Epithalon vitamin b6 conjugated derivative, and preparation method and application thereof

Abstract

The application discloses an Epithalon vitamin B6 coupled derivative and a preparation method and application thereof, and belongs to the technical field of medicines and cosmetics. The structure of the Epithalon vitamin B6 coupled derivative is: the Epithalon vitamin B6 coupled derivative has improved skin barrier passing ability and stability compared with vitamin B6 and Epithalon, has good stability, good transdermal effect, excellent health promotion and disease prevention functions such as sleep aid, and solves the defects of instability, poor permeability and inconvenient use of Epithalon and vitamin B6 in the prior art, and meanwhile, the Epithalon vitamin B6 coupled derivative has the functions of Epithalon and vitamin B6, and solves the technical problems of the need for separate addition of Epithalon and vitamin B6 in functional cosmetics and health products, the inactivation of Epithalon and vitamin B6 due to environmental influence, and short action time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine and cosmetics, and particularly relates to an Epithalon vitamin B6 coupled derivative, a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already commonly known in the art.

[0003] Epitalon, also known as Epithalon or AEDG peptide (Ala-Glu-Asp-Gly), is a synthetic peptide derived from the natural substance epithalamin in the pineal gland of the brain, first discovered by scientist Vladimir Khavinson and his team in the 1980s, known as the "Fountain of Youth" peptide. It is mainly known for its anti-aging and telomerase activator effects, which can stimulate the pineal gland to regulate its own function and brain function, and promote the production of telomerase to maintain telomere length and delay cell aging. Its biological effects include in vitro induction of human cell telomerase activity to elongate telomeres, promote heterochromatin depolymerization of aged lymphocytes, reduce chromosome aberration rate in aging mice, increase antioxidant enzyme activity in aging rats, and increase telomere length in blood cells of patients in a certain age group, restore pineal gland secretion of melatonin function, etc. Epitalon can activate telomerase, effectively delay the shortening process of telomeres, 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 effects of Epitalon, which can promote neuronal differentiation, which is essential for the development and function maintenance of the nervous system. Epitalon increases the expression of neuronal-related genes by promoting neuronal differentiation, providing protection for the normal operation of the nervous system. In animal models with hereditary retinal pigment degeneration, it can prolong the integrity of the retina, which is of great significance for protecting vision and preventing retinal diseases. In terms of regulating the endocrine system, Epitalon also plays an active role. It can increase the secretion of melatonin in the pineal gland of aged primates. Melatonin is a hormone secreted by the pineal gland, which plays a key role in regulating sleep-wake cycles, biological clocks and various physiological functions of the body. By increasing the secretion of melatonin, Epitalon improves the quality of sleep in animals, which promotes the health and normal physiological function of the body. These mechanisms of Epitalon are interrelated and synergistic. In the telomerase activation mechanism, it directly intervenes in the core process of cell aging, laying the foundation for other effects. Its antioxidant effect not only plays a role in anti-tumor, but also plays an important role in the overall protection of cells, reducing free radical damage to cells, maintaining normal cell function, regulating gene expression and protein synthesis through histone binding, which involves DNA interaction, further promoting neural differentiation gene transcription, thus playing a key role in neuroprotection. At the same time, it regulates the endocrine system, especially the secretion of melatonin, which is closely related to the overall physiological state of cells and the regulation of biological clocks.

[0004] Pyridoxine is an important water-soluble vitamin, belonging to the B6 family of vitamins, and plays a crucial role in the physiological processes of the human body. As a coenzyme, pyridoxine is involved in various amino acid metabolic reactions. It is the coenzyme for many transaminases, which play a key role in the transamination of amino acids, i.e., the transfer of an amino group from one amino acid to another α-keto acid, forming a new amino acid and α-keto acid. Pyridoxine also plays an important role in glycogen metabolism. It is the coenzyme for glycogen phosphorylase, which catalyzes the breakdown of glycogen into glucose-1-phosphate, a key step in glycogenolysis. When blood glucose levels are low, glycogen is broken down into glucose to maintain blood glucose stability, and pyridoxine ensures that glycogen can be broken down in a timely manner to provide energy for the body by participating in the catalytic reaction of glycogen phosphorylase. Pyridoxine is essential for the normal functioning of the nervous system. It is involved in the synthesis of various neurotransmitters, such as serotonin (5-hydroxytryptamine), dopamine, and gamma-aminobutyric acid (GABA). In the process of serotonin synthesis, pyridoxine, in the form of pyridoxal phosphate, acts as a coenzyme in the conversion of tryptophan to 5-hydroxytryptophan, and further to serotonin. Neurotransmitters play a key role in regulating mood, cognitive function, sleep, and other aspects, so 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 the coenzyme for certain fatty acid synthase enzymes, participating 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 energy balance in the body, the integrity of cell membranes, and cardiovascular health, among other things. 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 and B lymphocytes. At the same time, it can also regulate the inflammatory response by inhibiting the production of pro-inflammatory cytokines and promoting the secretion of anti-inflammatory cytokines to maintain the balance of the immune system. Normal function of the immune system is crucial for preventing infection and disease. Pyridoxine has certain antioxidant capacity. It can participate in the body's antioxidant defense system by participating in the synthesis of some antioxidant enzymes or acting as a coenzyme in antioxidant reactions to scavenge free radicals. Free radicals are highly active molecules that can cause damage to cellular DNA, proteins, and lipids, leading to cell aging and disease. Pyridoxine helps protect cells from damage by free radicals through its antioxidant effect.

[0005] However, the two active substances have certain shortcomings affecting their application. Epitalon is a natural amino acid polypeptide without modification, 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, reducing its efficacy; it is difficult to pass through the skin barrier due to its large polarity and strong hydrophilicity, resulting in low bioavailability; non-invasive drug delivery routes (such as oral, transdermal) are difficult to achieve, and it can only be administered by injection, nasal spray and other invasive methods, making it inconvenient to use and reducing user convenience and compliance. Pyridoxine has poor stability, is easily chemically reacted under alkaline conditions, is easily oxidized in aqueous solution and oxidized faster under alkaline conditions, and is easily decomposed under light and heating conditions. There is no report on solving the above-mentioned shortcomings of Epitalon and pyridoxine at the same time. SUMMARY

[0006] In order to solve the problems of the prior art, the purpose of the present application is to provide an Epithalon vitamin B6 coupled derivative, a preparation method and application thereof. The Epithalon vitamin B6 coupled derivative provided by the present application has the efficacy of Epithalon and vitamin B6 at the same time, and has improved ability to pass through the skin barrier and stability compared with vitamin B6 and natural amino acid Epithalon. It has good stability, good transdermal effect, excellent health promotion and disease prevention functions such as sleep aid, solves the defects of instability, poor permeability and inconvenience of use of Epithalon and vitamin B6 in the prior art, and also solves the technical problems of the need for separate addition of Epithalon and vitamin B6 in functional cosmetics and health products, easy inactivation by environmental factors and short action time.

[0007] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0008] In a first aspect of the present application, an Epithalon vitamin B6 coupled derivative is provided, and the structure of the Epithalon vitamin B6 coupled derivative is as follows:

[0009]

[0010] In a second aspect of the present application, a preparation method of the above-mentioned Epithalon vitamin B6 coupled derivative is provided, comprising:

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

[0012] The structure of the Epithalon with a protective group is as follows:

[0013]

[0014] In a third aspect of the present application, there is provided use of the Epithalon vitamin B6 conjugated derivative as described above in the preparation of a product having any one or more of the following (1) to (9) functions:

[0015] (1) helping to enhance immunity;

[0016] (2) helping to improve sleep;

[0017] (3) helping to resist aging;

[0018] (4) helping to repair skin;

[0019] (5) helping to improve skin moisture condition;

[0020] (6) helping to resist inflammation;

[0021] (7) helping to resist oxidation;

[0022] (8) helping to protect nerves;

[0023] (9) helping to improve mood.

[0024] In a fourth aspect of the present application, there is provided a product comprising the Epithalon vitamin B6 conjugated derivative as described above.

[0025] The present application has the following advantages:

[0026] The Epithalon and vitamin B6 coupled derivative is prepared by covalently bonding Epithalon and 3,4-dipalmitoyl pyridoxine to form an ester bond. In structure, Epithalon and vitamin B6 are covalently connected by an ester bond and are modified by two palmitic acid chains. For Epithalon, the fatty acid chain modification can protect it from proteolytic enzyme degradation, reduce the charge, enhance its ability to pass through the skin barrier, make it suitable for various administration routes, and prolong its activity time on the skin surface and after entering the skin. For pyridoxine, the fatty acid chain modification greatly improves its stability. In terms of synergy, under the action of skin esterase, the coupled derivative can slowly release Epithalon and vitamin B6, which synergistically promote health and prevent diseases. In cosmetics and health products, there is a synergistic effect in terms of anti-aging, sleep aid, skin repair and moisturizing, enhanced immunity, anti-inflammatory and antioxidant, neuroprotection, improved mood, etc., which can comprehensively improve the product efficacy. Compared with the prior art, the Epithalon vitamin B6 coupled derivative of the present application has improved ability to pass through the skin barrier and stability, solves the defects of Epithalon and vitamin B6 in the prior art, such as instability, poor permeability, and inconvenience to use, and solves the problems of functional cosmetics and health products, such as the need to add Epithalon and vitamin B6 separately, the inactivation of Epithalon and vitamin B6 due to environmental influence, and the short action time. In the preparation process, the synthesis route of the present application has the characteristics of high atom economy, high yield and environmental friendliness. BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings, which form a part of the present description, illustrate the present application and together with the written description serve to explain the application. In the drawings, which are not necessarily to scale, like elements are represented by like reference numerals.

[0028] Figure 1 Mass spectrum of the Epithalon with a protecting group prepared for Example 1 of the present application;

[0029] Figure 2 Mass spectrum of the Epithalon vitamin B6 coupled derivative prepared for Example 4 of the present application. DETAILED DESCRIPTION

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

[0031]

[0032] The Epithalon vitamin B6 conjugated derivative provided by the application is characterized in that Epithalon and vitamin B6 are covalently connected by an ester bond and contain two palmitic acid chains. The fatty acid chain modification can protect Epithalon from proteolytic enzyme degradation, reduce the charge of Epithalon, improve the ability of Epithalon to pass through the skin barrier, and improve the stability and permeability of Epithalon, and is suitable for various administration routes. The fatty acid chain modification greatly improves the stability of pyridoxine. In terms of effect, the Epithalon vitamin B6 conjugated derivative slowly releases Epithalon and vitamin B6 under the action of skin esterase after reaching the skin surface and entering the skin, and the two substances synergistically act to prolong the active time. The fatty acid chain increases the hydrophobicity of Epithalon, improves the ability of Epithalon to pass through the cell membrane and the stratum corneum, and is more easily absorbed by the skin, thereby improving the efficacy, and non-invasive administration such as oral administration and transdermal administration can be achieved, and the skin permeability and absorption are improved. Epithalon and vitamin B6 have a synergistic effect in cosmetics and health products, which is manifested in the aspects of anti-aging (Epitalon prolongs cell lifespan, and pyridoxine has antioxidant effect), sleep aid (Epitalon and vitamin B6 synergistically regulate the secretion of melatonin), skin repair and moisturizing (Epitalon promotes cell regeneration, and pyridoxine participates in metabolism), immune enhancement, anti-inflammatory and antioxidant effects, and neuroprotection, mood improvement, and the like. The use of Epithalon and vitamin B6 together can more comprehensively improve the efficacy of the product and increase the value of the product in promoting health and preventing diseases. The Epithalon vitamin B6 conjugated derivative described in the application has improved ability to pass through the skin barrier and stability compared with vitamin B6 and natural amino acid Epithalon, has good stability, good transdermal effect, excellent health-promoting and disease-preventing 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 of use. At the same time, the Epithalon vitamin B6 conjugated derivative has the functions of Epithalon and vitamin B6, and solves the technical problems of Epithalon and vitamin B6 in functional cosmetics and health products, such as the need for separate addition, easy inactivation due to environmental influence, and short action time.

[0033] The application further provides a preparation method of the Epithalon vitamin B6 conjugated derivative.

[0034] The Epithalon vitamin B6 conjugated derivative is obtained by coupling the Epithalon with a protective group with 3,4-dipalmitoyl pyridoxine ester and then removing the protective group.

[0035] The structure of the Epithalon with a protective group is as follows:

[0036]

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

[0038] The protected Epithalon, 3,4-dipalmitoyl pyridoxine and catalyst are added into an organic solvent, and the temperature is lowered to 0-10℃; the dehydrating agent is added under the control of temperature lower than 20℃, and the temperature is raised to 20-30℃, to obtain an intermediate product; the protected group is removed by acid to obtain the Epithalon vitamin B6 coupling derivative.

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

[0040]

[0041] In some embodiments of the present application, the molar ratio of the protected Epithalon, 3,4-dipalmitoyl pyridoxine, catalyst and 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 application, the catalyst includes but is not limited to 4-dimethylaminopyridine.

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

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

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

[0046] In some embodiments of the present application, the preparation method of the protected Epithalon comprises the following steps:

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

[0048] In some embodiments of the present application, 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-terminal to the N-terminal, and obtaining the fully protected linear crude peptide after cleavage and drying.

[0049] In some embodiments of the present application, the preparation method of the fully protected linear crude peptide comprises:

[0050] swelling the CTC resin in dichloromethane;

[0051] reacting the first amino acid Fmoc-Gly-OH with the swollen CTC resin to obtain Fmoc-Gly-CTC resin, removing the Fmoc protecting group to obtain NH2-Gly-CTC resin;

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

[0053] adding the activated Fmoc-L-Asp(tBu)-OH to the NH2-Gly-CTC resin for condensation, removing the Fmoc protecting group after the condensation is completed, continuing to condense Fmoc-L-Glu(tBu)-OH, removing the Fmoc protecting group after the condensation is completed, and finally condensing Boc-L-Ala-OH, and obtaining the fully-protected linear crude peptide after cleavage and concentration to dryness.

[0054] In some embodiments of the present application, the CTC resin has a degree of substitution of 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, i.e. 1.5-1.8 mmol of active reaction sites are contained in each gram of CTC resin. The amount of other ingredients (such as amino acids, condensing agents, etc.) used in the preparation process can be determined according to the content of active reaction sites in the resin used.

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

[0057] In some embodiments of the present application, the reaction conditions for the reaction of the 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 for reaction to obtain Fmoc-Gly-CTC resin.

[0058] 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 application, the operation of removing the Fmoc protecting group comprises: adding 20% piperidine / N,N-dimethylformamide solution containing 1M 2-hydroximino ethyl cyanoacetate to the Fmoc-Gly-CTC resin, removing the Fmoc twice at 10-30°C, and the reaction time is 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 application, 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 application, the activation is carried out by adding the amino acid and the condensing agent into dimethylformamide, and 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 with a molar ratio of 1:1.

[0062] It should be noted that the activation methods for different amino acids are the same, which are all carried out by adding the amino acid and the condensing agent into dimethylformamide.

[0063] In some embodiments of the present application, the reaction condition of the condensation is 10-30°C for 2-5h.

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

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

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

[0067] Preferably, the reverse phase high performance liquid chromatography for purifying the fully protected linear crude peptide is carried out by using a C18 preparation column (50*250mm, 10μm).

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

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

[0070] (1) helping to enhance immunity;

[0071] (2) helping to improve sleep;

[0072] (3) helping to resist aging;

[0073] (4) helping to repair skin;

[0074] (5) helping to improve skin moisture condition;

[0075] (6) helping to resist inflammation;

[0076] (7) helping to resist oxidation;

[0077] (8) helping to protect nerves;

[0078] (9) helping to improve mood.

[0079] The application also provides a product comprising the Epithalon vitamin B6 conjugated derivative.

[0080] It should be noted that the active ingredient in the product can only be the Epithalon vitamin B6 conjugated derivative, or the Epithalon vitamin B6 conjugated derivative and other active ingredients, and those skilled in the art can select appropriate active ingredients according to actual needs.

[0081] The Epithalon vitamin B6 conjugated derivative can be orally taken, topically applied or injected, and those skilled in the art can select appropriate excipients according to conventional methods, and prepare the Epithalon vitamin B6 conjugated derivative and the excipients into appropriate dosage forms or use forms.

[0082] In some embodiments of the application, the dosage form of the product includes injections, ointments, powder injections, liniments, dressings and liquid preparations.

[0083] In some embodiments of the application, the product includes cosmetics, and when it is a cosmetic, the product includes but is not limited to cosmetic water, emulsion, essence, gel, foundation, cream and mask, etc., and the application range of the cosmetic includes but is not limited to facial washing, body washing and head washing.

[0084] The Epithalon vitamin B6 coupled derivative described in the present application can be used to configure cosmetics and health products with functions of anti-aging, sleep aid, and skin repair, etc. Therefore, in some embodiments of the present application, the product comprises a health product.

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

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or raw materials used in the present application can be purchased through conventional channels, and unless otherwise specified, the reagents or raw materials used in the present application are used in accordance with conventional methods in the art or in accordance with the product instructions. In addition, any method and material similar or equivalent to those described can be applied to the method of the present application. The preferred implementation methods and materials described herein are only for demonstration. Unless otherwise specified, the percentages described in the present application are mass percentages.

[0087] The English abbreviations of substances appearing in the claims and specification of the present application correspond to the following Chinese meanings:

[0088]

[0089]

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

[0091]

[0092] Preparation of Example 1 Epithalon with a protecting group

[0093] 1. Swelling of the resin

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

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

[0096] Dissolve Fmoc-Gly-OH (3 equivalents, 15 mmol) and DIEA (6 equivalents, 30 mmol) in an appropriate amount of dichloromethane, add to the resin (1 equivalent), and react at 25°C for 2 hours. After the reaction is completed, wash with an appropriate amount of dimethylformamide three times, and dry the solvent to obtain Fmoc-Gly-CTC resin.

[0097] 3. Removal of Fmoc protecting group

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

[0099] 4. Activation of amino acids

[0100] Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH were respectively added to an appropriate amount of dimethylformamide, 15 mmol each, and activated with DIC (15 mmol) / HOBt (15 mmol) 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 was added to the NH2-Gly-CTC resin for condensation, and after the condensation was completed, the Fmoc protecting group was removed, and Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH were successively condensed, and after the condensation was completed, the Fmoc protecting group was removed. The condensation of amino acids was carried out at 25°C for 2 hours, and the reaction progress was monitored by ninhydrin color reaction. After the condensation of Boc-L-Ala-OH, Boc-LAla-Glu(tBu)-L-Asp(tBu)-Gly-OH-resin was obtained.

[0103] 6. Cleavage of peptide resin

[0104] A cleavage reagent (trifluoroethanol: dichloromethane = 1:4, by volume) was added to the peptide resin in a volume of 50 mL, and the reaction was carried out at 25°C for 3 hours. The filtrate was concentrated to dryness under reduced pressure to obtain a fully protected linear crude peptide

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

[0106] 7. Purification of crude peptide with protecting group

[0107] The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 preparative column (50*250 mm, 10 μm), and the mobile phase A was 0.1% trifluoroacetic acid in water, and the mobile phase B was 0.1% trifluoroacetic acid in acetonitrile. After concentration and lyophilization, Epithalon with protecting group was obtained, 2.3 g, with a yield of 76.2%, a purity of 96.8%, and a mass spectrum as shown in Figure 1 .

[0108] Preparation of protected Epithalon of Example 2

[0109] 1. Swelling of the resin

[0110] 31.6 g (1 eq, 50 mmol) of CTC resin (substitution degree 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 eq, 150 mmol) and DIEA (6 eq, 300 mmol) were dissolved in an appropriate amount of dichloromethane and added to the resin (1 eq), and reacted at 25°C for 2 hours. After the reaction was completed, the resin was washed with an appropriate amount of dimethylformamide three times, and the solvent was removed by suction to obtain Fmoc-Gly-CTC resin.

[0113] 3. Removal of Fmoc protecting group

[0114] 400 mL of 20% PIP / DMF solution containing 1M Oxyama Pure was added at 25°C 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 removed by suction to obtain NH2-Gly-CTC resin.

[0115] 4. Activation of amino acid

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

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

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

[0119] 6. Cleavage of peptide resin

[0120] To the peptide resin, 50 mL volume of cleavage reagent (trifluoroethanol: dichloromethane = 1:4, volume ratio) was added, and the reaction was carried out at 25°C for 5 hours, and then filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the fully protected linear crude peptide

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

[0122] 7. Purification of the crude peptide with a protecting group

[0123] The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 preparation column (50*250mm, 10μm), 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 lyophilization, 23.3g of the protected Epithalon was obtained, with a yield of 77.2% and a purity of 97.1%.

[0124] Example 3. Preparation of the protected Epithalon

[0125] 1. Swelling of the resin:

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

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

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

[0129] 3. Removal of the Fmoc protecting group

[0130] 800mL of 1M OxymaPure-containing 20% PIP / DMF solution was added at 25°C 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 removed by suction to obtain NH2-Gly-CTC resin.

[0131] 4. Activation of the amino acid

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

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

[0134] 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 condensed. After the condensation was completed, the Fmoc protecting group was removed and finally Boc-L-Ala-OH was condensed. The condensation of amino acids was carried out at 25°C for 3 hours. The reaction progress was monitored by ninhydrin color reaction. After the condensation of Boc-L-Ala-OH, Boc-L Ala-Glu(tBu)-L-Asp(tBu)-Gly-OH-resin was obtained.

[0135] 6. Cleavage of peptide resin

[0136] To the peptide resin, 50 mL of cleavage reagent (trifluoroethanol: dichloromethane = 1:4, volume ratio) was added and reacted at 25°C for 5 hours. The filtrate was concentrated to dryness under reduced pressure to obtain the fully protected linear crude peptide

[0137] Boc-L Ala-Glu(tBu)-L-Asp(tBu)-Gly-OH 64.0 g.

[0138] 7. Purification of crude peptide with protecting group

[0139] The crude peptide was purified by reverse phase high performance liquid chromatography using C18 preparative column (50*250 mm, 10 μm). The mobile phase A was 0.1% trifluoroacetic acid in water and the mobile phase B was 0.1% trifluoroacetic acid in acetonitrile. After concentration and lyophilization, the protected Epithalon was obtained, 47.2 g, with a yield of 78.3% and a purity of 97.9%.

[0140] Example 4. Preparation of vitamin B6 coupled derivative of Epithalon

[0141] Into a 100 mL reaction flask, 2.0 g (3.3 mmol) of the protected Epithalon prepared in Example 3, 2.6 g of 3,4-dipalmitoyl pyridoxal ester (4.0 mmol, 1.2 equivalents relative to the protected Epithalon), 0.04 g of 4-dimethylaminopyridine (0.33 mmol, 0.1 equivalents relative to the protected Epithalon), and 20 mL of dimethylformamide were added, and the temperature was lowered to 0-10 °C. While controlling the temperature below 20 °C, 0.8 g of N,N'-dicyclohexyl carbodiimide (4.0 mmol, 1.2 equivalents relative to the protected Epithalon) was added in portions, the temperature was raised to 20-30 °C, and the reaction was carried out at this temperature for 16 hours. The reaction was completed as determined by TLC. The reaction solution was released into 100 mL of water, and 20 mL of dichloromethane was used to extract the reaction solution three times. The organic phase was combined, dried over 15 g of anhydrous sodium sulfate, and concentrated under reduced pressure to dryness.

[0142] After 20 mL of dichloromethane was added, 10 mL of trifluoroacetic acid was added, and the reaction was carried out at 20-30 °C for 4 hours. The reaction was completed as determined by TLC. The oil obtained by concentrating under reduced pressure at 30-35 °C was added dropwise to 40 mL of methyl tert-butyl ether, and the solid that precipitated was filtered to obtain the crude peptide. The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 preparative column (50*250 mm, 10 μm) with mobile phase A of 0.1% trifluoroacetic acid in water and mobile phase B of 0.1% trifluoroacetic acid in acetonitrile. After concentration and lyophilization, 2.7 g of the Epithalon vitamin B6 conjugate derivative was obtained at a yield of 79.5% and a purity of 98.3%. The mass spectrum is shown in FIG. 2. Figure 2

[0143] Example 5. Preparation of an Epithalon vitamin B6 conjugate derivative

[0144] Into a 100 mL reaction flask, 20 mL of dimethylformamide, 2.6 g of 3,4-dipalmitoyl pyridoxal ester (4.0 mmol, 1.2 equivalents relative to the protected Epithalon), and 1.7 g of triphenylphosphine (6.6 mmol, 2.0 equivalents relative to the protected Epithalon) were added, and the temperature was lowered to 0-10 °C. While controlling the temperature below 20 °C, 1.0 g of diisopropyl azodicarboxylate (5.0 mmol, 1.5 equivalents relative to the protected Epithalon) was added dropwise, and the reaction was carried out at 20-30 °C for 1 hour. Then, 2.0 g (3.3 mmol) of the protected Epithalon prepared in Example 3 was added in portions 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. The reaction was completed as determined by TLC.

[0145] ​After stirring for 4 hours, the reaction solution was released into 100 mL of water, extracted with 20 mL x 3 dichloromethane, and the organic phase was combined. The organic phase was dried with 15 g of anhydrous sodium sulfate and concentrated under reduced pressure to dryness. After dissolving in 20 mL of dichloromethane, 10 mL of trifluoroacetic acid was added, and after stirring at 20-30°C for 4 hours, the reaction was detected by TLC. After being concentrated under reduced pressure to an oil at 30-35°C, the oil was dropped into 40 mL of methyl tert-butyl ether, and the solid precipitated was filtered to obtain the crude peptide. The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 preparation column (50*250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid in water and mobile phase B being 0.1% trifluoroacetic acid in acetonitrile. After concentration and lyophilization, 2.8 g of Epithalon vitamin B6 coupled derivative was obtained, with a yield of 83.6% and a purity of 98.9%.

[0146] Example 6 Preparation of Epithalon vitamin B6 coupled derivative

[0147] Into a 2 L 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. While controlling the temperature below 20°C, 16.3 g of N,N'-dicyclohexyl carbodiimide (79.2 mmol, 1.2 equivalents relative to the protected Epithalon) was added in batches, the temperature was raised to 20-30°C, and the reaction was carried out at this temperature for 16 hours. After the reaction was detected by TLC, the reaction solution was released into 2000 mL of water, extracted with 400 mL x 3 dichloromethane, and the organic phase was combined. The organic phase was dried with 300 g of anhydrous sodium sulfate and concentrated under reduced pressure to dryness.

[0148] After stirring at 20-30°C for 4 hours, the reaction was detected by TLC. After being concentrated under reduced pressure to an oil at 30-35°C, the oil was dropped into 800 mL of methyl tert-butyl ether, and the solid precipitated was filtered to obtain the crude peptide. The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 preparation column (50*250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid in water and mobile phase B being 0.1% trifluoroacetic acid in acetonitrile. After concentration and lyophilization, 54.8 g of Epithalon vitamin B6 coupled derivative was obtained, with a yield of 81.6% and a purity of 98.1%.

[0149] Experimental Example 1 Light and heat stability experiment

[0150] The light and heat stability experiment was performed on the Epithalon vitamin B6 conjugated derivative prepared in the above Example 6, and pyridoxine hydrochloride was used for comparison, and the results showed that the stability of the Epithalon vitamin B6 conjugated derivative was significantly better than that of pyridoxine hydrochloride.

[0151] The test method and results are shown below:

[0152] Light stability test: Epithalon vitamin B6 conjugated derivative and pyridoxine hydrochloride aqueous solutions were prepared with a concentration of 0.5% (w / v), and the pH was adjusted to 6.5-6.8. The solutions were sealed in glass ampoules. At the same time, they were placed in a test box under the condition of illumination of 4500 lx ± 500 lx for 1 day, 5 days, and 10 days. The samples were detected by HPLC (high performance liquid chromatography) to determine the degree of decrease in compound content, and the results are shown in Table 1.

[0153] Table 1 Light stability of Epithalon vitamin B6 conjugated derivative and pyridoxine hydrochloride

[0154]

[0155]

[0156] Heat stability test: Epithalon vitamin B6 conjugated derivative and pyridoxine hydrochloride aqueous solutions were prepared with a concentration of 0.5% (w / v), and the pH was adjusted to 6.5-6.8. The solutions were sealed in glass ampoules. At the same time, they were placed in a 45-50°C vacuum drying oven for 30 days and 60 days. The samples were detected by HPLC (high performance liquid chromatography) to determine the degree of decrease in compound content, and the results are shown in Table 2.

[0157] Table 2 Heat stability of Epithalon vitamin B6 conjugated derivative and pyridoxine hydrochloride

[0158]

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

[0160] Experimental Example 2 Plasma stability experiment

[0161] The plasma stability experiment was performed on the Epithalon vitamin B6 conjugated derivative prepared in the above Example 6, and Epithalon was used for comparison, and the results showed that the stability of the Epithalon vitamin B6 conjugated derivative was significantly better than that of Epithalon.

[0162] Test method and results are shown as follows:

[0163] Plasma stability test: dilute the mouse plasma containing Epithalon and the Epithalon vitamin B6 conjugated derivative prepared in Example 6 (select 8-week-old male ICR mice, select sodium heparin as an anticoagulant) to 0.01 g / mL, incubate at 37°C, take 100 μL every 12 hours, and end after 36 hours. Add 4% phosphoric acid to the sample for protein precipitation, vortex mix for 3 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C. Take the supernatant 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 derivative and Epithalon

[0165]

[0166] By comparison of the above stability tests, the plasma stability of the Epithalon vitamin B6 conjugated derivative of the application is much higher than that of Epithalon.

[0167] Experimental Example 3

[0168] This experimental example detects 1) in vitro cytotoxicity experiment and stimulation experiment, 2) transdermal experiment and 3) hair growth activity test of the test sample.

[0169] I. Experimental grouping and test sample

[0170] At room temperature, 10% propylene glycol, 10% pentylene glycol, 5% glycerol and 0.05% ethylenediaminetetraacetic acid disodium as a matrix were added to 5 equal parts of sterile water, respectively, as experimental group 1 to experimental group 4 and blank group 5. Then, 0.5% Epithalon, pyridoxine hydrochloride, a mixture of Epithalon and pyridoxine hydrochloride, and the Epithalon vitamin B6 conjugated derivative prepared in Example 6 were added to experimental group 1 to experimental group 4, respectively. After the same amount of sterile water was added to blank group 5, they were all stirred to complete dissolution, 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 conjugated derivative stock solution, and blank group 5.

[0171] II. In vitro cytotoxicity experiment and stimulation experiment

[0172] 1. In vitro cytotoxicity experiment

[0173] According to GB T16886.5-2017 / ISO10993-5:2009 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity tests", the Epithalon vitamin B6 coupled derivative prepared in Example 6 is subjected to in vitro cytotoxicity test to verify the biocompatibility. The MTT cytotoxicity test evaluation method is selected, the cell line is L929 cells, 10% DMSO solution is the positive control group, 3% fetal calf serum medium is the negative control group, and the test group is the 3% fetal calf serum medium extraction solution added with the sample to be tested, which is cultured at 37℃ for 24 hours.

[0174] The specific experimental steps are as follows: the L929 cells subcultured for 48 hours are prepared into a cell suspension of 1×10 5 cells / mL with a cell culture solution for standby; the cell suspension of 1×10 5 cells / mL is inoculated in a 96-well cell culture plate (100 μL / well), and is placed in a 37℃, 5% CO2 incubator for culture for 24 hours to form a semi-confluent monolayer; after 24 hours, the original culture solution is discarded, and then 100 μL of sample extraction solution, positive control solution and negative control solution are added respectively, 5 wells for each group, and are placed in a 37℃, 5% CO2 incubator for culture for 24 hours. 50 μL of MTT solution is added to each well, and then incubation is continued at 37℃, 5% CO2 incubator for 2 hours.

[0175] The MTT solution is discarded, 100 μL of DMSO solution is added to each well, the plate is shaken, and the absorbance at 570 nm is measured on a microplate photometer. The cell proliferation rate (RGR, %) is calculated, and the cytotoxicity is evaluated according to the standard in the following table.

[0176] Table 4 In vitro cytotoxicity grading table

[0177]

[0178] Table 5 In vitro cytotoxicity test results

[0179] Sample Toxicity level Experiment group 4 1 Positive control group 5

[0180] The experimental results are shown in Table 5, and the Epithalon vitamin B6 coupled derivative prepared in the application is grade 1, which indicates that it has good biocompatibility and does not produce toxicity and cause toxic residues.

[0181] 2. Irritation test

[0182] Take 15 healthy rabbits, body weight of about 2 kg, randomly divided into 3 groups, 5 in each group, 24 hours before the experiment to remove the hair on both sides of the back skin of rabbits, 24 hours after the hair removal, detect whether the skin of the hair removal area is injured, the skin of the rabbit is not suitable for skin irritation experiment, the samples of experimental group 4 and blank group 5 are applied 3 times a day, and the application is continuously applied for 7 days, the experimental results are observed, and the experimental results are shown in table 6.

[0183] Table 6: results of skin irritation experiment

[0184]

[0185] Note: "+" indicates that the rabbit has skin redness, inflammation and congestion; "++" indicates that the rabbit has skin redness, inflammation and congestion, and has an increasing trend; "-" indicates that the rabbit has no skin redness, inflammation and congestion.

[0186] From the experimental results of table 6, it can be seen that the sample of experimental group 4 has no skin irritation, i.e. the Epithalon vitamin B6 coupled derivative of the present application has no skin irritation.

[0187] Three, transdermal experiment

[0188] The vertical Franz diffusion cell method is used for in vitro mouse skin transdermal experiment. The abdominal skin of SD male rats is fixed between the receiving chamber and the supply chamber, and the experimental group 1, experimental group 2, experimental group 3 and experimental group 4 are taken in the supply chamber, and the physiological saline containing 2% sodium dodecyl sulfate and 20% ethanol is used as the receiving liquid, which is stirred and diffused at 37℃ with 300 rpm. After 24 hours, sample is taken, HPLC (high performance liquid chromatography) is used to detect the compound, and the cumulative penetration amount and penetration rate of different Epithalon, pyridoxine and skin non-irritating are calculated. The experimental data is shown in table 7.

[0189] Table 7: cumulative penetration amount of test sample in different experimental groups

[0190]

[0191] From table 7, it can be seen that Epithalon is difficult to penetrate the stratum corneum of the skin, so its bioavailability is low, pyridoxine has slightly stronger transdermal ability, Epithalon and pyridoxine synergistically promote transdermal absorption, and the penetration rate of the skin non-irritating of the present application after 24 hours can reach 48.6%.

[0192] Four, evaluation of sleep quality improvement effect

[0193] The subjective sleep quality of each experimental group is evaluated by Pittsburgh Sleep Quality Index (PSQI) scale: it is composed of 19 self-evaluation items and 5 evaluation items by others, the 19th self-evaluation item and the 5 evaluation items by others are not scored. The remaining 18 items scored are divided into 7 dimensions: sleep quality, sleep time, sleep time, sleep efficiency, sleep disorder, sleep promoting drug and daytime dysfunction. Each dimension is scored by 0-3, and the cumulative score of each dimension is the total score of PSQI, which is in the range of 0-21, the score > 7 indicates poor sleep quality, the score ≤ 7 indicates good sleep quality, and when the score decreases by ≥ 3 before and after the test, it is considered that the subjective sleep is improved, and the results are shown in Table 8.

[0194] Table 8 Sleep improvement test results

[0195]

[0196]

[0197] As can be seen from the test results in Table 8, pure use of pyridoxine hydrochloride and Epithalon stock solution has no sleep improvement effect, Epithalon and pyridoxine have a synergistic effect on sleep improvement, the Epithalon vitamin B6 coupled derivative prepared in the application has a significant sleep improvement effect, and the score decreases by 8 before and after the test, and the sleep improvement effect is better.

[0198] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An Epithalon vitamin B6 conjugated derivative, characterized in that, The structure of the Epithalon vitamin B6 coupled derivative is as follows: 。 2. A process for the preparation of the Epithalon vitamin B6 conjugated derivative according to claim 1, characterized by, The preparation method comprises the following steps: The Epithalon vitamin B6 coupled derivative is obtained by coupling the protected Epithalon and 3,4-dipalmitoyl pyridoxine and then removing the protection group; The structure of the protected Epithalon is as follows: 。 3. The production method according to claim 2, wherein The preparation method comprises the following steps: The preparation method comprises the following steps: The intermediate product is obtained by adding the protected Epithalon, 3,4-dipalmitoyl pyridoxine and a catalyst into an organic solvent, cooling to 0-10 DEG C, adding a dehydrating agent under the condition of temperature control lower than 20 DEG C, and then heating to 20-30 DEG C; 。 4. The production method according to claim 3, wherein The structure of the intermediate product is as follows:

5. The production method according to claim 4, wherein The molar ratio of the protected Epithalon, 3,4-dipalmitoyl pyridoxine, the catalyst and the dehydrating agent is 1:(1-1.5):(0.05-0.15):(1-1.5).

6. The production method according to claim 3, wherein The molar ratio of the protected Epithalon, 3,4-dipalmitoyl pyridoxine, the catalyst and the dehydrating agent is 1:1.2:0.1:1.

2.

7. The production method according to claim 3, wherein The catalyst comprises 4-dimethylaminopyridine.

8. The production method according to claim 3, wherein The dehydrating agent comprises N,N'-dicyclohexyl carbodiimide.

9. The production method according to claim 3, wherein The organic solvent comprises dimethylformamide.

10. The production method according to any one of claims 2 to 9, characterized by, The acid comprises trifluoroacetic acid. The preparation method of the protected Epithalon comprises the following steps:

11. The production method according to claim 10, wherein The preparation method of the full-protected linear crude peptide comprises the following steps: taking 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-terminal to the N-terminal, cleaving and drying to obtain the full-protected linear crude peptide.

12. The production method according to claim 11, wherein The preparation method of the full-protected linear crude peptide comprises the following steps: taking 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-terminal to the N-terminal, cleaving and drying to obtain the full-protected linear crude peptide. The preparation method of the full-protected linear crude peptide comprises the following steps: The CTC resin is swelled in dichloromethane; The first amino acid Fmoc-Gly-OH is reacted with the swelled CTC resin to obtain Fmoc-Gly-CTC resin, and the Fmoc protection group is removed to obtain NH2-Gly-CTC resin; Fmoc-L-Asp(tBu)-OH, Fmoc-L-Glu(tBu)-OH and Boc-L-Ala-OH are activated respectively; 13. The production method according to claim 12, wherein After the activated Fmoc-L-Asp(tBu)-OH is added to the NH2-Gly-CTC resin for condensation, the Fmoc protection group is removed, Fmoc-L-Glu(tBu)-OH is continuously condensed, the Fmoc protection group is removed after the condensation is completed, and finally Boc-L-Ala-OH is condensed, and after cleavage and concentration to dryness, the full-protected linear crude peptide is obtained. The degree of substitution of the CTC resin is 1.5-1.8 mmol / g.

14. The production method according to claim 12, wherein The molar ratio of 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.

15. The production method according to claim 12, wherein The activation is carried out by adding amino acids and a condensing agent into dimethylformamide, and the molar ratio of the amino acids to the condensing agent is (2-5):(2-5), and the condensing agent is a mixed solution of N, N'-diisopropyl carbodiimide and 1-hydroxybenzotriazole at a molar ratio of 1:

1.

16. The production method according to claim 12, wherein The cleavage operation comprises: adding a cleavage reagent in an amount of 10-15 times the volume of the resin into the reaction system, and reacting at 10-30°C for 2-5 hours.

17. The production method according to claim 16, wherein The cleavage reagent is a mixed solution of trifluoroethanol and dichloromethane at a volume ratio of 1:

4.

18. The production method according to claim 10, wherein The operation of purification comprises: purifying by reverse phase high performance liquid chromatography, and the mobile phase A is a trifluoroacetic acid aqueous solution, and the mobile phase B is a trifluoroacetic acid acetonitrile solution.

19. The production method according to claim 18, wherein The reverse phase high performance liquid chromatography is carried out by using a C18 preparation column.

20. The production method according to claim 18, wherein The mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution.

21. Use of the Epithalon vitamin B6 conjugated derivative of claim 1 in the preparation of a product having any one or more of the following functions (1)-(9): (1) helping to enhance immunity; (2) helping to improve sleep; (3) helping to resist aging; (4) helping to repair the skin; (5) helping to improve the moisture condition of the skin; (6) helping to resist inflammation; (7) helping to resist oxidation; (8) helping to protect the nerves; (9) helping to improve the mood.

22. A product, characterized in that The product comprises the Epithalon vitamin B6 conjugated derivative of claim 1.

23. The product of claim 22, wherein The dosage form of the product comprises injections, ointments, powder injections, liniments, dressings and liquid preparations.

24. The product of claim 22, wherein, The product is a cosmetic, and the application range of the cosmetic comprises facial washing, body washing and head washing.

25. The product of claim 22, wherein, The product is a health product. The product is a cosmetic, and the application range of the cosmetic comprises facial washing, body washing and head washing. The product is a health product.

Images