active components, and the obtained results are as Figure 2 and shown in Table 2.

[0088] Among them, the proportion of the addition amount of the single active component is the same as that of the composition of Example 1-2, the mass ratio of the two active components is the same as the corresponding mass ratio of Example 1-2, and the total proportion of the two active components is the same as the total proportion of the composition of Example 1-2.

[0089] Detection method: When the cell confluence reaches about 80%, digest with trypsin and adjust the cell density to 1×10 5Cells were inoculated evenly at a density of cells / mL into 96-well plates, 0.1 mL per well. After 24 h, the old culture medium was aspirated completely, and then serum-free medium was added for continued culture for 24 h to synchronize the cells. The old culture medium was discarded, and 0.1 mL of serum-free medium containing samples at different concentrations was added. At the same time, a control group and blank wells (containing only culture medium without cells) were set up. After 48 h of culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the culture was continued for 3 h. After the culture was completed, the culture medium in the wells was carefully aspirated, 150 μL of dimethyl sulfoxide was added to each well, and the plate was placed on a shaker and shaken at a low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm using a microplate reader.

[0090] Cell viability % = [OD (sample) - OD (blank)] / [OD (control) - OD (blank)] × 100%.

[0091] Table 2 Comparison Table of Examples 1-2 and Single or Two Active Components

[0092]

[0093]

[0094] Combined with Table 2 and Figure 2 It can be seen that, compared with the single-component cell proliferation performance of decapeptide-4, the hair care products of Examples 1-2 of the present invention can achieve the same efficacy as decapeptide-4 at 160 μg / mL under the condition of a very low concentration of 10 μg / mL; compared with the single-component cell proliferation performance of Poria cocos sclerotium extract, the hair care products of Examples 1-2 of the present invention can achieve the same efficacy as Poria cocos sclerotium extract at 80 μg / mL under the condition of a low concentration of 20 μg / mL, and even exceed it; compared with the single-component cell proliferation performance of Ginkgo biloba extract, the hair care products of Examples 1-2 of the present invention can achieve the same efficacy as Ginkgo biloba extract at 40 μg / mL under the condition of a low concentration of 20 μg / mL.

[0095] Compared with the two-component cell proliferation performance of decapeptide-4 and Poria cocos sclerotium extract, the hair care products of Examples 1-2 of the present invention can achieve the same efficacy as the two components of decapeptide-4 and Poria cocos sclerotium extract at 80 μg / mL under the condition of a very low concentration of 20 μg / mL; compared with the two-component cell proliferation performance of Poria cocos sclerotium extract and Ginkgo biloba extract, the hair care products of Examples 1-2 of the present invention can achieve the same efficacy as Poria cocos sclerotium extract and Ginkgo biloba extract at 40 μg / mL under the condition of a low concentration of 20 μg / mL; compared with the two-component cell proliferation performance of decapeptide-4 and Ginkgo biloba extract, the hair care products of Examples 1-2 of the present invention can achieve the same efficacy as decapeptide-4 and Ginkgo biloba extract at 40 μg / mL under the condition of a low concentration of 20 μg / mL.

[0096] That is, the hair products of Embodiments 1-2 of the present invention, when compared with single or dual active components, can promote cell proliferation at low concentrations in combination, effectively improving cell activity.

[0097] Performance Test 3: Expression of Key Growth Factors

[0098] The hair products prepared in Embodiments 1-2 of the present invention were subjected to performance tests for the expression of key growth factors with single or two active components, and the obtained results are shown in Table 3.

[0099] Detection method: The dermal papilla cell suspension was inoculated into a T25 culture flask at a density of 5×10 6 cells / bottle, and after culturing for 24 h, the cells were incubated with different sample solutions for 24 h. During detection, the cells were digested, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were washed twice with pre-cooled PBS. 1 mL of RNA extraction solution was added to the cell pellet, and the cells were blown and broken. 250 μL of chloroform was added, and the mixture was thoroughly mixed and allowed to stand for 3 min. Centrifuged at 12000 rpm for 10 min at 4 °C, the supernatant was collected and transferred to a new centrifuge tube, 0.8 times the volume of isopropanol was added and mixed well, and it was placed in a -20 °C refrigerator for 15 min. Centrifuged at 12000 rpm for 10 min at 4 °C, and the white precipitate at the bottom of the tube was collected, which was the sample RNA. According to the operation instructions of the reverse transcription and RT-qPCR kits, the expression level of dermal papilla cell mRNA was detected. The cells were incubated with different samples for 24 h, and the effect on the activity of alkaline phosphatase (ALP) was measured.

[0100] Table 3 Comparison Table of Embodiments 1-2 with Single or Two Active Components

[0101]

[0102] Combined with Table 3, it can be seen that the hair products of Embodiments 1-2 (40 μg / mL) up-regulate the expression of growth factors HGF and IGF-1 in dermal papilla cells, indicating that the composition can positively regulate hair follicle hair growth; TGF-β is related to hair growth or apoptosis of catagen follicle cells, and down-regulation of TGF-β can prevent hair loss; the activity of alkaline phosphatase (ALP) helps to maintain the hair follicle induction ability of dermal papilla cells, and the scalp care composition can significantly increase the expression of ALP.

[0103] Performance Test 4: 5α-Reductase Inhibition

[0104] The hair products prepared in Embodiments 1-2 of the present invention were subjected to 5α-reductase inhibition performance tests with single or two active components, and the obtained results are as Figure 3 shown in Table 4.

[0105] Detection method: Add 500 μL of phosphate buffer solution, 100 μL of 50% ethanol, 150 μL of testosterone, and 200 μL of crude enzyme extract of type II 5α-reductase into each reaction tube, and then add 250 μL of NADPH solution. Place the reaction tubes in a 37 °C incubator and react in the dark for 1 h. Add 2.5 mL of anhydrous methanol at 4 °C to terminate the enzymatic reaction. Centrifuge each reaction solution at 10000 rpm for 5 min, collect the supernatant, filter it with a 0.22 μm filter membrane, pipette 200 μL of the filtrate into a 96-well plate, and measure the absorbance value of each sample well at 340 nm using an enzyme-labeled instrument to determine the effect of finasteride positive control and the sample on type II 5α-reductase.

[0106] Table 4 Comparison Table of Examples 1-2 and Single or Two Active Components

[0107]

[0108] Combined Figure 3 With Table 4, it can be seen that for the hair care products of Examples 1-2 of the present invention, whether compared with single active components or dual active components, the combination of the three can improve the inhibitory ability at low concentrations.

[0109] Performance Test 5: Inhibiting the Expression of Inflammatory Factors Related to DHT-Induced Typical Hair Loss

[0110] The hair care products prepared in Examples 1-2 of the present invention and single or two active components were subjected to a test for inhibiting the expression of inflammatory factors related to DHT-induced typical hair loss, and the obtained results are as shown in Figure 4 、 Figure 5 and Tables 5 and 6.

[0111] Detection method: Inoculate the dermal papilla cell suspension into a 12-well plate at a density of 1×10 6 cells / well, after culturing for 24 h, incubate the cells with dihydrotestosterone and different sample solutions for 24 h, collect the cell supernatant, and determine the effect of the sample on the production of cell inflammatory factors according to the operation instructions of the ELISA kit.

[0112] Table 5 Comparison Table of Examples 1-2 and Single or Two Active Components (Inhibition Rate)

[0113]

[0114]

[0115] Table 6 Comparison Table of Examples 1-2 and Single or Two Active Components (Inhibition Rate)

[0116]

[0117] Combined Figure 4 andFigure 5 As can be seen from Table 5 and Table 6, for the hair products of Examples 1-2 of the present invention, regardless of comparison with single active components or dual active components, the compositions formed by their three-component combination can enhance the inhibitory ability at low concentrations.

[0118] Performance Test 6: Inhibiting the Expression of Genes Related to DHT-Induced Typical Male Pattern Hair Loss

[0119] The hair products prepared in Examples 1-2 of the present invention and single or two active components were subjected to detection of inhibiting the expression of genes related to DHT-induced typical male pattern hair loss. The obtained results are as Figure 6 、 Figure 7 shown in Table 7 and Table 8.

[0120] Detection method: The dermal papilla cell suspension was inoculated into a T25 culture flask at a density of 5×10 6 cells / bottle, and after culturing for 24 h, the cells were incubated with the sample solution for 24 h. During detection, the cells were digested, centrifuged at 1500 rpm for 5 min to discard the supernatant, and the cells were washed twice with pre-cooled PBS. 1 mL of RNA extraction solution was added to the cell pellet, and the cells were blown and broken. 250 μL of chloroform was added, and the mixture was thoroughly mixed and allowed to stand for 3 min. Centrifuged at 12000 rpm for 10 min at 4 °C, the supernatant was collected and transferred to a new centrifuge tube, 0.8 times the volume of isopropanol was added and mixed evenly, and it was placed in a -20 °C refrigerator for 15 min. Centrifuged at 12000 rpm for 10 min at 4 °C, and the white precipitate at the bottom of the tube was collected, which was the sample RNA. According to the operation instructions of the reverse transcription and RT-qPCR kits, the relative expression levels of SRD5A2 and P53 in dermal papilla cells were detected.

[0121] Table 7 Comparison Table of Examples 1-2 and Single or Two Active Components (Inhibition Rate)

[0122]

[0123]

[0124] Table 8 Comparison Table of Examples 1-2 and Single or Two Active Components (Inhibition Rate)

[0125]

[0126] Combined with Figure 6 、 Figure 7 Table 7 and Table 8, it can be seen that for the hair products of Examples 1-2 of the present invention, regardless of comparison with single active components or dual active components, their three-component combination can inhibit the expression of SRD5A2 mRNA and p53 mRNA at low concentrations.

[0127] Performance Test 7: Inhibiting the Intracellular ROS Level in Dermal Papilla Cells Induced by H2O2

[0128] The hair products prepared in the above Examples 1-2 of the present invention were tested for their performance in inhibiting the ROS level in dermal papilla cells induced by H2O2 with single or two active components. The obtained results are as Figure 8 , Figure 9 and Table 9 show.

[0129] Detection method: After digesting and counting the cells in the logarithmic growth phase, inoculate them into a 6-well plate at a density of 1.5×10 5 cells / mL. After culturing for 24 h, incubate the cells with the sample solution for 24 h. According to the instructions of the reactive oxygen species kit, dilute DCFH-DA 1:1000 to 10 μM. After incubating with the drug for 4 h, remove the culture medium, add 1 mL of the diluted DCFH-DA solution to each well, and incubate in the incubator for 20 min. Then take it out and wash the cells three times with serum-free DMEM medium. Observe and photograph under a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). Use trypsin to digest and collect the cells, count the cell suspension and inoculate it into a black-edged 96-well plate at a density of 5×10 4 cells / mL for real-time fluorescence detection.

[0130] Table 9 Comparison table of Examples 1-2 with single or two active components (inhibition rate)

[0131]

[0132] Combined with Figure 8 , Figure 9 and Table 9, it can be seen that the hair products of Examples 1-2 of the present invention, whether compared with single active components or dual active components, the combination of the three can significantly reduce the ROS content in dermal papilla cells at low concentrations and shows a concentration dependence.

[0133] Performance test 8: Inhibition of β-galactosidase activity

[0134] The hair products prepared in the above Examples 1-2 of the present invention were tested for their performance in inhibiting β-galactosidase activity with single or two active components. The obtained results are as Figure 10 , Figure 11 and Table 10 show.

[0135] Detection method: Inoculate the dermal papilla cell suspension into a 12-well plate at a density of 1×10 6 cells / well, culture for 24 h, then incubate the cells with dihydrotestosterone and different samples for 24 h, and operate according to the instructions of the β-galactosidase test kit. Calculate the positive cell staining rate according to the following formula:

[0136]

[0137] Table 10 Comparison Table of Examples 1-2 with Single or Two Active Components (Inhibition Rate)

[0138]

[0139]

[0140] Combined Figure 10 and Figure 11 From Table 10, it can be seen that for the hair care products of Examples 1-2 of the present invention, whether compared with single active components or dual active components, the combination of the three can improve the ability to inhibit the activity of β-galactosidase in dermal papilla cells at low concentrations, thereby delaying cell / hair follicle aging.

[0141] Performance Test 9: Promoting the Expression of Vascular Endothelial Growth Factor in Dermal Papilla Cells

[0142] The hair care products prepared in Examples 1-2 of the present invention and single or two active components were subjected to a performance test on promoting the expression of vascular endothelial growth factor in dermal papilla cells, and the obtained results are as Figure 12 shown in

[0143] Detection method: After the cells were cultured for 24 h and adhered, the complete culture medium was aspirated, and the bottom of the plate was rinsed twice with PBS. 2 mL of freshly prepared drug was added to each well, a blank group and different sample groups were set, and the cells were cultured for another 48 h. The concentration of vascular endothelial growth factor in the culture medium was measured according to the operation manual of the ELISA kit.

[0144] Table 11 Comparison Table of Examples 1-2 with Single or Two Active Components (Enhancement Rate)

[0145]

[0146] Combined Figure 12 and Table 11, it can be seen that for the hair care products of Examples 1-2 of the present invention, whether compared with single active components or dual active components, the combination of the three can significantly increase the VEGF level at low concentrations.

[0147] Performance Test 10: Water Solubility Stability Test

[0148] The hair care products prepared in Examples 1-2 of the present invention were left standing at -18 °C, -4 °C, room temperature, under light, 45 °C, and 40 °C for 22 days, and then their appearances were observed. The obtained results are as Figures 13 to 18 shown. From this figure, it can be seen that after standing for a period of time under different environmental conditions, their appearances remain unchanged, indicating strong stability.

[0149] Example 2

[0150] The hair product of Example 2 includes the raw materials shown in Table 12.

[0151] Table 12 Raw material composition of the hair product of Example 2

[0152] Serial number Raw material Content / % 1 Poria cocos sclerotium extract 0.432 2 Ginkgo biloba leaf extract 0.528 3 Decapeptide-4 0.024 4 1,3-Butanediol 80 5 Myristoyl pentapeptide-4 0.01 6 Water To 100

[0153] The hair product is prepared by the following steps:

[0154] (1) Add the poria cocos sclerotium extract to 1,3-butanediol, and heat and stir at 40 °C until the poria cocos sclerotium extract is completely dissolved to obtain a mixed solution;

[0155] (2) Add water to the mixed solution in step (1) to obtain a mixed solution;

[0156] (3) Add the ginkgo biloba extract, decapeptide-4, and myristoyl pentapeptide-4 to the solution in step (2), and stir and dissolve to obtain the hair product.

[0157] Example 3

[0158] The hair product of Example 3 includes the raw materials shown in Table 13.

[0159] Table 13 Raw material composition of the hair product of Example 3

[0160]

[0161]

[0162] The hair product is prepared by the following steps:

[0163] (1) Add the poria cocos sclerotium extract to the alcohol solution, and heat and stir at 40 °C until the poria cocos sclerotium extract is completely dissolved to obtain a mixed solution;

[0164] (2) Add water and Tween 20 to the mixed solution in step (1) to obtain a mixed solution;

[0165] (3) Add the ginkgo biloba extract and decapeptide-4 to the solution in step (2), and stir and dissolve to obtain the hair product.

[0166] Example 4

[0167] The hair product of Example 4 includes the raw materials shown in Table 14.

[0168] Table 14 Raw material composition of the hair product of Example 4

[0169] Serial number Raw material Content / % 1 Poria cocos sclerotium extract 2 2 Ginkgo biloba leaf extract 2.875 3 Decapeptide-4 0.125 4 1,3-Butanediol 88 5 Tween 20 0.5 6 Sodium sulfite 0.5 7 Water To 100

[0170] The hair product is prepared by the following steps:

[0171] (1) Add the extract of Poria cocos sclerotium to 1,3-butanediol, heat and stir at 50 °C until the extract of Poria cocos sclerotium is completely dissolved to obtain a mixed solution.

[0172] (2) Add water, Tween 20 and sodium sulfite to the mixed solution in step (1) to obtain a mixed solution.

[0173] (3) Add the ginkgo extract and decapeptide-4 to the solution in step (2), stir and dissolve to obtain the hair care product.

[0174] Example 5

[0175] The hair care product of Example 5 includes the raw materials shown in Table 15.

[0176] Table 15 Composition of Raw Materials of the Hair Care Product of Example 5

[0177]

[0178] The hair care product is prepared by the following steps:

[0179] (1) Add soybean lecithin and cholesterol to ethanol, heat to 55 °C, stir until completely dissolved to obtain mixed solution I.

[0180] (2) Dissolve polysorbate-80 and half of the water to obtain mixed solution II, slowly drop mixed solution I into mixed solution II, and stir for 30 - 50 min to obtain a milky white suspension.

[0181] (3) Perform ethanol evaporation treatment on the above milky white suspension, and after the system temperature drops to room temperature, add the anti-hair loss composition, preservative and the remaining water, stir to make the system uniform, and finally perform homogenization treatment to obtain the liposomal hair care product.

[0182] In addition to the above examples, it should be noted that the water-soluble hair care products of the present invention may also contain extended peptides, solubilizers, antioxidants, etc., including but not limited to the specific types of substances defined in the present invention, and the same technical effects can be achieved. At the same time, for the liposomal hair care products, the same technical effects can be obtained within the scope of the raw materials defined in the invention, and thus no further experiments will be carried out for verification.

Claims

1. A method for preparing decapeptide-4, characterized in that: The steps include: (1) Synthesizing peptide resin using the Fmoc solid phase method and cleaving it to obtain a crude peptide; (2) Dissolving the crude polypeptide and freeze-drying the polypeptide to obtain polypeptide powder; (3) After the polypeptide powder is dissolved, decapeptide-4 is obtained after oxidation, purification and freeze-drying; Wherein, in step (1), the use of Fmoc solid phase method to synthesize polypeptide resin comprises the following steps: (a) dichlororesin is soaked in dichloromethane, washed and dried, and then Fmoc-L-Cys(Trt)-OH is added, and methanol is used to cap the head; piperidine is then added to react and remove the protecting group; (b) adding Fmoc-L-Tyr(tbu)-OH and HOBT to step (a) for condensation reaction; then adding piperidine to react and remove the protecting group; (c) Follow the steps to sequentially connect Fmoc-L-Met-OH, Fmoc-L-Glu (otbu) -OH, Fmoc-L-Leu-OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-L-Leu-OH, Fmoc-L-Asp (otbu) -OH, and Fmoc-L-Cys (Trt) -OH amino acids for coupling reaction, remove the Fmoc protecting group, wash, and dry to obtain a polypeptide resin.

2. A composition for preventing hair loss, characterized in that: The anti-hair loss composition is composed of decapeptide-4 prepared by the preparation method according to claim 1, Poria cocos sclerotium extract and Ginkgo biloba leaf extract, and the mass ratio of the three is 1: (15-20): (20-25).

3. A hair product, characterized in that: The method comprises 0.5-5% of the anti-hair loss composition according to claim 2, 80-88% of an alcohol solution and the remainder of water; the alcohol solution is butylene glycol, glycerol, 1,3-butylene glycol, 1,3-propanediol, propylene glycol, pentanediol, 1,2-hexanediol, isopentylene glycol or ethanol.

4. The hair product according to claim 3, characterized in that: The hair product also includes 0.005-0.1% of extended peptides, and the extended peptides include at least one of myristoyl pentapeptide-4, biotin tripeptide-1, acetyl tetrapeptide-3, octapeptide-2, decapeptide-18, oligopeptide-54, oligopeptide-71, decapeptide-10, myristoyl dipeptide-13, myristoyl pentapeptide-17, myristoyl pentapeptide-16 or copper peptide.

5. The hair product according to claim 3 or 4, characterized in that: The hair product further comprises 0.3-1.0% of a solubilizer, wherein the solubilizer comprises LRI, Tween 20, Tween 40, Tween 60, Span 20 or PEG-40.

6. The hair product according to claim 5, characterized in that: The hair product further comprises 0.01-0.5% of an antioxidant, wherein the antioxidant comprises sodium sulfite, sodium bisulfite, sodium pyrosulfite, butylated hydroxytoluene, tocopherol, butylated hydroxyanisole or tert-butylhydroquinone.

7. A hair product, characterized in that: The method comprises 0.5-5% of the anti-hair loss composition according to claim 3, 1-2% of soy lecithin, 0.4-0.6% of cholesterol, 15-18% of ethanol, 0.2-0.5% of polysorbate-80, 0.5-1.5% of a preservative and the balance of water.

8. The hair product according to claim 3, characterized in that: The hair product is prepared by the following steps: (1) dissolving the Poria cocos sclerotium extract in an alcohol solution, heating and stirring at 40-50° C. until the Poria cocos sclerotium extract is completely dissolved, to prepare a solution; (2) adding water to the mixed solution of step (1) to prepare a solution; (3) Adding the ginkgo leaf extract and decapeptide-4 into the solution of step (2), stirring and dissolving to prepare the hair product.

9. The hair product according to claim 7, characterized in that: The hair product is prepared by the following steps: (1) Add soybean lecithin and cholesterol to ethanol, raise the temperature to 50-55°C, and stir until completely dissolved to prepare mixed solution I; (2) Dissolve polysorbate 80 and a portion of water to obtain a mixed solution II, slowly drop the mixed solution I into the mixed solution II, and stir for 30-50 minutes to obtain a milky white suspension; (3) The milky white suspension is subjected to ethanol evaporation treatment, and after the system temperature drops to room temperature, the anti-hair loss composition, preservative and remaining water are added, and the system is stirred to make the system uniform. Finally, the liposome hair product is prepared by homogenization treatment.

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