Epihalon vitamin B6 coupling derivative as well as preparation method and application thereof

Abstract

The invention discloses an Epihalon vitamin B6 coupling derivative as well as a preparation method and application thereof, and belongs to the technical field of medicines and cosmetics. The structure of the Epihalon vitamin B6 coupling derivative is # imgabs0. Compared with vitamin B6 and Epihalon, the Epihalon vitamin B6 coupling derivative has the advantages that the ability of passing through a skin barrier and the stability are improved, the Epihalon vitamin B6 coupling derivative has good stability and a good transdermal effect, and has excellent functions of promoting health and preventing diseases, such as helping sleep; according to the present invention, the defects of instability, poor permeability, inconvenient use and the like of the Epihalon and the vitamin B6 in the prior art are solved, the Epihalon and the vitamin B6 have effects of the Epihalon and the vitamin B6, and the technical problems that the Epihalon and the vitamin B6 in the functional cosmetics and the health-care products need to be added separately, are easily affected by the environment and are inactivated, are short in action time and the like are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and cosmetics, and specifically relates to an Epithalon vitamin B6 coupled derivative and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Epitalon, also known as Epithalon or AEDG peptide (Ala-Glu-Asp-Gly), is a synthetic peptide derived from epithalamin, a natural substance in the pineal gland of the brain. It was first discovered by scientist Vladimir Khavinson and his team in the 1980s and is known as the "fountain of youth" peptide. It is mainly known for its anti-aging and role as a telomerase activator. It can stimulate the pineal gland to regulate its own function and brain function, and promote the production of telomerase, thereby maintaining telomere length and delaying cell aging. Its biological effects include inducing telomerase activity in human cells in vitro to elongate telomeres and promote heterochromatin depolymerization in elderly lymphocytes. In vivo animal studies can reduce the chromosome aberration rate of aged mice and increase the antioxidant enzyme activity of aged rats. Human clinical studies have shown that it can increase telomere length in blood cells of patients in certain age groups and restore the function of the pineal gland to secrete melatonin. Epitalon can activate telomerase, effectively delay the process of telomere shortening, and inhibit cell aging. Animal studies have shown that it significantly prolongs the lifespan of fruit flies, mice and rats. Neuroprotection is one of the important functions of Epitalon. It can promote neuronal differentiation. Neurons are the basic components of the nervous system, and their differentiation is crucial to the development and function maintenance of the nervous system. Epitalon increases the expression of neuron-related genes by promoting neuronal differentiation, thus providing guarantee for the normal operation of the nervous system. In animal models with hereditary retinitis pigmentosa, it can prolong the functional integrity of the retina, which is of great significance for protecting vision and preventing retinal diseases. Epitalon also plays a positive role in regulating the endocrine system. It can increase the secretion of melatonin in the pineal gland of elderly primates. Melatonin is a hormone secreted by the pineal gland, which plays a key role in regulating the sleep-wake cycle, biological clock and various physiological functions of the body. By increasing the secretion of melatonin, Epitalon improves the sleep quality of animals and promotes the health of animals and the normal maintenance of physiological functions. These mechanisms of action of Epitalon are interrelated and synergistic. In the telomerase activation mechanism, it directly intervenes in the core process of cell aging and lays the foundation for other effects. Its antioxidant effect is not only effective in anti-tumor, but also plays an important role in the overall protection of cells, reducing free radical damage to cells and maintaining normal cell function. It regulates gene expression and protein synthesis by binding to histones. This process involves DNA interaction and further promotes the transcription of neural differentiation genes, thus playing a key role in neuroprotection. At the same time, its regulation of the endocrine system, especially its influence on melatonin secretion, is closely related to the overall physiological state of cells and biological clock regulation.

[0004] Pyridoxine is an important water-soluble vitamin, a member of the vitamin B6 family, and plays a vital role in the physiological processes of the human body. Pyridoxine participates in a variety of amino acid metabolic reactions as a coenzyme. It is a coenzyme of many transaminases, which play a key role in the transamination of amino acids, that is, transferring the amino group of one amino acid to another α-keto acid to form new amino acids and α-keto acids. Pyridoxine also plays an important role in glycogen metabolism. It is a coenzyme of glycogen phosphorylase, which catalyzes the decomposition of glycogen into glucose-1-phosphate, which is a key step in glycogen decomposition. When blood sugar levels are low, glycogen is decomposed into glucose to maintain blood sugar stability. Pyridoxine ensures that glycogen can be decomposed in time to provide energy for the body by participating in the catalytic reaction of glycogen phosphorylase. Pyridoxine is essential for the normal function of the nervous system. It is involved in the synthesis of a variety of neurotransmitters, such as serotonin (5-hydroxytryptamine), dopamine, γ-aminobutyric acid (GABA), etc. In the synthesis of serotonin, pyridoxine, in the form of pyridoxal phosphate, participates in the conversion of tryptophan into 5-hydroxytryptophan and then into serotonin as a coenzyme. Neurotransmitters play a key role in regulating emotions, cognitive functions, sleep, etc., so an adequate supply of pyridoxine is important for maintaining the health and normal function of the nervous system. Pyridoxine is also involved in lipid metabolism. It is a coenzyme of certain fatty acid synthases and participates in the synthesis of fatty acids. At the same time, it may regulate lipid metabolism by affecting the expression of genes related to fat metabolism. Normal lipid metabolism is very important for maintaining the body's energy balance, cell membrane integrity, and cardiovascular health. Pyridoxine has a regulatory effect on the immune system. It can enhance the activity of immune cells, such as promoting the proliferation and differentiation of T lymphocytes and B lymphocytes. At the same time, it can also regulate inflammatory responses and maintain the balance of the immune system by inhibiting the production of proinflammatory cytokines and promoting the secretion of anti-inflammatory cytokines. The normal function of the immune system is essential for preventing infections and diseases. Pyridoxine has a certain antioxidant capacity. It can participate in the body's antioxidant defense system and remove free radicals by participating in the synthesis of some antioxidant enzymes or participating in antioxidant reactions as a coenzyme. Free radicals are highly active molecules that can damage the cell's DNA, proteins, and lipids, leading to cell aging and disease. Pyridoxine helps protect cells from free radical damage through its antioxidant effect.

[0005] However, both of the above active substances have certain shortcomings that affect their application. Epitalon is an unmodified natural amino acid polypeptide, which is easily degraded by proteases in the body, resulting in a very short half-life, usually only a few minutes to a few hours, which reduces its efficacy; it has high polarity and strong hydrophilicity, making it difficult to pass through the skin barrier, resulting in low bioavailability; non-invasive routes of administration (such as oral and transdermal) are difficult to achieve, and it can usually only be administered through invasive methods such as injection and nasal spray, which makes application inconvenient and reduces the user's convenience and compliance. Pyridoxine has poor stability and is prone to chemical reactions under alkaline conditions, resulting in the destruction of its molecular structure. It is easily oxidized in aqueous solution when exposed to air and oxidizes faster under alkaline conditions. It is easy to decompose under light and heating conditions. There is currently no report that simultaneously solves the above-mentioned shortcomings of Epitalon and pyridoxine. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide an Epithalon vitamin B6 coupled derivative and a preparation method and application thereof. The Epithalon vitamin B6 coupled derivative provided by the present invention has the effects of Epithalon and vitamin B6 at the same time. Compared with vitamin B6 and Epithalon of natural amino acids, the ability and stability of the Epithalon to pass through the skin barrier are improved, the derivative has good stability and good transdermal effect, and has excellent health promotion and disease prevention functions such as sleep aid. The defects of Epithalon and vitamin B6 in the prior art, such as instability, poor permeability, and inconvenience in use, are solved. The derivative also solves the technical problems that Epithalon and vitamin B6 in functional cosmetics and health products need to be added separately, are easily inactivated by environmental influences, and have a short action time.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides an Epithalon vitamin B6 coupled derivative, the structure of the Epithalon vitamin B6 coupled derivative is as follows:

[0009]

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned Epithalon vitamin B6 coupled derivative, comprising:

[0011] Epithalon with a protective group is coupled with 3,4-dipalmitoyl pyridoxine ester and then deprotected to obtain Epithalon vitamin B6 coupled derivative;

[0012] The structure of the Epithalon with a protecting group is shown below:

[0013]

[0014] The third aspect of the present invention provides a use of the above-mentioned Epithalon vitamin B6 coupled derivative in the preparation of a product having any one or more of the following functions (1)-(9);

[0015] (1) Helps to enhance immunity;

[0016] (2) Helps improve sleep;

[0017] (3) Helps in anti-aging;

[0018] (4) Helps repair skin;

[0019] (5) Helps improve skin moisture status;

[0020] (6) Helps fight inflammation;

[0021] (7) Helps in anti-oxidation;

[0022] (8) Helps protect nerves;

[0023] (9) Helps improve mood.

[0024] The fourth aspect of the present invention provides a product, which comprises the above-mentioned Epithalon vitamin B6 coupled derivative.

[0025] The beneficial effects of the present invention are:

[0026] The present invention covalently bonds Epithalon with 3,4-dipalmitoyl pyridoxine ester to form an ester bond, thereby obtaining an Epithalon vitamin B6 coupling derivative. Structurally, Epithalon is covalently connected to vitamin B6 with an ester bond and is modified with two palmitic acid chains. For Epithalon, fatty acid chain modification can protect it from proteolytic degradation, reduce the charge, enhance its ability to pass through the skin barrier, make it suitable for a variety of administration routes, and prolong its active time on the skin surface and after entering the skin. For pyridoxine, fatty acid chain modification greatly improves its stability. In terms of synergistic effect, under the action of skin esterase, the coupling derivative can slowly release Epithalon and vitamin B6, and the two synergistically play a role in promoting health and preventing diseases. In cosmetics and health products, it has synergistic effects in anti-aging, sleep aid, skin repair and moisturizing, enhancing immunity, anti-inflammatory and anti-oxidation, neuroprotection, and improving mood, which can comprehensively improve the efficacy of the product. Compared with the prior art, the Epithalon vitamin B6 coupled derivative of the present invention has improved ability and stability in passing through the skin barrier, solves the defects of the prior art that Epithalon and vitamin B6 are unstable, poorly permeable, and inconvenient to use, and also solves the problem that Epithalon and vitamin B6 in functional cosmetics and health products need to be added separately, are easily inactivated by environmental influences, and have a short action time. In terms of preparation technology, the synthetic route of the present invention has the characteristics of high atom economy, high yield, and environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 The mass spectrum of Epithalon with a protective group prepared in Example 1 of the present invention;

[0029] Figure 2 This is the mass spectrum of the Epithalon vitamin B6 coupled derivative prepared in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] The present invention provides an Epithalon vitamin B6 coupled derivative, the structure of the Epithalon vitamin B6 coupled derivative is as follows:

[0031]

[0032] In the structure of the Epithalon vitamin B6 coupled derivative provided by the present invention, Epithalon and vitamin B6 are covalently linked by an ester bond and are modified with two palmitic acid chains. Fatty acid chain modification can protect Epithalon from proteolytic degradation, reduce its charge, improve its ability to pass through the skin barrier, stability and permeability, and is suitable for a variety of administration routes; fatty acid chain modification greatly improves the stability of pyridoxine. From the effect point of view, the Epithalon vitamin B6 coupled derivative slowly releases Epithalon and vitamin B6 under the action of skin esterase on the skin surface and after entering the skin, and the two work synergistically to prolong the active time. The fatty acid chain increases its hydrophobicity, improves its ability to pass through the cell membrane and the stratum corneum of the skin, is more easily absorbed and utilized by the skin, improves efficacy, and can also achieve non-invasive administration such as oral, transdermal, etc., to improve skin penetration and absorption. Epithalon and vitamin B6 have synergistic effects in cosmetics and health products, which are reflected in anti-aging (Epitalon prolongs cell life, pyridoxine is antioxidant), sleep aid (Epitalon and vitamin B6 synergistically regulate the secretion of melatonin), skin repair and moisturizing (Epitalon promotes cell regeneration, pyridoxine participates in metabolism), immunity enhancement, anti-inflammatory and antioxidant, neuroprotection, mood improvement, etc. The synergistic use of Epithalon and vitamin B6 can more comprehensively enhance the efficacy of the product and increase its value in promoting health and preventing diseases. Compared with vitamin B6 and Epithalon of natural amino acids, the Epithalon vitamin B6 coupled derivative of the present invention has improved ability to pass through the skin barrier and stability, has good stability, good transdermal effect, and has excellent health promotion and disease prevention functions such as sleep aid, and solves the defects of Epithalon and vitamin B6 in the prior art such as instability, poor permeability, and inconvenience in use. At the same time, it has the efficacy of Epithalon and vitamin B6, and also solves the technical problems that Epithalon and vitamin B6 in functional cosmetics and health products need to be added separately, are easily inactivated by environmental influences, and have a short action time.

[0033] The present invention also provides a method for preparing the above-mentioned Epithalon vitamin B6 coupled derivative, comprising:

[0034] Epithalon with a protective group is coupled with 3,4-dipalmitoyl pyridoxine ester and then deprotected to obtain Epithalon vitamin B6 coupled derivative;

[0035] The structure of the Epithalon with a protecting group is shown below:

[0036]

[0037] In some embodiments of the present invention, the preparation method comprises:

[0038] Epithalon with a protective group, 3,4-dipalmitoyl pyridoxine and a catalyst are added to an organic solvent and the temperature is lowered to 0-10°C; a dehydrating agent is added while the temperature is controlled below 20°C, and the temperature is raised to 20-30°C to react to obtain an intermediate product, and the protective group is removed with an acid to obtain an Epithalon vitamin B6 coupling derivative;

[0039] The structure of the intermediate product is as follows:

[0040]

[0041] In some embodiments of the present invention, the molar ratio of the epithalon with a protecting group, 3,4-dipalmitoyl pyridoxine, the catalyst and the dehydrating agent is 1:(1-1.5):(0.05-0.15):(1-1.5), preferably 1:1.2:0.1:1.2.

[0042] In some embodiments of the present invention, the catalyst includes but is not limited to 4-dimethylaminopyridine.

[0043] In some embodiments of the present invention, the dehydrating agent includes but is not limited to N,N'-dicyclohexylcarbodiimide.

[0044] In some embodiments of the present invention, the organic solvent includes but is not limited to dimethylformamide.

[0045] In some embodiments of the present invention, the acid includes but is not limited to trifluoroacetic acid.

[0046] In some embodiments of the present invention, the method for preparing the Epithalon with a protecting group comprises the following steps:

[0047] The solid phase synthesis method is adopted, CTC resin is used as a solid phase synthesis carrier, and the amino acid residues protected by substituents are sequentially condensed according to the amino acid sequence from the C-terminus to the N-terminus of the main chain of Epithalon to obtain a fully protected linear crude peptide, which is purified and freeze-dried to obtain Epithalon with a protective group.

[0048] In some embodiments of the present invention, the method for preparing the fully protected linear crude peptide comprises: using CTC resin as a solid phase synthesis carrier, sequentially condensing Fmoc-Gly-OH, Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH from the C-terminus to the N-terminus, and obtaining a fully protected linear crude peptide after cleavage and drying.

[0049] In some embodiments of the present invention, the method for preparing the fully protected linear crude peptide comprises:

[0050] Take CTC resin and swell it in dichloromethane;

[0051] The first amino acid Fmoc-Gly-OH is reacted with the swollen CTC resin to obtain Fmoc-Gly-CTC resin, and the Fmoc protecting group is removed to obtain NH2-Gly-CTC resin;

[0052] Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH were activated respectively;

[0053] The activated Fmoc-L-Asp(tBu)-OH was added to NH2-Gly-CTC resin for condensation. After the condensation was completed, the Fmoc protecting group was removed, and Fmoc-L-Glu(tBu)-OH was continued to be condensed. After the condensation was completed, the Fmoc protecting group was removed, and finally Boc-L-Ala-OH was condensed. After cleavage and concentration to dryness, a fully protected linear crude peptide was obtained.

[0054] In some embodiments of the present invention, the degree of substitution of the CTC resin is 1.5-1.8 mmol / g.

[0055] It should be noted that the term degree of substitution refers to how many millimoles of active reaction sites are loaded in each resin. For example, the degree of substitution is 1.5-1.8 mmol / g, that is, each gram of CTC resin contains 1.5-1.8 mmol of active reaction sites. The amount of other ingredients (such as amino acids, condensing agents, etc.) used in the preparation process can be determined based on the content of active reaction sites in the resin used.

[0056] In some embodiments of the present invention, the CTC resin is swelled in dichloromethane for 1-2 hours.

[0057] In some embodiments of the present invention, the reaction conditions of the reaction of Fmoc-Gly-OH and the swollen CTC resin are as follows: Fmoc-Gly-OH and diisopropylethylamine are dissolved in an organic solvent, and the swollen CTC resin is added to react to obtain Fmoc-Gly-CTC resin.

[0058] Wherein, the molar ratio of Fmoc-Gly-OH, diisopropylethylamine and CTC resin is (2-5):(4-8):1, preferably 3:6:1.

[0059] In some embodiments of the present invention, the operation of removing the Fmoc protecting group includes: adding a 20% piperidine / N,N-dimethylformamide solution containing 1M ethyl 2-oximecyanoacetate to the Fmoc-Gly-CTC resin at 10-30°C, removing Fmoc twice, with reaction times of 5 minutes and 10 minutes respectively; then washing the resin with dimethylformamide until the pH reaches about 7.

[0060] In some embodiments of the present invention, the molar ratio of the amino acids Fmoc-Gly-OH, Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH to the resin is (2-5):1.

[0061] In some embodiments of the present invention, the activation is performed by adding an amino acid and a condensing agent to dimethylformamide for activation, the molar ratio of the amino acid to the condensing agent is (2-5):(2-5), and the condensing agent is a mixed solution of N,N'-diisopropylcarbodiimide and 1-hydroxybenzotriazole in a molar ratio of 1:1.

[0062] It should be noted that when activating different amino acids, the activation method is the same, that is, the amino acid and the condensation agent are added to dimethylformamide for activation.

[0063] In some embodiments of the present invention, the condensation reaction conditions are 10-30° C. for 2-5 h.

[0064] In some embodiments of the present invention, the cleavage operation comprises: adding a cleavage reagent of 10-15 times the volume of the resin to the reaction system, and reacting at 10-30° C. for 2-5 hours; further preferably, the cleavage reagent is a mixed solution of trifluoroethanol and dichloromethane in a volume ratio of 1:4.

[0065] It should be noted that after the reaction of adding the cleavage reagent is completed, the mixture is filtered and the filtrate is concentrated to dryness to obtain a crude peptide with a protected group, Boc-LAla-Glu(tBu)-L-Asp(tBu)-Gly-OH, that is, a fully protected linear crude peptide.

[0066] In some embodiments of the present invention, the purification operation includes: purification by reverse phase high performance liquid chromatography, wherein mobile phase A is trifluoroacetic acid aqueous solution, and mobile phase B is trifluoroacetic acid acetonitrile solution.

[0067] Preferably, a C18 preparative column (50*250 mm, 10 μm) is used to purify the fully protected linear crude peptide by reverse phase high performance liquid chromatography.

[0068] Preferably, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution.

[0069] The present invention also provides a use of the above-mentioned Epithalon vitamin B6 coupled derivative in the preparation of a product having any one or more of the following functions (1)-(9):

[0070] (1) Helps to enhance immunity;

[0071] (2) Helps improve sleep;

[0072] (3) Helps in anti-aging;

[0073] (4) Helps repair skin;

[0074] (5) Helps improve skin moisture status;

[0075] (6) Helps fight inflammation;

[0076] (7) Helps in anti-oxidation;

[0077] (8) Helps protect nerves;

[0078] (9) Helps improve mood.

[0079] The present invention also provides a product, which comprises the Epithalon vitamin B6 coupled derivative.

[0080] It should be noted that the active ingredient in the product may be only Epithalon vitamin B6 coupled derivatives, or may be Epithalon vitamin B6 coupled derivatives and other active ingredients. Those skilled in the art may choose to add appropriate active ingredients according to actual needs.

[0081] The Epithalon vitamin B6 coupled derivatives of the present invention can be taken orally, applied externally or injected. Those skilled in the art can select and add appropriate excipients according to conventional methods to prepare the Epithalon vitamin B6 coupled derivatives of the present invention and the excipients into a suitable dosage form or usage form.

[0082] In some embodiments of the present invention, the dosage forms of the product include injections, ointments, powder injections, liniments, dressings and liquid preparations.

[0083] In some embodiments of the present invention, the product includes cosmetics. When used as cosmetics, the product includes but is not limited to lotions, emulsions, essences, gels, foundations, creams and facial masks, etc. The application scope of the cosmetics is but is not limited to facial washing and maintenance, body washing and maintenance, and head washing and maintenance.

[0084] The Epithalon vitamin B6 coupled derivatives of the present invention can be used to prepare cosmetics and health products with anti-aging, sleep-aiding and skin-repairing functions. Therefore, in some embodiments of the present invention, the products include health products.

[0085] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0086] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. If not otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equal to the recorded content can be applied to the method of the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes. If not otherwise specified, the percentages described in the present invention are all percentages by mass.

[0087] The corresponding Chinese meanings of the English abbreviations of substances appearing in the claims and specification of the present invention are shown in the following table:

[0088]

[0089]

[0090] In the following examples, the synthetic route of Epithalon vitamin B6 coupled derivatives is as follows:

[0091]

[0092] Example 1 Preparation of Epithalon with Protecting Group

[0093] 1. Swelling of resin

[0094] 3.16 g (1 equivalent, 5 mmol) of CTC resin (substitution degree is 1.58 mmol / g) was added to 40 mL of dichloromethane to swell the resin for 1.5 hours.

[0095] 2. Preparation of Fmoc-Gly-CTC resin

[0096] Fmoc-Gly-OH (3 equivalents, 15 mmol) and DIEA (6 equivalents, 30 mmol) were dissolved in an appropriate amount of dichloromethane, added to the resin (1 equivalent), and reacted at 25°C for 2 hours. After the reaction was completed, it was washed three times with an appropriate amount of dimethylformamide, and the solvent was drained to obtain Fmoc-Gly-CTC resin.

[0097] 3. Removal of Fmoc protecting group

[0098] At 25°C, 40 mL of 20% PIP / DMF solution containing 1 M OxymaPure was added to remove the Fmoc protecting group twice, with reaction times of 5 minutes and 10 minutes respectively. The resin was then washed with dimethylformamide until the pH reached about 7, and the solvent was drained to obtain NH2-Gly-CTC resin.

[0099] 4. Activation of amino acids

[0100] 15 mmol each of Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH and DIC (15 mmol) / HOBt (15 mmol) were added to an appropriate amount of dimethylformamide and activated at 25°C for 5 minutes.

[0101] 5. Coupling of amino acids with NH2-Gly-CTC resin

[0102] The activated Fmoc-L-Asp(tBu)-OH is added to NH2-Gly-CTC resin for condensation. After the condensation is completed, the Fmoc protecting group is removed, and Fmoc-L-Glu(tBu)-OH and are continuously condensed. After the condensation is completed, the Fmoc protecting group is removed, and finally Boc-L-Ala-OH is condensed. The amino acid condensation reaction is carried out at 25°C for 2 hours, and the reaction progress is monitored by ninhydrin color reaction. After condensation of Boc-L-Ala-OH, Boc-LAla-Glu(tBu)-L-Asp(tBu)-Gly-OH-resin is obtained.

[0103] 6. Cleavage of Peptide Resin

[0104] Add 50 mL of cleavage reagent (trifluoroethanol: dichloromethane = 1:4, volume ratio) to the peptide resin, react at 25 ° C for 3 hours, filter, and concentrate the filtrate to dryness under reduced pressure to obtain a fully protected linear crude peptide.

[0105] Boc-LAla-Glu(tBu)-L-Asp(tBu)-Gly-OH 3.1g.

[0106] 7. Purification of crude peptides with protected groups

[0107] The crude peptide was purified by reverse phase HPLC using a C18 preparative column (50*250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 2.3 g of Epithalon with a protective group was obtained, with a yield of 76.2% and a purity of 96.8%. The mass spectrum is shown in FIG. Figure 1 shown.

[0108] Example 2 Preparation of Epithalon with Protecting Group

[0109] 1. Swelling of resin

[0110] 31.6 g (1 equivalent, 50 mmol) of CTC resin (substitution degree is 1.58 mmol / g) was added to 400 mL of dichloromethane to swell the resin for 2 hours.

[0111] 2. Preparation of Fmoc-Gly-OH-resin

[0112] Fmoc-Gly-OH (3 equivalents, 150 mmol) and DIEA (6 equivalents, 300 mmol) were dissolved in an appropriate amount of dichloromethane, added to the resin (1 equivalent), and reacted at 25°C for 2 hours. After the reaction was completed, it was washed three times with an appropriate amount of dimethylformamide, and the solvent was drained to obtain Fmoc-Gly-CTC resin.

[0113] 3. Removal of Fmoc protecting group

[0114] At 25°C, 400 mL of 20% PIP / DMF solution containing 1M OxymaPure was added to remove the Fmoc protecting group twice, with reaction times of 5 minutes and 10 minutes respectively. The resin was then washed with dimethylformamide until the pH reached about 7, and the solvent was drained to obtain NH2-Gly-CTC resin.

[0115] 4. Activation of amino acids

[0116] 150 mmol each of Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH and DIC (150 mmol) / HOBt (150 mmol) were added to an appropriate amount of dimethylformamide and activated at 25°C for 5 minutes.

[0117] 5. Coupling of amino acids with NH2-Gly-CTC resin

[0118] The activated Fmoc-L-Asp(tBu)-OH is added to NH2-Gly-CTC resin for condensation. After the condensation is completed, the Fmoc protecting group is removed, and Fmoc-L-Glu(tBu)-OH and are continuously condensed. After the condensation is completed, the Fmoc protecting group is removed, and finally Boc-L-Ala-OH is condensed. The amino acid condensation reaction is carried out at 25°C for 2.5 hours, and the reaction progress is monitored by ninhydrin color development reaction. After condensation of Boc-L-Ala-OH, Boc-LAla-Glu(tBu)-L-Asp(tBu)-Gly-OH-resin is obtained.

[0119] 6. Cleavage of Peptide Resin

[0120] Add 50 mL of cleavage reagent (trifluoroethanol: dichloromethane = 1:4, volume ratio) to the peptide resin, react at 25 ° C for 5 hours, filter, and concentrate the filtrate to dryness under reduced pressure to obtain a fully protected linear crude peptide.

[0121] Boc-LAla-Glu(tBu)-L-Asp(tBu)-Gly-OH 32.3g.

[0122] 7. Purification of crude peptides with protected groups

[0123] The crude peptide was purified by reverse phase HPLC using a C18 preparative column (50*250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 23.3 g of Epithalon with a protective group was obtained, with a yield of 77.2% and a purity of 97.1%.

[0124] Example 3 Preparation of Epithalon with Protecting Group

[0125] 1. Swelling of resin:

[0126] 63.3 g (1 equivalent, 100 mmol) of CTC resin (substitution degree is 1.58 mmol / g) was added to 800 mL of dichloromethane to swell the resin for 2 hours.

[0127] 2. Preparation of Fmoc-Gly-OH-resin

[0128] Fmoc-Gly-OH (3 equivalents, 300 mmol) and DIEA (6 equivalents, 600 mmol) were dissolved in an appropriate amount of dichloromethane, added to the resin (1 equivalent), and reacted at 25°C for 3 hours. After the reaction was completed, it was washed three times with an appropriate amount of dimethylformamide, and the solvent was drained to obtain Fmoc-Gly-CTC resin.

[0129] 3. Removal of Fmoc protecting group

[0130] At 25°C, 800 mL of 20% PIP / DMF solution containing 1 M OxymaPure was added to remove the Fmoc protecting group twice, with reaction times of 5 minutes and 10 minutes respectively. The resin was then washed with dimethylformamide until the pH reached about 7, and the solvent was drained to obtain NH2-Gly-CTC resin.

[0131] 4. Activation of amino acids

[0132] 300 mmol each of Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH and DIC (300 mmol) / HOBt (300 mmol) were added to an appropriate amount of dimethylformamide and activated at 25°C for 5 minutes.

[0133] 5. Coupling of amino acids with NH2-Gly-CTC resin

[0134] The activated Fmoc-L-Asp(tBu)-OH is added to NH2-Gly-CTC resin for condensation. After the condensation is completed, the Fmoc protecting group is removed, and Fmoc-L-Glu(tBu)-OH and are continuously condensed. After the condensation is completed, the Fmoc protecting group is removed, and finally Boc-L-Ala-OH is condensed. The amino acid condensation reaction is carried out at 25°C for 3 hours, and the reaction progress is monitored by ninhydrin color development reaction. After condensation of Boc-L-Ala-OH, Boc-LAla-Glu(tBu)-L-Asp(tBu)-Gly-OH-resin is obtained.

[0135] 6. Cleavage of Peptide Resin

[0136] Add 50 mL of cleavage reagent (trifluoroethanol: dichloromethane = 1:4, volume ratio) to the peptide resin, react at 25 ° C for 5 hours, filter, and concentrate the filtrate to dryness under reduced pressure to obtain a fully protected linear crude peptide.

[0137] Boc-LAla-Glu(tBu)-L-Asp(tBu)-Gly-OH64.0g.

[0138] 7. Purification of crude peptides with protected groups

[0139] The crude peptide was purified by reverse phase HPLC using a C18 preparative column (50*250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 47.2 g of Epithalon with a protective group was obtained, with a yield of 78.3% and a purity of 97.9%.

[0140] Example 4 Preparation of Epithalon Vitamin B6 Conjugated Derivatives

[0141] Add 2.0g (3.3mmol) of Epithalon with protective group prepared in Example 3, 2.6g of 3,4-dipalmitoyl pyridoxine ester (4.0mmol, 1.2 equivalents relative to Epithalon with protective group), 0.04g of 4-dimethylaminopyridine (0.33mmol, 0.1 equivalents relative to Epithalon with protective group) and 20mL of dimethylformamide to a 100mL reaction bottle, cool to 0-10°C, control the temperature below 20°C, add 0.8g of N,N'-dicyclohexylcarbodiimide (4.0mmol, 1.2 equivalents relative to Epithalon with protective group) in batches, heat to 20-30°C and react at this temperature for 16 hours, and the reaction is complete by TLC detection. Release the reaction solution into 100mL of water, extract with 20mL×3 dichloromethane, combine the organic phases, dry the organic phases with 15g of anhydrous sodium sulfate, and concentrate to dryness under reduced pressure.

[0142] Add 20 mL of dichloromethane to dissolve, then add 10 mL of trifluoroacetic acid, stir at 20-30°C for 4 hours, and then detect the completion of the reaction by TLC. After concentrating to an oily substance under reduced pressure at 30-35°C, drop the oil into 40 mL of methyl tert-butyl ether, stir during the addition, filter the precipitated solid to obtain a crude peptide, and then use a C18 preparative column (50*250 mm, 10 μm) to purify the crude peptide by reverse-phase high performance liquid chromatography. Mobile phase A is 0.1% trifluoroacetic acid / water solution, and mobile phase B is 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and lyophilization, 2.7 g of Epithalon vitamin B6 coupled derivative was obtained, with a yield of 79.5% and a purity of 98.3%. The mass spectrum is as shown below. Figure 2 shown.

[0143] Example 5 Preparation of Epithalon Vitamin B6 Conjugated Derivatives

[0144] To a 100 mL reaction bottle, 20 mL of dimethylformamide, 2.6 g of 3,4-dipalmitoyl pyridoxine ester (4.0 mmol, 1.2 equivalents to Epithalon with a protected group) and 1.7 g of triphenylphosphine (6.6 mmol, 2.0 equivalents to Epithalon with a protected group) were added, the temperature was lowered to 0-10°C, 1.0 g of diisopropyl azodicarboxylate (5.0 mmol, 1.5 equivalents to Epithalon with a protected group) was added dropwise while controlling the temperature below 20°C, after stirring for 1 hour, 2.0 g (3.3 mmol) of Epithalon with a protected group prepared in Example 3 was added in batches while controlling the temperature below 20°C, the temperature was raised to 20-30°C and the reaction was carried out at this temperature for 16 hours, and the reaction was completed after TLC detection.

[0145] After adding 5 mL of purified water to the reaction system and stirring for 4 hours, the reaction solution was released into 100 mL of water, extracted with 20 mL × 3 dichloromethane, and the organic phases were combined. The organic phases were dried with 15 g of anhydrous sodium sulfate and concentrated to dryness under reduced pressure; 20 mL of dichloromethane was added to dissolve, and then 10 mL of trifluoroacetic acid was added. After stirring at 20-30 ° C for 4 hours, the reaction was completed by TLC detection. After being concentrated to an oily substance at 30-35 ° C under reduced pressure, the oily substance was dropped into 40 mL of methyl tert-butyl ether, stirred during the dropping process, and the precipitated solid was filtered to obtain a crude peptide, and then a C18 preparative column (50*250 mm, 10 μm) was used to purify the crude peptide by reverse phase high performance liquid chromatography, and the mobile phase A was 0.1% trifluoroacetic acid / water solution, and the mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 2.8 g of Epithalon vitamin B6 coupling derivative was obtained, with a yield of 83.6% and a purity of 98.9%.

[0146] Example 6 Preparation of Epithalon Vitamin B6 Conjugated Derivatives

[0147] To a 2L reaction bottle, 40.0 g (66 mmol) of the protected Epithalon prepared in Example 3, 51.2 g of 3,4-dipalmitoyl pyridoxine ester (79.2 mmol, 1.2 equivalents relative to the protected Epithalon), 0.4 g of 4-dimethylaminopyridine (3.3 mmol, 0.1 equivalents relative to the protected Epithalon) and 400 mL of dimethylformamide were added, and the temperature was lowered to 0-10°C, and 16.3 g of N,N'-dicyclohexylcarbodiimide (79.2 mmol, 1.2 equivalents relative to the protected Epithalon) was added in batches while controlling the temperature below 20°C. The temperature was raised to 20-30°C and reacted at this temperature for 16 hours. The reaction was completed by TLC detection, and the reaction solution was released into 2000 mL of water, extracted with 400 mL×3 dichloromethane, and the organic phases were combined, dried over 300 g of anhydrous sodium sulfate, and concentrated to dryness under reduced pressure.

[0148] 400 mL of dichloromethane was added to dissolve, and then 200 mL of trifluoroacetic acid was added. After stirring at 20-30°C for 4 hours, the reaction was completed by TLC detection. After being concentrated to an oily substance at 30-35°C under reduced pressure, the oily substance was dripped into 800 mL of methyl tert-butyl ether, and stirred during the dripping process. The precipitated solid was filtered to obtain a crude peptide, and then a C18 preparative column (50*250 mm, 10 μm) was used to purify the crude peptide by reverse phase high performance liquid chromatography. The mobile phase A was 0.1% trifluoroacetic acid / water solution, and the mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 54.8 g of Epithalon vitamin B6 coupling derivative was obtained, with a yield of 81.6% and a purity of 98.1%.

[0149] Experimental Example 1: Light and thermal stability test

[0150] The Epithalon vitamin B6 coupled derivative prepared in Example 6 was subjected to light and heat stability tests and compared with pyridoxine hydrochloride. The results showed that the Epithalon vitamin B6 coupled derivative was significantly more stable than pyridoxine hydrochloride.

[0151] The test methods and results are as follows:

[0152] Strong light stability test: Prepare an aqueous solution of Epithalon vitamin B6 coupled derivative and pyridoxine hydrochloride at a concentration of 0.5% (w / v), adjust the pH value to 6.5-6.8, and seal the solution in a glass ampoule. Place it in a test box at an illumination of 4500lx±500lx for 1 day, 5 days, and 10 days. Take a sample and use HPLC (high performance liquid chromatography) to detect the degree of decrease in the content of the compound. The results are shown in Table 1.

[0153] Table 1 Photostability of Epithalon vitamin B6 conjugated derivatives and pyridoxine hydrochloride

[0154]

[0155]

[0156] Thermal stability test: An aqueous solution of Epithalon vitamin B6 coupled derivative and pyridoxine hydrochloride was prepared at a concentration of 0.5% (w / v), and the pH value was adjusted to 6.5-6.8. The solution was sealed in a glass ampoule. At the same time, it was placed in a vacuum drying oven at 45-50°C for 30 days and 60 days. Samples were taken and tested for the degree of decrease in the compound content by HPLC (high performance liquid chromatography). The results are shown in Table 2.

[0157] Table 2 Thermal stability of Epithalon vitamin B6 conjugated derivatives and pyridoxine hydrochloride

[0158]

[0159] Through the above stability test comparison, the light and heat environment stability of the Epithalon vitamin B6 coupled derivative of the present invention is much higher than that of pyridoxine hydrochloride.

[0160] Experimental Example 2 Plasma stability experiment

[0161] The Epithalon vitamin B6 coupled derivative prepared in Example 6 was subjected to a plasma stability test and compared with Epithalon. The results showed that the Epithalon vitamin B6 coupled derivative was significantly more stable than Epithalon.

[0162] The test methods and results are as follows:

[0163] Plasma stability test: Mouse plasma containing Epithalon and Epithalon vitamin B6 conjugated derivative prepared in Example 6 was diluted (8-week-old male ICR mice were selected, and sodium heparin was selected as the anticoagulant) to 0.01 g / mL, incubated at 37°C, and 100 μL was sampled every 12 hours, and the test was terminated after 36 hours. 4% phosphoric acid was added to the sample for protein precipitation, vortexed for 3 minutes, and centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was taken for content determination in LC-MS / MS, and the results are shown in Table 3.

[0164] Table 3 Plasma stability of Epithalon vitamin B6 conjugated derivatives and Epithalon

[0165]

[0166] Through the above stability test comparison, the plasma stability of the Epithalon vitamin B6 coupled derivative of the present invention is much higher than that of Epithalon.

[0167] Experimental Example 3

[0168] This experimental example tests the test samples in 1) in vitro cytotoxicity test and irritation test, 2) skin permeation test and 3) hair growth activity test.

[0169] 1. Experimental groups and test samples

[0170] At room temperature, 10% propylene glycol, 10% pentanediol, 5% glycerol and 0.05% disodium ethylenediaminetetraacetate were added to 5 equal parts of sterile water as matrices, respectively, as experimental groups 1 to 4 and blank group 5, respectively, and then 0.5% of Epithalon, pyridoxine hydrochloride, a mixture of Epithalon and pyridoxine hydrochloride, and the Epithalon vitamin B6 coupled derivative prepared in Example 6 were added to the experimental groups 1 to 4, respectively. After adding an equal amount of sterile water to the blank group 5, the mixtures were stirred until completely dissolved, and the pH value was adjusted to 5-7 to obtain experimental group 1 of Epithalon stock solution, experimental group 2 of pyridoxine hydrochloride stock solution, experimental group 3 of Epithalon and pyridoxine hydrochloride combination stock solution, experimental group 4 of Epithalon vitamin B6 coupled derivative stock solution, and blank group 5, respectively.

[0171] 2. In vitro cytotoxicity and irritation experiments

[0172] 1. In vitro cytotoxicity assay

[0173] According to GB T16886.5-2017 / ISO10993-5:2009 "Biological Evaluation of Medical Devices Part 5: In vitro Cytotoxicity Test", the Epithalon vitamin B6 conjugated derivative prepared in Example 6 was subjected to an in vitro cytotoxicity test to verify biocompatibility. The MTT cytotoxicity test evaluation method was used, L929 cells were selected as the cell line, 10% DMSO solution was used as the positive control group, 3% fetal calf serum culture medium was used as the negative control group, and the test group was added with 3% fetal calf serum culture medium extract of the test sample, and cultured at 37°C for 24 hours with shaking.

[0174] The specific experimental steps are as follows: L929 cells cultured for 48 hours were prepared with cell culture medium to produce 1×10 5 Cell suspension of 1×10 5 The 100 μL / mL cell suspension was inoculated into a 96-well cell culture plate (100 μL / well) and cultured in a 37°C, 5% CO2 incubator for 24 hours to form a semi-confluent monolayer; the original culture medium was discarded after 24 hours, and then 100 μL of sample extract, positive control solution, and negative control solution were added, 5 wells per group, and cultured in a 37°C, 5% CO2 incubator for 24 hours. 50 μL of MTT solution was added to each well, and then continued to incubate in a 37°C, 5% CO2 incubator for 2 hours.

[0175] The MTT solution was discarded, and 100 μL of DMSO solution was added to each well. The plate was shaken and placed on a microtiter plate photometer to measure the absorbance at 570 nm. The cell proliferation rate (RGR, %) was calculated and the cytotoxicity was evaluated according to the standards in the table below.

[0176] Table 4 In vitro cytotoxicity grading table

[0177]

[0178] Table 5 In vitro cytotoxicity test results

[0179] sample Toxicity level Experimental Group 4 1 Positive control group 5

[0180] The experimental results are shown in Table 5. The Epithalon vitamin B6 coupled derivatives prepared by the present invention are of Grade 1, which indicates that they all have good biocompatibility and will not produce toxicity or cause toxic residues.

[0181] 2. Stimulation test

[0182] Fifteen healthy rabbits, weighing about 2 kg, were randomly divided into three groups, with 5 rabbits in each group. The hair on both sides of the rabbits' backs was removed 24 hours before the experiment. The skin in the removed area was tested 24 hours after the removal to see if it was injured. Rabbits with injured skin were not suitable for skin irritation experiments. The samples of experimental group 4 and blank group 5 were applied three times a day for 7 consecutive days, and the experimental results were observed. The experimental results are shown in Table 6.

[0183] Table 6 Skin irritation test results

[0184]

[0185] Note: "+" indicates that the rabbits have skin redness, swelling, inflammation and congestion; "++" indicates that the rabbits have skin redness, swelling, inflammation and congestion, and there is an increasing trend; "-" indicates that the rabbits have no skin redness, swelling, inflammation and congestion.

[0186] From the experimental results in Table 6, it can be seen that the samples in Experimental Group 4 are not irritating to the skin, that is, the Epithalon vitamin B6 coupled derivatives of the present invention are not irritating to the skin.

[0187] 3. Transdermal Experiment

[0188] The vertical Franz diffusion cell method was used to conduct the permeation experiment of isolated rat skin. The abdominal skin of SD male rats was fixed between the receiving chamber and the supply chamber, and experimental group 1, experimental group 2, experimental group 3, and experimental group 4 were placed in the supply chamber. The physiological saline containing 2% sodium dodecyl sulfate and 20% ethanol by mass fraction was used as the receiving liquid and stirred and diffused at 300 speed at 37°C. Samples were taken after 24 hours, and the compounds were detected by HPLC (high performance liquid chromatography). The cumulative penetration amount and permeability of different epithalon, pyridoxine and non-irritating to the skin were calculated. The experimental data are shown in Table 7.

[0189] Table 7 Cumulative permeation of samples in different experimental groups

[0190]

[0191] As shown in Table 7, Epithalon has difficulty in penetrating the stratum corneum of the skin, so its bioavailability is low. Pyridoxine has a slightly stronger transdermal ability. Epithalon and pyridoxine synergistically promote transdermal absorption. The present invention has no irritation to the skin and the permeability can reach 48.6% after 24 hours.

[0192] 4. Evaluation of the effect of improving sleep quality

[0193] The Pittsburgh Sleep Quality Index (PSQI) scale was used to evaluate the subjective sleep quality of the above experimental groups: it consists of 19 self-assessment items and 5 other-assessment items, and the 19th self-assessment item and the 5 other-assessment items are not scored. The remaining 18 scored items are divided into 7 dimensions: sleep quality, sleep onset time, sleep time, sleep efficiency, sleep disorders, hypnotic drugs and daytime dysfunction. Each dimension is scored from 0 to 3, and the cumulative score of each dimension is the total PSQI score. In the range of 0 to 21 points, a score of >7 points indicates poor sleep quality, and a score of ≤7 points indicates good sleep quality. When the score before and after the test decreases by ≥3 points, it is considered that the subjective sleep has improved. The results are shown in Table 8.

[0194] Table 8 Sleep improvement test results

[0195]

[0196]

[0197] It can be seen from the test results in Table 8 that the use of pyridoxine hydrochloride and Epithalon stock solution alone has no effect on improving sleep, while Epithalon and pyridoxine can synergistically improve sleep. The Epithalon vitamin B6 coupled derivative prepared in the present application has a significant sleep improvement effect, and the score before and after the test decreased by 8 points, indicating a good sleep improvement effect.

[0198] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An Epithalon vitamin B6 coupled derivative, characterized in that: The structure of the Epithalon vitamin B6 conjugated derivative is as follows:

2. A method for preparing the Epithalon vitamin B6 coupled derivative according to claim 1, characterized in that: include: Epithalon with a protective group is coupled with 3,4-dipalmitoyl pyridoxine ester and then deprotected to obtain Epithalon vitamin B6 coupled derivative; The structure of the Epithalon with a protecting group is shown below:

3. The preparation method according to claim 2, characterized in that: The preparation method comprises: Epithalon with a protective group, 3,4-dipalmitoyl pyridoxine and a catalyst are added to an organic solvent and the temperature is lowered to 0-10°C; a dehydrating agent is added while the temperature is controlled below 20°C, and the temperature is raised to 20-30°C to react to obtain an intermediate product, and the protective group is removed with an acid to obtain an Epithalon vitamin B6 coupling derivative; The structure of the intermediate product is as follows:

4. The preparation method according to claim 3, characterized in that: The molar ratio of the epithalon with a protective group, 3,4-dipalmitoyl pyridoxine, the catalyst and the dehydrating agent is 1:(1-1.5):(0.05-0.15):(1-1.5), preferably 1:1.2:0.1:1.2; Preferably, the catalyst comprises 4-dimethylaminopyridine; Preferably, the dehydrating agent comprises N,N'-dicyclohexylcarbodiimide; Preferably, the organic solvent comprises dimethylformamide; Preferably, the acid comprises trifluoroacetic acid.

5. The preparation method according to any one of claims 2 to 4, characterized in that: The preparation method of the Epithalon with a protecting group comprises the following steps: The solid phase synthesis method is adopted, CTC resin is used as a solid phase synthesis carrier, and the amino acid residues protected by substituents are sequentially condensed according to the amino acid sequence from the C-terminus to the N-terminus of the main chain of Epithalon to obtain a fully protected linear crude peptide, which is purified and freeze-dried to obtain Epithalon with a protective group.

6. The preparation method according to claim 5, characterized in that: The preparation method of the fully protected linear crude peptide comprises: using CTC resin as a solid phase synthesis carrier, sequentially condensing Fmoc-Gly-OH, Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH from the C-terminus to the N-terminus, and obtaining the fully protected linear crude peptide after cleavage and drying; Preferably, the method for preparing the fully protected linear crude peptide comprises: Take CTC resin and swell it in dichloromethane; The first amino acid Fmoc-Gly-OH is reacted with the swollen CTC resin to obtain Fmoc-Gly-CTC resin, and the Fmoc protecting group is removed to obtain NH2-Gly-CTC resin; Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH were activated respectively; The activated Fmoc-L-Asp(tBu)-OH was added to NH2-Gly-CTC resin for condensation. After the condensation was completed, the Fmoc protecting group was removed, and Fmoc-L-Glu(tBu)-OH was continued to be condensed. After the condensation was completed, the Fmoc protecting group was removed, and finally Boc-L-Ala-OH was condensed. After cleavage and concentration to dryness, a fully protected linear crude peptide was obtained.

7. The preparation method according to claim 6, characterized in that: The degree of substitution of the CTC resin is 1.5-1.8 mmol / g; Preferably, the molar ratio of the amino acids Fmoc-Gly-OH, Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH to the resin is (2-5):1; Preferably, the activation is performed by adding an amino acid and a condensing agent into dimethylformamide for activation, the molar ratio of the amino acid to the condensing agent is (2-5):(2-5), and the condensing agent is a mixed solution of N,N'-diisopropylcarbodiimide and 1-hydroxybenzotriazole in a molar ratio of 1:1; Preferably, the cleavage operation comprises: adding a cleavage reagent of 10-15 times the volume of the resin to the reaction system, and reacting at 10-30° C. for 2-5 hours; further preferably, the cleavage reagent is a mixed solution of trifluoroethanol and dichloromethane in a volume ratio of 1:

4.

8. The preparation method according to claim 5, characterized in that: The purification operation comprises: purifying by reverse phase high performance liquid chromatography, wherein mobile phase A is trifluoroacetic acid aqueous solution, and mobile phase B is trifluoroacetic acid acetonitrile solution; Preferably, the fully protected linear crude peptide is purified by reverse phase high performance liquid chromatography using a C18 preparative column; Preferably, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution.

9. Use of the Epithalon vitamin B6 coupled derivative according to claim 1 in the preparation of a product having any one or more of the following functions (1) to (9); (1) Helps to enhance immunity; (2) Helps improve sleep; (3) Helps in anti-aging; (4) Helps repair skin; (5) Helps improve skin moisture status; (6) Helps fight inflammation; (7) Helps in anti-oxidation; (8) Helps protect nerves; (9) Helps improve mood.

10. A product, characterized in that The product comprises the Epithalon vitamin B6 conjugated derivative according to claim 1; Preferably, the dosage forms of the product include injections, ointments, powder injections, liniments, dressings and liquid preparations; Preferably, the product is a cosmetic, and the application scope of the cosmetic includes facial care, body care and head care; Preferably, the product is a health product.

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