Theanine composition, preparation method and application thereof

Abstract

The application discloses a theanine composition and a preparation method and application thereof, relates to the field of cocrystal technology, and the theanine composition comprises a cocrystal compound containing theanine and arginine. The preparation method of the theanine composition comprises the following steps: S1, providing arginine, stirring and dissolving the arginine under a heating condition by using a solvent to obtain an arginine solution; S2, adding excessive theanine into the solution obtained in S1 and continuing to heat and stir to obtain a mixed solution; S3, continuing to react under an inert atmosphere, recrystallizing after filtration and reducing to low temperature to obtain the cocrystal compound of theanine and arginine; and the application of the theanine composition, the application of the cocrystal compound containing theanine and arginine in the preparation of cosmetics. The theanine and amino acid are simply mixed in the prior art, and the effect is not obviously improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of co-crystals, and particularly relates to a theanine composition and a preparation method and application thereof. BACKGROUND

[0002] Theanine, also known as N-ethyl-L-glutamine, is a characteristic arginine of tea leaves and one of the flavoring substances of tea leaves. Theanine has a moisturizing effect in skin care products, can be added to moisturizing skin care products to help the skin hold moisture and maintain the water content of the skin surface; it also has the effect of a nutrient agent, can supplement nutrients to skin layer cells, increase the activity of cells, and regulate the skin; at the same time, it can be used as an anti-wrinkle agent, has a promoting effect on the generation of collagen, maintains skin elasticity, resists wrinkles, and gives the skin a young and fresh vitality. Theanine can also be used in anti-inflammatory and repairing skin care products, and experiments show that it has an anti-apoptosis effect.

[0003] In actual application, theanine has weak efficacy when used alone. The existing technology often improves the comprehensive effect of theanine by compounding amino acids, but the effect is not obviously improved when theanine and amino acids are simply mixed. Therefore, the existing technology still needs to be improved and developed.

[0004] As disclosed in the prior art, a theanine amino acid salt, a preparation method and an application thereof are disclosed, wherein the specific structure of the theanine amino acid salt and the preparation method thereof are disclosed. The theanine amino acid is simply mixed to form the theanine amino acid salt, and the effect is not obviously improved. SUMMARY

[0005] To solve the above problems, the primary purpose of the present application is to provide a theanine composition, which can solve the problem that the effect is not obviously improved when theanine and amino acids are simply mixed in the prior art.

[0006] Another purpose of the present application is to provide a preparation method of the theanine composition, which is simple and has good comprehensive effect.

[0007] Still another purpose of the present application is to provide an application of the theanine composition, which has good application effect.

[0008] To achieve the above purposes, the technical solutions of the present application are as follows.

[0009] The present application provides a theanine composition, which comprises a co-crystal compound of theanine and arginine, and the structural formula of the co-crystal compound of theanine and arginine is as shown in formula (I). Figure 1

[0010] ​Theanine is a characteristic amino acid in tea, which is synthesized in the roots of tea plants by the action of theanine synthetase from glutamic acid and ethylamine. It is an important substance for the taste of tea, mainly showing fresh and sweet, and is the main component of tea for generating a refreshing and sweet taste. Theanine has a great effect on skin care products, has excellent moisturizing effect, can be added to moisturizing skin care products, helps the skin to hold water, maintains the water content of the skin surface, and has very good effect; it also has the effect of a nutrient agent, can supplement nutrients to skin cells, increase cell activity, and regulate the skin; at the same time, it can be used as an anti-wrinkle agent, has a promoting effect on the generation of collagen, maintains skin elasticity, resists wrinkles, and gives the skin a young and fresh vitality. Theanine can also be used in anti-inflammatory and repair skin care products, experiments show that it has an anti-apoptotic effect, can promote the healing of skin damage, has a good effect on anti-inflammatory and anti-inflammatory of facial skin, and promotes the healing of acne marks.

[0011] L-arginine is a coded amino acid in protein synthesis and is one of the eight essential amino acids for the human body. In cosmetics and skin care products, arginine mainly functions as a pH regulator and a moisturizing agent. The risk coefficient of arginine is 1, which is relatively safe. In cosmetics, arginine is mainly used for the conditioning of dry skin and has a synergistic effect with fruit acid and trehalose, which have moisturizing effects, to maintain skin moisture and smooth skin.

[0012] A co-crystal compound refers to a crystal formed by the combination of two or more precursors under certain conditions through the action of hydrogen bonds, other non-covalent bonds, electrostatic interactions, or hydrophobic interactions, and is a new solid material with biological activity.

[0013] In the present application, the theanine arginine forms a uniform and stable crystal solid with an ordered triclinic crystal system through intermolecular hydrogen bonds and intermolecular electrostatic interactions. The presence of intermolecular hydrogen bonds causes the electron cloud density of theanine and arginine to shift, resulting in changes in physicochemical properties. Experiments have found that the co-crystal compound has the advantages of good water solubility, high permeability, low irritation, small use amount, low effective concentration, good moisturizing effect, and strong antioxidant capacity, and is particularly suitable for moisturizing, anti-aging, and anti-wrinkle product formulations. The theanine and arginine form a hydrogen bond network and other intermolecular forces, and are arranged in a regular monoclinic crystal system co-crystal compound in a certain order. This new co-crystal compound material does not affect the functional groups and performance of the precursors, while improving their solubility, bioavailability, and reducing irritation, etc., greatly improving the performance of the raw materials, and providing great technical support for the research, development, and utilization of difficult-to-dissolve raw materials in the pharmaceutical and cosmetic industries.

[0014] Further, the composition also comprises an efficacy component. When the co-crystal compound of theanine and arginine and the efficacy component are combined, the composition is mild and low irritation, has better stability and biocompatibility, has the effects of whitening, antioxidation and penetration promotion, and can effectively improve the penetration and human body absorption of the co-crystal compound of theanine and arginine. Moreover, the efficacy component in the composition also retains the original structure, and thus also endows the composition with the effects of whitening and moisturizing, anti-wrinkle, antioxidation, anti-inflammation and collagen production promotion.

[0015] Further, the efficacy component comprises ergothioneine. Specifically, the structure of the co-crystal compound of theanine and arginine and ergothioneine is as shown in the following formula: Figure 2 The ergothioneine is chemically named as 2-mercaptohistidine trimethyl inner salt, and is the only natural 2-thioimidazole amino acid known so far. The human epidermis is composed of keratinocytes, melanocytes and fibroblasts, and all the three kinds of cells can express the transporter ETT of ergothioneine, and thus can absorb and accumulate ergothioneine. The ergothioneine has an absorption wavelength similar to DNA in the ultraviolet absorption range, and can effectively prevent the damage caused by ultraviolet radiation; meanwhile, the ergothioneine has a strong antioxidation ability, can make 70% of the cells still have reducing property under the condition of 18 hours of continuous ultraviolet irradiation, and can also inhibit the formation of various active oxidants, lipid peroxidation reaction and apoptosis of the irradiated cells. In addition, the ergothioneine has good chemical stability, can be accumulated in the epidermal cells and is non-toxic.

[0016] As an inner salt, the ergothioneine lacks one hydrogen on the hydroxyl group. When the ergothioneine is combined with the co-crystal of theanine and arginine to form the composition, the hydrogen on the co-crystal shifts to the ergothioneine, and the ergothioneine as the efficacy component can greatly improve the bioactivity and bioavailability of the co-crystal compound of theanine and arginine.

[0017] Further, the efficacy component also comprises one or more of cysteine, ascorbic acid glucoside, hydroxysafflor yellow A, glutathione and VC ethyl ether.

[0018] Further, the mass ratio of the co-crystal compound of theanine and arginine and the efficacy component is 1:0.01-1:5. For example, the mass ratio of the co-crystal compound and the efficacy component can be 1:0.1, 1:1, 1:2, 1:3, 1:4, 1:5, etc. It should be noted that the formation of the ternary supermolecular structure of the efficacy component and the co-crystal compound can have a synergistic effect with the co-crystal compound, and further enhances the antioxidation ability of the co-crystal compound of theanine and arginine. Therefore, the amount of the co-crystal compound of theanine and arginine and other efficacy components can be adjusted according to the final performance of the composition.

[0019] The present application also provides a preparation method of the co-crystal compound of theanine and arginine, which comprises the following steps:

[0020] S1, providing arginine, dissolving arginine in solvent under heating condition to obtain arginine solution; in step S1, arginine is an organic compound containing basic amino group and acidic carboxyl group, and the specific structural formula is as shown in Figure 3 The arginine needs to be dissolved in solvent under the condition of heating and stirring. Preferably, the heating temperature is 40-60℃, and the heating rate is 0.1-5℃ / min.

[0021] S2, adding excess theanine to the solution obtained in S1 and continuing to heat and stir to obtain a mixed solution; in step S2, theanine is usually in powder form, and the specific structure is N-ethyl-L-glutamine (N-Ethyl-L-glutamin), and the structural formula is as shown in Figure 4 .

[0022] S3, under inert atmosphere, continuing to react under heating, and after filtration, recrystallizing at low temperature to obtain a co-crystal compound of theanine and arginine. In this step, inert gas is introduced, and the mixture solution is continuously stirred and heated. Preferably, the inert atmosphere includes helium, neon, argon, nitrogen and the like. Since the solution needs to be heated to a higher temperature in the subsequent step, the mixture solution needs to be heated under inert atmosphere. Specifically, the mixture solution is continuously heated under stirring. The heating temperature is 60-100℃, the heating rate is 0.1-5℃ / min, and the reaction time is 1-24h. During the heating process, theanine reacts with arginine to form a co-crystal molecule. During the cooling process, the co-crystal molecule solidifies and crystallizes to obtain a crude product, which can be purified to obtain a co-crystal of theanine and arginine. The cooling rate is 0.1-5℃ / min. Here, normal temperature refers to 25℃.

[0023] Further, the solvent includes one or more of methanol, ethanol, isopropanol, N-methyl pyrrolidone, tetrahydrofuran, benzene, toluene, petroleum ether, n-hexane, acetone, dioxane, N,N-dimethylformamide, ethyl acetate or acetonitrile.

[0024] Further, the molar ratio of theanine to arginine is 2-3:1. The ratio of theanine to arginine in the mixed solution can be determined as needed. Generally, the ratio of reactants can be determined according to the structure of the reactants. Theanine has one nitrogen atom and one oxygen atom, and arginine has at least one carboxyl group. Since the hydrogen atoms on the carboxyl group of arginine can form hydrogen bonds with the nitrogen atom and the oxygen atom of theanine respectively, a co-crystal is formed. Theanine powder is added to the solution obtained in S1 to perform the co-crystallization reaction in the subsequent steps.

[0025] Further, the heating temperature is 40-100℃, and the reaction time is 1-24h. Further, in S3, the filtrate is cooled to a low temperature for recrystallization to obtain a co-crystal compound of theanine and arginine, specifically, the solution obtained by the heating and reaction is filtered, the solution is concentrated to a supersaturated state under vacuum, and recrystallization is performed after cooling to room temperature, and the recrystallized product is filtered, washed, and dried to obtain the co-crystal of theanine and arginine.

[0026] Further, the recrystallization temperature in S3 is less than 5℃.

[0027] Further, the method further comprises S4: using a high-pressure pump to pump the mixed solution of the co-crystal compound of theanine and arginine and the efficacy component into a high-pressure homogenizer, and fully reacting under an inert gas, and obtaining a composition by decompression concentration. The composition is a uniform, nature-stable solid powder state.

[0028] More specifically, S4 comprises:

[0029] S41, stirring and mixing the co-crystal compound of theanine and arginine and the efficacy component in a container to obtain a mixed solution of the composition after adding a solvent.

[0030] S42, under an inert gas atmosphere, pumping the mixed solution obtained in S41 into a high-pressure homogenizer through a homogenization valve, and obtaining a composition by decompression concentration.

[0031] In step S41, the solvent comprises one or more of water, methanol, ethanol, isopropanol, N-methylpyrrolidone, tetrahydrofuran, benzene, toluene, petroleum ether, n-hexane, acetone, dioxane, N,N-dimethylformamide, ethyl acetate, or acetonitrile.

[0032] In step S42, the inert gas is introduced, and the mixed solution is decompressed and concentrated after passing through the high-pressure homogenization valve. In an embodiment, the inert atmosphere comprises helium, neon, argon, nitrogen, or the like. The pressure of the homogenization valve of the high-pressure homogenizer is 500-1000 bar, and finally the decompression concentration is performed by a rotary evaporator, and the composition is obtained by oven drying. The decompression concentration pressure is -0.1-0 MPa, and the oven temperature is 50-80℃.

[0033] It should be noted that the high-pressure homogenizer is a high-pressure reciprocating pump as a power transmission and material conveying mechanism, which delivers liquid materials or solid particles carried by liquid to the homogenizing valve (high-pressure homogenizing cavity) part. The material to be processed is subjected to strong shearing, impact, cavitation and turbulent vortex action under high pressure during passing through the homogenizing valve, so that the liquid material or solid particles carried by liquid is super-fined. The homogenizing valve receives the high-pressure liquid material delivered by the manifold, and completes the tasks of superfine crushing, emulsification and homogenization. The high-pressure homogenizer can make the solution state material flow at high speed through the cavity with special internal structure (high-pressure homogenizing cavity / homogenizing valve) under high pressure, so that the material undergoes a series of changes in physical, chemical and structural properties, and finally achieves the effect of homogenization.

[0034] Further, the efficacy component includes one or more of ergothioneine, cysteine, ascorbic acid glucoside, hydroxylopol, glutathione, and VC ethyl ether. Preferably, the efficacy component uses ergothioneine.

[0035] The present application also provides a tea amino acid composition and application, and application of a co-crystal compound containing tea amino acid and arginine in cosmetic preparation.

[0036] The present application has the advantages that: compared with the prior art, the tea amino acid arginine co-crystal compound obtained in the present application improves the physicochemical properties of the product without changing the chemical structure of tea amino acid and arginine, and the tea amino acid arginine co-crystal compound has better stability, solubility and applicability than tea amino acid. The physicochemical properties of the two precursors are improved while the original functional groups are retained, and the product efficacy development is maximized. The tea amino acid arginine co-crystal has good effects when added to emulsion, cream and other stay-on products and facial cleanser, shampoo and shower gel and other rinse-off products.

[0037] In addition, the tea amino acid arginine co-crystal compound obtained by the present application retains the efficacy and advantages of tea amino acid and arginine. The tea amino acid arginine co-crystal is arranged in a regular monoclinic crystal system co-crystal compound in a certain order through hydrogen bond network and other intermolecular forces. Compared with simple mixing, the structure has more excellent efficacy improvement.

[0038] The present application provides a preparation method of tea amino acid composition, i.e. a preparation method of tea amino acid arginine co-crystal compound. The preparation steps are simple, the yield is high, the cost is saved, and a tea amino acid arginine co-crystal compound with good thermal stability can be obtained. Generally, tea amino acid arginine salt is obtained by mixing and evaporating, and it is difficult to generate tea amino acid arginine co-crystal. In the method of the present application, preparation is carried out at a relatively high temperature, so that the obtained product has good thermal stability and does not affect the activity when added at any stage of cosmetic preparation.

[0039] The composition containing the theanine arginine co-crystal compound provided by the present application has low irritation, obvious efficacy, and good antioxidant, wrinkle improvement, and skin tone uniformity. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The structural formula of the theanine arginine co-crystal compound of the present application is shown in the following formula.

[0041] Figure 2 The structural formula of the theanine arginine ergothioneine composition of the present application is shown in the following formula.

[0042] Figure 3 The structural formula of arginine of the present application is shown in the following formula.

[0043] Figure 4 The structural formula of theanine of the present application is shown in the following formula.

[0044] Figure 5 The nuclear magnetic hydrogen spectrum of the theanine arginine co-crystal compound in the present application is shown in the following figure.

[0045] Figure 6 The nuclear magnetic hydrogen spectrum (1H-NMR) of the theanine arginine ergothioneine composition in the present application is shown in the following figure.

[0046] Figure 7 The XRD spectrum of the theanine arginine co-crystal in the present application is shown in the following figure.

[0047] Figure 8 The broken line graph of the theanine arginine co-crystal compound and the theanine unit area permeation amount in the present application is shown in the following figure.

[0048] Figure 9 The relative survival rate trend of human skin fibroblasts when the composition in the present application is used is shown in the following figure. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. Specific Example 1

[0051] A preferred method for preparing a theanine arginine co-crystal is as follows:

[0052] Step 1: Take 0.01 mol of arginine and place it in a reactor. Add an appropriate amount of ethanol to the reactor and stir and heat to 50°C. After dissolving and clarifying, take 0.02 mol of theanine and add it to the reactor. After adding, introduce inert gas into the reactor to protect the reaction. Seal the reactor and slowly warm it to 65°C. React for 2 h.

[0053] Second step: filter the solution and concentrate the filtrate to supersaturation (evaporate about 4 / 5 of the volume of the solution). Place the supersaturated solution in a low temperature of 2-5°C and wait for crystallization. Filter the co-crystal product, wash the product thoroughly, and repeat the recrystallization 2-3 times. Dry the final product at 50°C for more than 24 hours. The purity of the theanine arginine co-crystal is greater than 99%. The yield is 90.2%. Specific embodiment 2

[0055] First step: weigh 0.01 mol of arginine and place it in a reactor. Add an appropriate amount of ethanol to the reactor and stir and heat to 50°C. After dissolving and clarifying, weigh 0.02 mol of theanine and add it to the reactor. After adding, protect the reaction by purging the reactor with inert gas, seal the reactor, and slowly warm it to 75°C. React for 2 hours.

[0056] Second step: concentrate the co-crystal solution to supersaturation (evaporate about 4 / 5 of the volume of the solution). Place the supersaturated solution in a low temperature of 2-5°C and wait for crystallization. Filter the co-crystal product, wash the product thoroughly, and repeat the recrystallization 2-3 times. Dry the final product at 50°C for more than 24 hours. The purity of the theanine arginine co-crystal is greater than 99%. The yield is 95.6%. Specific embodiment 3

[0058] The theanine arginine co-crystal obtained in embodiment 2 was characterized by nuclear magnetic resonance hydrogen spectrum (1H-NMR), and the results are shown in Figure 5 The experiment selected D2O as the test solvent. From the nuclear magnetic hydrogen spectrum, the 6 hydrogen atoms of the 3 methylene groups of arginine, the 6 hydrogen atoms of the 3 methylene groups of theanine, and the 3 hydrogen atoms of the methyl group of theanine can be clearly found. The remaining is a small amount of residual reagent peak, and no obvious impurity peak and hydrogen shift peak are found. Therefore, the theanine arginine does not form an ionic salt, but a co-crystal compound. In the theanine arginine co-crystal compound, theanine and arginine exist in a 1:1 molecular ratio, the purity is more than 99%, and the structural formula is shown in Figure 1 . Specific embodiment 4

[0060] The theanine arginine co-crystal obtained in embodiment 2 was characterized by powder diffraction. Powder diffraction is a technique for structural characterization of powder or microcrystalline samples using X-ray, neutron or electron diffraction. When verifying the supramolecular structure by X-ray powder diffraction, first uniformly spread the sample into the sample cell, compactly process, set the wide-angle diffraction-Co target (5-85°), the scanning speed is 8° / min, the scanning time is set to 10 min, and after the scanning is completed, data collection is performed. The results are shown in Figure 2 .

[0061] FromFigure 7 As can be seen, the powder diffraction spectrum of the theanine-arginine co-crystal compound is compared with the diffraction spectrum of the theanine and arginine monomers, a new and strong characteristic peak appears near 2θ = 25-26° on the powder diffraction spectrum of the theanine-arginine co-crystal compound, indicating that the theanine and arginine form a new crystal structure. Specific embodiment 5

[0063] A preferred method for preparing a composition:

[0064] 0.01 mol of the theanine-arginine co-crystal compound and 0.001 mol of ergothioneine were weighed into a beaker, and an appropriate amount of water was added to the beaker and stirred. After dissolving and clarifying, inert gas was introduced into the solution while the mixed solution was pumped through the high-pressure homogenizer pump to the high-pressure homogenizer through the homogenizer valve, and the pressure of the homogenizer valve was set to 800 Bar. After homogenization, the water was removed by rotary evaporation under reduced pressure at a pressure of -0.1 MPa, and the composition was obtained by drying at 60°C. Specific embodiment 6

[0066] The theanine-arginine-ergothioneine composition obtained in Example 5 was characterized by nuclear magnetic resonance hydrogen spectrum (1H-NMR), as shown in Figure 6 , no obvious impurity peak was observed except a small amount of residual reagent peak, and a broad peak appeared at 2.90-3.30 ppm, which was generated by active hydrogen transfer, so the theanine-arginine and ergothioneine formed an ionic salt and interacted with each other. The structural formula of the composition is shown in Figure 2 .

[0067] The theanine-arginine co-crystal compound and the composition were also evaluated for efficacy:

[0068] Specific test example 1

[0069] Transdermal test:

[0070] The theanine-arginine co-crystal compound prepared in Example 1 was formulated into a 20% theanine-arginine co-crystal compound solution (experimental group), and a 10% theanine solution was prepared according to the same amount of substance (control group), and the transdermal effect of the above solutions was tested, and the specific test method was as follows:

[0071] I. The back skin of a piglet was carefully stripped of the subcutaneous fat layer and connective tissue, washed with physiological saline, and placed in physiological saline for standby.

[0072] II. The Franz cell method was used for transdermal experiment, and the exposed skin area in the diffusion cell in the Franz diffusion device was 3.14 cm2, and the receiving chamber volume was 7.5 mL.

[0073] III. Take 400 μL of the prepared experimental group and control group solutions respectively, as the exposed skin surface in the diffusion cell, and add 7.5 mL of physiological saline receiving solution to the receiving cell, and place in a 32±1℃ constant temperature water bath, with a stirring speed of 300 rad / min.

[0074] IV. Subcutaneous sample collection: take 1 mL of the receiving solution at 0.5 h, immediately after sampling, supplement 1 mL of the receiving solution into the receiving chamber, and collect the sample at 2 h.

[0075] V. Treatment of the non-permeated part on the skin: after 2 h (sample) permeation, repeatedly wash the piglet skin surface with physiological saline solution for 3 times, and then fix the volume to 2.0 mL.

[0076] VI. Treatment of the residual part in the skin: after 2 h (sample) permeation, cut the piglet skin into pieces and place in a 2.0 mL EP tube, fix the volume to 2.0 mL with physiological saline, and ultrasonic for 30 minutes;

[0077] VII. After filtration through a 0.22 μm microporous filter, use high performance liquid chromatography evaporative light scattering detector (HPLC-ELSD) to rapidly determine the content of theanine.

[0078] The calculation formula of the cumulative transdermal permeation amount is as follows:

[0079] Qn = CnVn + ΣCi*Vi (i = 1 …… n-1);

[0080] In the formula, Qn is the transdermal permeation amount per unit area, in μg; Cn is the sample concentration measured at the nth sampling point, in μg·mL-1; Vn is the volume of the receiving liquid in the receiving chamber, 7.5 mL, in mL; Ci is the drug concentration in the receiving liquid at the 1st to n-1th sampling; and Vi is the volume of each sampling, 1.0 mL;

[0081] Figure 8 is the broken line graph of the transdermal permeation amount per unit area, from which Figure 8 It can be seen that, at the same concentration, the transdermal absorption test of the theanine arginine co-crystal compound has a theanine unit area permeation amount greater than that of the theanine monomer at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, six different time points, which means that the theanine arginine co-crystal compound has a better bioavailability than the theanine monomer.

[0082] Specific test example 2

[0083] DPPH free radical scavenging:

[0084] The present application tests the antioxidant (DPPH free radical scavenging) efficacy of the theanine monomer, theanine arginine mixture, theanine arginine co-crystal compound, and the composition of the theanine arginine co-crystal compound.

[0085] 1. Principle of the assay: DPPH is a stable free radical in organic solvents, its alcoholic solution is purple, and must be stored in low temperature and dark, so it can accept an electron or hydrogen ion, with a maximum absorption at a wavelength of 517 nm. In the presence of free radical scavengers, the single electron of DPPH is captured, and its color becomes lighter, the absorbance at the maximum absorption wavelength decreases, and the degree of decrease is linear, the decrease in absorbance level indicates the increase in antioxidant activity, thereby evaluating the antioxidant capacity of the test sample. The antioxidant capacity is expressed by the inhibition rate, the greater the inhibition rate, the stronger the antioxidant activity.

[0086] 2. Preparation of DPPH reagent: 0.0600 g of DPPH powder (molecular weight about 394) was accurately weighed and placed in a 250 mL volumetric flask, dissolved with appropriate amount of 95% ethanol to 250 mL, and a concentration of 0.06 mmol / L of DPPH reagent was obtained.

[0087] 3. Preparation of positive control: Vitamin E was dissolved and diluted with 95% ethanol to a series of concentration gradients of 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, 0.01 mg / mL to verify the test system.

[0088] 4. Sample preparation: Theanine, theanine arginine mixture, theanine arginine co-crystal, and the combination of theanine arginine co-crystal compounds were diluted with water to multiple levels of concentration samples of 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.8 mg / mL, 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, etc.

[0089] 5. According to Table 1, using 10 mL test tubes, sample tubes (T), sample background (T0), DPPH tubes (C) and solvent background (C0) were set up, 3 parallel tubes were set up for each sample tube (T) of each test concentration, and 3 parallel tubes were also set up for DPPH tubes (C). In sample tubes (T) and sample background (T0), 1 mL of sample solution of the same concentration was added. In all test tubes (T, T0, C, C0), 3 mL of solvent water was added to make up, and mixed well. In sample tubes (T) and DPPH tubes (C), 1 mL of DPPH ethanol solution was added, and 95% ethanol was used instead of sample background (T0) and solvent background (C0), and gently shaken, and left to stand at room temperature for 5 minutes. Each reaction solution was transferred into a 1 cm cuvette, and the absorbance was measured at 517 nm.

[0090] Table 1: Sample preparation

[0091]

[0092] Table 2: Test data and results

[0093]

[0094] Calculation method:

[0095] In the formula:

[0096] T: Absorbance of the sample tube, i.e., the absorbance of the solution after the sample reacts with DPPH;

[0097] T0: Sample background absorbance;

[0098] C: The average of the three absorbance values ​​of the DPPH tube, i.e., the absorbance value of the DPPH solution without the addition of sample;

[0099] C0: Background absorbance of the solution;

[0100] As shown in Table 2, at the same concentration (0.8 mg / mL), the DPPH radical scavenging rate of the theanine-arginine cocrystal compound composition is higher than that of the theanine-arginine cocrystal and higher than that of theanine monomer. At the same time, the DPPH radical scavenging rate of the theanine-arginine cocrystal compound is higher than that of theanine-arginine mixture.

[0101] Specific Test Example 3

[0102] ABTS+ free radical scavenging:

[0103] 1. Sample preparation:

[0104] Control group: 0.1 g / mL theanine solution stock solution

[0105] Experimental Group 1: 0.1 g / mL theanine-arginine cocrystal compound solution stock solution

[0106] Experimental Group 2: Stock solution of 0.1 g / mL theanine-arginine cocrystal compound

[0107] 2. Based on the sample characteristics and recommended dosage, set an appropriate mass concentration gradient, and prepare sample solutions for testing using PBS buffer as the solvent. Set up sample tubes (A... S ), Sample background (A) b ), sample blank tube (A0), and sample tube (A1) for each test concentration of each sample. S Three parallel tubes are required, and three parallel tubes are also required for the sample blank tube (A0). In the sample tube (A... S ) and sample background (A b Add 0.2 mL of the same concentration of sample solution to each of the sample tubes (A0 and A0), and add 0.2 mL of PBS buffer to the blank sample tube (A0). S) and 0.8 mL ABTS+working solution was added to sample blank tube (A0), sample background (A b ) and 0.8 mL PBS buffer solution was added. The reaction was carried out in dark for 6 min. The solution of each reaction tube was moved into 1 cm cuvette, and the absorbance was measured at 734 nm. The ABTS+radical scavenging rate was calculated according to the measured absorbance. As shown in Table 3:

[0108]

[0109]

[0110] As can be seen from Table 3, at the same concentration, the ABTS+radical scavenging rate of the composition of the theanine arginine co-crystal compound is much higher than that of the theanine arginine co-crystal compound and theanine. And the ABTS+radical scavenging rate of the composition of the theanine arginine co-crystal compound is greater than 50% at a concentration of 1 mg / mL.

[0111] Specific Test Example 4

[0112] Cytotoxicity Test

[0113] 1. Instruments and Reagents

[0114] Instruments: 96-well plate, enzyme-linked immunoassay instrument, cell incubator, inverted phase contrast microscope, centrifuge, 60 mm culture dish, cell culture bottle.

[0115] Reagents: PBS, cell culture medium, 0.25% trypsin, MTT solution, DMSO solution, ascorbic acid.

[0116] 2. Experimental Steps

[0117] 1) Cells were inoculated into a 96-well plate at a density of 1.5 x 104cells / well, 100 μL per well, and incubated for 24 h.

[0118] 2) After the cells were adhered for 24 h, the culture medium was discarded, 100 μL of the sample solution to be tested was added to each well, and a blank group and a zero setting group were set.

[0119] 3) After the cells were incubated for 24 h, 10 μL of MTT solution was added to each well, and the plate was placed in the incubator for 4 h.

[0120] 4) The culture medium was discarded, 100 μL of DMSO was added to each well, and the plate was placed on a shaker for 10-15 min. The absorbance was measured at a wavelength of 570 nm on an enzyme-linked immunosorbent assay instrument. The results were compared with the control sample without the test substance and calculated, as shown in Table 4.

[0121] Table 4: Results of the Effect of Theanine Arginine Composition on Human Skin Fibroblasts

[0122]

[0123] Figure 9 The relative survival rate trend of human skin fibroblasts of the composition of theanine arginine compound is from Figure 9 It can be seen that the composition of theanine arginine compound still has a cell relative survival rate of about 80% at an addition amount of 6 mg / mL, indicating that the composition of theanine arginine compound has good cell compatibility.

[0124] Specific test example 5

[0125] Fibroblast function promoting assay experiment:

[0126] 1. Instruments and reagents

[0127] Instruments: 96-well plate, enzyme-linked immunoassay instrument, cell incubator, inverted phase contrast microscope, centrifuge, 60 mm culture dish.

[0128] 2. Reagents: trypsin, skin fibroblasts, DMSO, PBS solution, MTT solution;

[0129] Experimental group: 10% ethanol aqueous solution of theanine arginine co-crystal compound composition with a mass fraction of 0.1%;

[0130] Control group: 10% ethanol aqueous solution of theanine with a mass fraction of 0.1%.

[0131] 3. Experimental steps

[0132] 1) Cell culture and treatment

[0133] Human skin fibroblasts were inoculated in culture bottles containing an appropriate amount of culture medium and incubated in a 37°C 5% carbon dioxide incubator. The cells were subcultured every 1-2 days and the original culture medium was discarded every 3-4 days when the cells reached 85%-90% confluence. The cells were washed with PBS for 2-3 times and then 0.25% trypsin was added for moderate digestion. After the trypsin was discarded, 10 ml of culture medium was added and the cells were gently blown with a pipette. 5 ml of cell suspension was taken and inoculated in a new culture bottle, which was then incubated in the incubator.

[0134] Take the fibroblasts in the logarithmic growth state, adjust the cell density to 1*106 / ml, inoculate in 96-well plates, culture for 24 h, and after 50%-60% confluence, starve for 12 h to achieve cell synchronization. At the same time, set up zero holes (culture medium, MTT, DMSO), sample holes (cells, experimental sample, culture medium, MTT, DMSO), control holes (cells, control sample, culture medium, MTT, DMSO), and positive control holes (cells, HSF growth factor of the same concentration, culture medium, MTT, DMSO), with 5 parallel holes in each group.

[0135] Incubate in a 37°C 5% carbon dioxide incubator for 16-48 h, and observe under an inverted microscope.

[0136] Add MTT solution to each well, and continue to culture for 4 h.

[0137] Terminate the culture, and carefully aspirate the culture medium in the hole.

[0138] Add DMSO to each well, and shake on a shaker at low speed for 10 min to fully dissolve the crystals. Measure the absorbance OD of each well at 490 nm.

[0139] 2) Result calculation

[0140] Take the average of the absorbance values of the 5 parallel holes in each group, compare the absorbance values between groups, and determine the proliferation-promoting ability of the sample. The experimental data are shown in Table 5.

[0141] Table 5: Absorbance values (OD values) of each hole in different groups

[0142]

[0143]

[0144] The effect of the sample on the MMP-1 level of the cells is shown in Table 5. The OD value of the sample hole is higher than that of the control hole, proving that the composition of the theanine arginine co-crystal compound has a better promotion ability on fibroblast proliferation than theanine, and further proving that the composition of the theanine arginine co-crystal compound containing ergothioneine has a better anti-aging effect.

[0145] Specific test example 6

[0146] Stimulating effect test of the composition.

[0147] In this example, the composition was subjected to human patch test.

[0148] Experimental method:

[0149] Experimental group: Prepare a composition with a mass concentration of 10%.

[0150] Control group: prepare 10% theanine.

[0151] Negative control: blank + filter.

[0152] Select qualified patch test materials, and use the closed patch test method. 0.020g-0.025g of the test substance is placed in the patch test material, and a low-sensitization adhesive tape is applied to the flexor of the forearm of the subject. After 24 hours, the test substance is removed, and the skin reaction is observed at 0.5, 24, and 48 hours after removal, respectively. The results are recorded according to the skin reaction grading standard in (2015 edition). The test results are shown in Table 6 below.

[0153] Table 6: Patch test results summary

[0154]

[0155]

[0156] Skin closed patch test skin reaction grading standard: 0-negative reaction; 1-suspected reaction, only weak erythema; 2-weak positive reaction (erythema reaction); erythema, infiltration, edema, and papules may be present; 3-strong positive reaction (vesicle reaction); erythema, infiltration, edema, papules, and vesicles; the reaction may exceed the test area; 4-strong positive reaction (vesicle reaction); erythema, infiltration, edema, papules, and vesicles; the reaction may exceed the test area.

[0157] As can be seen from Table 6, compared with the same concentration of theanine, 0 cases of adverse reactions occurred in 32 subjects using the composition, indicating that the composition has lower irritation and can be safely applied in daily chemical products.

[0158] The above is only a preferred embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A theanine composition, characterized in that, It contains a co-crystal compound of theanine and arginine; the structural formula of the co-crystal compound of theanine and arginine is shown below: ; The eutectic compound has the XRD pattern shown in Figure 7.

2. The theanine composition according to claim 1, characterized in that: The composition also includes active ingredients.

3. The theanine composition according to claim 2, characterized in that: The active ingredient includes ergothioneine.

4. The theanine composition according to claim 2, characterized in that: The active ingredients include one or more of cysteine, ascorbate glucoside, hydroxytyrosol, glutathione, and VC ethyl ether.

5. A method for preparing the theanine composition according to any one of claims 2 to 4, characterized in that: The method includes the following steps: S1. Provide arginine, and dissolve the arginine in a solvent under heating conditions to obtain an arginine solution; S2. Add excess theanine to the solution obtained in S1 and continue heating and stirring to obtain a mixed solution; S3. Under an inert atmosphere, the reaction was continued by heating to 65℃-75℃, filtered, and then recrystallized at a low temperature to obtain a co-crystal compound of theanine and arginine. S4: The mixed solution of the theanine-arginine cocrystal compound and the active ingredient is pumped to a high-pressure homogenizer using a high-pressure pump. The mixture is fully reacted under inert gas and then concentrated under reduced pressure to obtain the composition.

6. The method for preparing the theanine composition according to claim 5, characterized in that: The solvent includes one or more of methanol, ethanol, isopropanol, N-methylpyrrolidone, tetrahydrofuran, benzene, toluene, petroleum ether, n-hexane, acetone, dioxane, N,N-dimethylformamide, ethyl acetate, or acetonitrile.

7. The method for preparing the theanine composition according to claim 5, characterized in that: The process of filtering in S3 and then recrystallizing at low temperature to obtain the cocrystallized compound of theanine and arginine is specifically as follows: the solution obtained by continuing the reaction after heating is filtered, the solution is concentrated to a supersaturated state under vacuum, and then recrystallized at room temperature. After filtration, washing and drying, the cocrystallized theanine and arginine is obtained.

8. The method for preparing the theanine composition according to claim 5, characterized in that: The active ingredients include one or more of ergothioneine, cysteine, ascorbate glucoside, hydroxytyrosol, glutathione, and VC ethyl ether.

9. The use of the theanine composition according to any one of claims 1 to 4 in the preparation of cosmetics.

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