A polypeptide, its preparation method and uses

Abstract

The present invention relates to the field of biotechnology, and in particular to a polypeptide and a preparation method and use thereof, which has a basic peptide structure of DGlu-DAsp-DSer-DSer. Using the polypeptide of the present invention, the hair can enter the early stage of growth earlier and significantly promote hair growth.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide and a preparation method and use thereof. Background Art

[0002] As people age, their body functions age accordingly. The color of their hair gradually changes from black to white, and the number of hair decreases. This is a normal natural phenomenon. However, under today's fast-paced lifestyle and high work pressure, people's mental stress increases, resulting in gray hair and hair loss occurring earlier and earlier in middle-aged and even young people, which seriously affects their appearance and causes unnecessary psychological burden. Young people have become the main group of people with hair loss problems, especially those born after 1990. They face serious hair loss problems due to work pressure, living habits and other factors.

[0003] The trend of hair loss is obvious, and the age of hair loss of contemporary people is 20 years earlier than that of the previous generation. The number of men with hair loss is more than that of women, and androgenic alopecia (male baldness) is the most common hair loss disease in clinical practice. In addition, hair loss not only affects personal image, but also seriously affects mental health and quality of life. Due to hair loss, many young people feel inferior and even affect their self-confidence, which may lead to depression.

[0004] In the face of the problem of hair loss, a variety of treatment methods have emerged on the market, including drug therapy, hair transplantation, etc. Minoxidil and finasteride are the most commonly used drugs for the treatment of hair loss. Among them, minoxidil is more popular in the market because of its wide applicability and can be used by both men and women. With the development of technology, hair transplantation technology is also constantly improving. For example, barley microneedle hair transplantation technology, through the use of microneedles and no shaving technology, reduces the damage to hair follicles caused by surgery, increases the survival rate of hair follicles, thereby reducing the possibility of shedding, making the postoperative recovery period shorter, and patients can return to normal life faster. The current status of hair growth technology mainly includes traditional hair transplant surgery, new hair growth technologies such as microneedles, fractional lasers, cell therapy, etc., as well as market products for different needs.

[0005] ‌Hair transplant surgery‌: Hair transplant is seen as one of the means to save hair loss, although not everyone with hair loss chooses hair transplant because not everyone is suitable. Hair transplant surgery helps restore hair density by transplanting healthy hair follicles to the hair loss area. However, hair transplant surgery is not suitable for all cases, and post-operative care and maintenance are also important considerations. ‌New hair growth technology‌: With the development of science and technology, new hair growth technologies such as microneedles, fractional lasers, and cell therapy have begun to enter clinical applications. These technologies use laser pulses or cell regeneration principles to stimulate hair follicle growth and increase the number of hair and hair follicle units.

[0006] Minoxidil is a hair growth drug that has a hair growth effect, but it is also a drug used for hypertension. It has the effect of promoting the recovery of hair follicles, but it has poor safety, large side effects, and is addictive. Direct cessation of use will cause blood pressure fluctuations. Summary of the invention

[0007] The purpose of the present invention is to provide a polypeptide and a preparation method and use thereof, so as to solve the technical problems of poor safety, large side effects, dependence and blood pressure fluctuation caused by direct cessation of use of hair growth drugs in the prior art.

[0008] The invention discloses a polypeptide having a basic peptide structure of DGlu-DAsp-DSer-DSer.

[0009] Furthermore, the basic peptide structure is repeated 1-20 times.

[0010] Furthermore, its structure is H2N-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-OH.

[0011] A method for preparing a polypeptide comprises the following steps:

[0012] S1. Carrying out condensation reaction of amino acids in resin;

[0013] S2. dissociating the peptide from the resin and removing the amino acid side chain protecting groups to obtain a crude peptide;

[0014] S3. After purification, the product is obtained.

[0015] Furthermore, the raw materials in step S1 are D-glutamic acid, D-aspartic acid and D-serine.

[0016] Furthermore, the specific steps of step S1 are as follows: dissolving O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid coupling reagent (TBTU), raw materials and protected amino acids in N,N-dimethylformamide (DMF);

[0017] The obtained reaction solution was added to the resin, and then N,N-diisopropylethylamine (DIEA) was added, and N2 was introduced for the first blowing, and the detection was performed by ninhydrin reaction;

[0018] After the reaction, the solvent was removed and the resin was washed with DMF;

[0019] Then, a 20% piperidine solution in DMF was added to the resin, and N2 was introduced for a second blow;

[0020] Remove the solvent and wash the resin with DMF to complete the coupling of the amino acid.

[0021] The above reaction procedure is repeated until the condensation reaction of all protected amino acids is completed.

[0022] Furthermore, the protected amino acid is Gln (glutamine).

[0023] Furthermore, the first blowing time is 80-100 minutes, and the second blowing time is 20-40 minutes.

[0024] Furthermore, the specific steps of removing the amino acid side chain protecting groups in step S2 are as follows:

[0025] Add the resin to the lysate and stir;

[0026] Then the resin and lysate are extracted, and the peptide is fully analyzed;

[0027] The polypeptide was filtered and washed to obtain a crude peptide.

[0028] Furthermore, the lysate comprises, by weight, 86% trifluoroacetic acid (TFA), 5% 1,2-ethanedithiol (EDT), 5% thioanisole, 3% phenol and 2% pure water.

[0029] Furthermore, the peptide was fully precipitated by adding ether.

[0030] The invention discloses an application of a polypeptide in preparing a care product.

[0031] Furthermore, the care product is a hair growth care product.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. Using the polypeptide of the present invention can enter the early stage of hair growth earlier and significantly promote hair growth;

[0034] 2. The purity of the polypeptide prepared by the preparation method of the present invention is about 85.6%;

[0035] 3. The biological polypeptide of the present invention is safe for epidermal application and subcutaneous injection.

[0036] 4. The biological polypeptide of the present invention is conducive to the design of sequence chiral D-type amino acid sequence, and its site can prevent calcium cell influx, inhibit the expression of hair growth inhibitory factors in TGF-β1 and DICKKOPF WNT signaling pathway, inhibit lactic acid metabolites, provide a good environment for the growth of hair papilla cells and hair follicle stem cells, adjust the growth cycle of hair follicle stem cells, and accelerate the entry into the growth phase. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only represent some embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0038] Figure 1 This is a graph showing the results of the mouse hair loss model of the present invention.

[0039] Figure 2 This is the HE tissue microscopic examination result of the present invention.

[0040] Figure 3 This is the immunohistochemistry result diagram of the present invention.

[0041] Figure 4 This is a diagram of the transdermal absorption results of the present invention.

[0042] Figure 5 This is a graph showing the stability test results of the polypeptide of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0044] Example 1

[0045] The method for preparing a polypeptide using the above device in this embodiment comprises the following steps:

[0046] Weigh the corresponding amount of TBTU, D-glutamic acid, D-aspartic acid, D-serine and protected amino acid Gln (glutamine) into a beaker and add DMF to dissolve;

[0047] The obtained reaction solution was added to the resin, and then DIEA was added, and N2 was blown for about 90 minutes, and the detection was performed by ninhydrin reaction;

[0048] After the reaction, the solvent was removed and the resin was washed three times with DMF;

[0049] Then, 20% piperidine in DMF solution was added to the resin, and N2 was introduced and continued to be blown for 30 minutes;

[0050] The solvent was removed and the resin was washed 6 times with DMF to complete the coupling of the amino acid.

[0051] The above reaction procedure was repeated twice until the condensation reaction of all protected amino acids was completed.

[0052] Lysis

[0053] Dissociate the peptide from the resin and remove the amino acid side chain protecting groups. The operation process is as follows:

[0054] Add the resin to the previously prepared lysis solution (86% TFA / 5% EDT / 5% thioanisole / 3% phenol / 2% pure water) and stir for 150 min;

[0055] The resin and lysate were extracted, and ether was added to allow the peptide to be fully precipitated;

[0056] The peptide was filtered using a Buchner funnel and washed with ether for 6 times to obtain a crude peptide;

[0057] After subsequent purification treatment, the polypeptide structure is H2N-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-OH, with a purity of about 85.6%.

[0058] The polypeptide is then combined with water, sodium polyglutamate, a penetration enhancer and pentylene glycol to prepare a care product.

[0059] Example 2

[0060] In this embodiment, as a preferred embodiment of the present invention, a method for preparing a polypeptide comprises the following steps:

[0061] The method for preparing a polypeptide using the above device in this embodiment comprises the following steps:

[0062] Weigh the corresponding amount of TBTU, D-glutamic acid, D-aspartic acid, D-serine and protected amino acid Gln (glutamine) into a beaker and add DMF to dissolve;

[0063] The obtained reaction solution was added to the resin, and then DIEA was added, and N2 was blown for about 90 minutes, and the detection was performed by ninhydrin reaction;

[0064] After the reaction, the solvent was removed and the resin was washed three times with DMF;

[0065] Then, 20% piperidine in DMF solution was added to the resin, and N2 was introduced and continued to be blown for 30 minutes;

[0066] The solvent was removed and the resin was washed 6 times with DMF to complete the coupling of the amino acid.

[0067] The above reaction procedure control is repeated three times until the condensation reaction of all protected amino acids is completed.

[0068] Lysis

[0069] Dissociate the peptide from the resin and remove the amino acid side chain protecting groups. The operation process is as follows:

[0070] Add the resin to the previously prepared lysis solution (86% TFA / 5% EDT / 5% thioanisole / 3% phenol / 2% pure water) and stir for 150 min;

[0071] The resin and lysate were extracted, and ether was added to allow the peptide to be fully precipitated;

[0072] The peptide was filtered using a Buchner funnel and washed with ether for 6 times to obtain a crude peptide;

[0073] After subsequent purification treatment, the structure of the polypeptide is H2N-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-OH, with a purity of about 88.3%.

[0074] The polypeptide is then combined with water, sodium polyglutamate, a penetration enhancer and pentylene glycol to prepare a care product.

[0075] Animal experiments

[0076] Mouse hair loss model (topical application)

[0077] Positive control group: imported minoxidil 5%

[0078] Low-dose group: 0.1% of the polypeptide in Example 1

[0079] High dose group: 1% of the polypeptide in Example 1

[0080] Injection group: 1% of the peptide in Example 1

[0081] The above percentages are by mass.

[0082] The external application group received the treatment 3 days a week, and the injection group received the treatment once a week.

[0083] Alopecia treatment research often evaluates the efficacy of candidate substances by establishing an androgenic alopecia animal model. The skin color of C57BL / 6 male mice changes with the hair cycle, which is easy to observe, so they are often used as model animals for studying hair growth. The study used C57BL / 6 male mice to construct an androgenic alopecia model. The depilatory agent is prepared by melting and mixing rosin and beeswax in a weight ratio of 1:1.

[0084] Heat the depilatory into liquid form. In order to avoid burning the animal, wait until the temperature is properly cooled to a level that does not solidify before applying the depilatory evenly to the animal's back hair. After the depilatory is condensed, gently tear the animal's back hair with your hands until the pink skin is exposed. If the hair is not completely removed, apply the depilatory again and remove the hair again.

[0085] Continuous transdermal administration was performed to observe the effects on skin color, length and quality of new hair, dermal thickness and number of hair follicles in C57BL / 6 male mice, and to evaluate the effect of promoting hair growth in model animals.

[0086] The days of skin darkening and hair growth in C57BL / 6 male mice in different drug administration groups were observed and recorded. The skin of the depilated area on the back of C57BL / 6 male mice was photographed on days 1, 3, 6, 9, 12, and 15. After shaving the hair, the back skin tissue was taken from the depilated area parallel to the spine, fixed in paraformaldehyde, and stained with H&E for microscopic examination and photography. The changes in dermal thickness and hair follicle number were analyzed. The results are shown in Table 1. Figure 1 and Figure 2 shown.

[0087] Immunohistochemical staining sections were dewaxed and placed in a repair box. They were then placed in a microwave oven at medium heat for 5 min, then off heat for 5 min, and then at low heat for 10 min. They were cooled, washed, blocked, and incubated with primary antibodies. They were dried and HRP-labeled secondary antibodies were added. They were incubated at room temperature, then developed with DAB, counterstained with hematoxylin, and finally dehydrated, sealed, and examined under a microscope. The results were as follows: Figure 3 shown.

[0088] from Figure 1 It can be seen that the minoxidil group promoted skin color change on the sixth day, entered the early stage of hair growth ahead of time, and had a significant effect on promoting hair growth. The low-dose, high-dose and injection groups promoted skin color change on the third day, entered the early stage of hair growth ahead of time, and had a significant effect on promoting hair growth.

[0089] from Figure 2 It can be seen that 7 days after hair removal treatment (early growth phase), the results of tissue microscopy showed that the number of hair follicles in the treatment groups (low-dose, high-dose, and injection groups) was significantly increased compared with the two control groups (negative control and positive control).

[0090] from Figure 3 It can be seen that 7 days after hair removal treatment (early growth stage), the expression of β-catenin in hair follicles still showed a higher positive rate in the treatment groups (low-dose, high-dose, and injection groups).

[0091] Transdermal absorption

[0092] Fluorescence labeling is a commonly used qualitative analysis method for evaluating the transdermal absorption of protein peptides. This method combines fluorescent markers such as fluorescein isothiocyanate (FITC) with recombinant collagen, and then applies the labeled peptide sample to mouse skin samples. The distribution and intensity of the fluorescent signal in the skin can be observed and analyzed using a fluorescence microscope to evaluate the transdermal absorption of the peptide sample.

[0093] The specific steps of the experimental protocol include:

[0094] Preparation of test substance: Combine the fluorescent marker (FITC) with the peptide sample to prepare the test substance.

[0095] Application of test substance: Apply the labeled peptide sample evenly on the intact skin of SD mouse skin animals and time it.

[0096] Sample collection: Skin samples of SD mice in the low-dose group were collected 4 hours after sample application and frozen sections were made.

[0097] Fluorescence microscopy: Use fluorescence microscopy to observe and analyze the distribution and intensity of fluorescent signals in the skin. Figure 4 shown.

[0098] from Figure 4 It can be seen that the formula of the topical preparation can help the active ingredients enter the mouse skin and has good transdermal absorption.

[0099] Formula stability test

[0100] For pharmaceutical preparations that are particularly sensitive to temperature, long-term testing is conducted under storage conditions close to the actual storage conditions of the drug, with the purpose of providing a basis for the shelf life of the preparation. The test product is a care product containing 0.1% of the polypeptide in Example 1, which is placed at a temperature of 25±2°C and a relative humidity of 60%±10% for 5 months, or at a temperature of 4°C±2°C and a relative humidity of 65%±5% for 5 months. Samples are taken twice, i.e., at 0 and 5 months, and the polypeptide content is tested with a focus on stability. The shelf life of the drug is determined, and the results are as follows: Figure 5 shown.

[0101] from Figure 5 It can be seen that the peptides in the 0.1% formula are very stable at 4°C and barely decompose for 150 days, and decompose less than 5% at 24°C for 150 days.

[0102] The above are the implementation methods listed in this embodiment, but this embodiment is not limited to the above optional implementation methods. Those skilled in the art can arbitrarily combine the above methods to obtain other various implementation methods. Anyone can derive other various forms of implementation methods under the inspiration of this embodiment. The above specific implementation methods should not be understood as limiting the scope of protection of this embodiment. The scope of protection of this embodiment shall be based on the definition in the claims, and the description can be used to interpret the claims.

Claims

1. A polypeptide, characterized in that: Its structure is H2N-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-DGlu-DAsp-DSer-DSer-OH.

2. The method for preparing a polypeptide according to claim 1, characterized in that: The following steps are included: S1. Carrying out condensation reaction of amino acids in resin; S2. dissociating the peptide from the resin and removing the amino acid side chain protecting groups to obtain a crude peptide; S3. After purification, the product is obtained.

3. The method for preparing a polypeptide according to claim 2, characterized in that: The raw materials in step S1 are D-glutamic acid, D-aspartic acid and D-serine.

4. The method for preparing a polypeptide according to claim 2, characterized in that: The specific steps of step S1 are to dissolve O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid coupling reagent, raw materials and protected amino acids in N , N - dimethylformamide; The obtained reaction solution was added to the resin, and then N,N-diisopropylethylamine was added and N2 was introduced for the first blowing, and the detection was performed by ninhydrin reaction; After the reaction was completed, the solvent was removed and N , N - washing the resin with dimethylformamide; Add to the resin N , N - dimethylformamide solution, introduce N2 for a second blow; Remove the solvent and use N , N - Wash the resin with dimethylformamide to complete the coupling of the amino acid; The above reaction procedure is repeated until the condensation reaction of all protected amino acids is completed.

5. The method for preparing a polypeptide according to claim 2, characterized in that: The specific steps of removing the amino acid side chain protecting groups in step S2 are: Add the resin to the lysate and stir; Then the resin and lysate are extracted, and the peptide is fully analyzed; The polypeptide was filtered and washed to obtain a crude peptide.

6. Use of a polypeptide in preparing a hair growth care product, characterized in that: The polypeptide is a polypeptide according to claim 1 or a polypeptide prepared by the method for preparing a polypeptide according to any one of claims 2 to 5.

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