An adenosine dihydroxysuccinate cocrystal, its preparation method and application

Abstract

The present invention provides a co-crystal of adenosine dihydroxysuccinate, a preparation method thereof, and an application thereof. In the co-crystal of adenosine dihydroxysuccinate, the molar ratio of dihydroxysuccinic acid molecules to adenosine molecules is 1:(1-4). Without changing the structure of adenosine itself, dihydroxysuccinic acid is combined with adenosine to improve the water solubility of adenosine. Adenosine, which is hardly soluble in water, is prepared into a water-soluble co-crystal of adenosine dihydroxysuccinate, and the water solubility of the co-crystal of adenosine dihydroxysuccinate can reach 4%. It not only does not destroy the efficacy of dihydroxysuccinic acid and adenosine monomers, but also the two work synergistically to exert better effects, and has better effects than monomers and simple mixtures in many aspects such as antibacterial, antioxidant, anti-aging, hair care and anti-hair loss, and has good application prospects in skin care and hair care products.

Description

Technical Field

[0001] The present invention relates to the field of eutectic technology and daily chemical industry, and particularly relates to a adenosine dihydroxysuccinate eutectic, a preparation method thereof and an application thereof. Background Art

[0002] Adenosine is an active substance with a basic structure of nucleoside and purine. It is a nucleoside formed by adenine binding to D-ribose through a β-glycosidic bond and is widely present in all types of cells. It can be either a free or a combined nucleoside in nucleic acids. As a natural nucleotide, adenosine is an intermediate product of the body's metabolism and one of the important active ingredients in the body. By participating in the synthesis and degradation of ATP (adenosine triphosphate) and ADP (adenosine diphosphate), it plays a key role in the energy metabolism process in cells and is an important energy carrier in biochemical reactions. Adenosine can inhibit inflammatory reactions and the activation of immune cells, help maintain the body's internal homeostasis, and can also act on vascular smooth muscle to cause vasodilation, thereby affecting the vasodilation and constriction states of blood vessels and regulating blood flow. Due to its important physiological activity, adenosine has important application potential as a nutritional adjuvant in skin care products. However, adenosine has poor solubility, is slightly soluble in water, and it is difficult to add a large amount in the conventional formulations of skin care products. Moreover, it is almost insoluble in ethanol and cannot be solubilized with alcohol, which limits its use scenarios.

[0003] Dihydroxysuccinic acid, also known as tartaric acid, has the chemical formula C4H6O6 and belongs to polybasic organic acids. It naturally exists in various fruits, especially grapes and lemons. Dihydroxysuccinic acid is a white crystal powder or needle-shaped crystal with a typical sour taste and is used in both the food industry and the pharmaceutical field. In the food industry, tartaric acid is commonly used as an acidity regulator, antioxidant, and stabilizer for tartrates, and is also used as a cleaner and detergent. In the pharmaceutical field, dihydroxysuccinic acid and its salts can be used in the preparation of therapeutic drugs, such as the preparation of antacids and injections.

[0004] In recent years, eutectic formation has become a feasible strategy for improving the stability and bioavailability of drug active ingredients. By co-crystallizing the active ingredient with a coformer, a new solid form with unique properties compared to the active ingredient is formed, thereby improving pharmaceutical formulations and enhancing their stability and bioavailability. However, eutectic formation is unpredictable and in fact is not always possible. In addition, there is no way to predict the specific properties of its eutectics before the compounds are formed. Finding suitable coformers and appropriate conditions to obtain specific eutectics requires a great deal of creative work from researchers.

[0005] Chinese Patent CN117143164A, "An Adenosine Cocrystal and Its Preparation Method and Application", discloses an adenosine cocrystal formed by adenosine and ectoine or nicotinamide in a molar ratio of 1:0.2 - 16.9, which has a higher solubility compared to adenosine. However, the water solubility of the obtained adenosine cocrystal is generally around 1.5%, the application scenarios are still relatively limited, and the beauty effect still needs to be further improved.

[0006] Therefore, it is of great significance to provide an adenosine dihydroxysuccinate cocrystal with high water solubility and excellent beauty effect, as well as its preparation method and application. Summary of the Invention

[0007] In view of the problems of the existing adenosine cocrystals, such as low water solubility, relatively limited application scenarios, and the need for further improvement in beauty effect, the present invention provides an adenosine dihydroxysuccinate cocrystal, its preparation method and application. Without changing the structure of adenosine itself, the water solubility of adenosine is increased, and the adenosine that is hardly soluble in water is prepared into a water-soluble adenosine dihydroxysuccinate cocrystal, so that the water solubility of the adenosine dihydroxysuccinate cocrystal can reach 4%. It not only does not destroy the efficacy of dihydroxysuccinic acid and adenosine monomers, but also the two work synergistically to exert better effects, and has better effects than monomers and simple mixtures in many aspects such as antibacterial, antioxidant, anti-aging, hair care and anti-hair loss, and has good application prospects in skin care and hair care products.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] An adenosine dihydroxysuccinate cocrystal, wherein the molar ratio of dihydroxysuccinic acid molecules to adenosine molecules is 1:(1 - 4).

[0010] Adenosine is a purine nucleoside, a compound formed by connecting the N-9 of adenine and the C-1 of D-ribose through a β-glycosidic bond. In essence, it is an endogenous purine nucleotide, mainly formed by dephosphorylation of adenosine triphosphate, and has always been used as a functional ingredient for dilating blood vessels and promoting blood circulation. When hair follicles become smaller or are in the resting phase, adenosine can emit an energy signal of cAMP, stimulate anti-hair loss factors, and reduce hair loss. Adenosine can not only stimulate hair growth, increase the production rate of FGF-7 factor and hair, but also directly act on dermal papilla cells to increase the growth rate of new hair and existing hair.

[0011] Organic eutectics are crystalline single-phase materials composed of two or more components in a certain stoichiometric ratio, and the components are connected by non-covalent interactions (such as hydrogen bonds, halogen bonds, π-π and charge transfer interactions, etc.). Eutectic engineering provides an opportunity to achieve non-covalent synthesis of functional molecules, and its physicochemical properties are determined by the intermolecular interactions and relative packing structures between donors and acceptors. Eutectic components self-assemble through hydrogen bonds or non-covalent bonds with saturation and directionality (such as van der Waals forces of aromatic hydrocarbons or benzene rings, π-π conjugation and halogen bonds), without the need to break existing covalent bonds. While retaining the function of the active substance itself, it can modify the physicochemical properties of the active substance, such as improving stability, reducing irritation, increasing solubility, and improving bioavailability.

[0012] Dihydroxysuccinic acid is a carboxylic acid that exists in many plants, such as grapes and tamarinds, and is also one of the main organic acids in wine. Dihydroxysuccinic acid has the properties of dissolving cutin and astringency, which helps to promote the activation of skin cells. When combined with adenosine to prepare adenosine dihydroxysuccinate eutectic, it can greatly improve the solubility of adenosine and exert better efficacy.

[0013] Furthermore, the molar ratio of dihydroxysuccinic acid to adenosine in the adenosine dihydroxysuccinate eutectic is 1:1; the eutectic has an X-ray powder diffraction pattern expressed in 2θ angle values, including the following 2θ angle values: 7.44°±0.2°, 10.94°±0.2°, 11.79°±0.2°, 13.70°±0.2°, 14.74°±0.2°, 15.28°±0.2°, 16.39°±0.2°, 18.84°±0.2°; in the infrared spectrum of the eutectic, there are characteristic broad peaks at 2500 cm -1 -3500 cm -1 and characteristic peaks at 1706 cm -1 , 1606 cm -1 , 1063 cm -1 .

[0014] Furthermore, the molar ratio of dihydroxysuccinic acid to adenosine in the adenosine dihydroxysuccinate eutectic is 1:4; the eutectic has an X-ray powder diffraction pattern expressed in 2θ angle values, including the following 2θ angle values: 7.49°±0.2°, 10.79°±0.2°, 11.89°±0.2°, 13.68°±0.2°, 14.94°±0.2°, 15.47°±0.2°, 16.22°±0.2°, 18.87°±0.2°.

[0015] Furthermore, the water solubility of the eutectic is 4%.

[0016] Another object of the present invention is to provide a preparation method for two kinds of adenosine dihydroxysuccinate eutectics.

[0017] A preparation method of the adenosine dihydroxysuccinate cocrystal according to any one of the foregoing, comprising the following steps:

[0018] S1. Mix dihydroxysuccinic acid and adenosine evenly, and dropwise add a solvent to obtain a dihydroxysuccinic acid-adenosine-solvent mixture;

[0019] S2. Ball-mill the dihydroxysuccinic acid-adenosine-solvent mixture described in S1 at 25-35 Hz for 10-30 times, with the single grinding time being 30-60 s and the intermittent time being 30 s; during the intermittent time, start ultrasonic waves with a frequency of 25-45 KHz; after the ball-milling is completed, take out the sample and dry it to obtain the adenosine dihydroxysuccinate cocrystal.

[0020] The ball-milling method can significantly reduce the reaction activation energy, refine the crystal grains, greatly improve the activity of the powder and improve the uniformity of particle distribution. The preparation of cocrystals by the ball-milling method is simple in operation and easy to control. It can induce the interaction between organic molecules through mechanical force, promote the formation of cocrystals, and has the advantages of simple operation, high yield, strong structure regulation ability, high cost-effectiveness, environmental friendliness, rapid synthesis, structural diversity and great application potential.

[0021] Furthermore, the molar ratio of the dihydroxysuccinic acid to adenosine described in S1 is 1:(1-4); the mass ratio of the dihydroxysuccinic acid and adenosine mixture to the solvent described in S1 is 1:(0.01-0.1).

[0022] Preferably, since the ball-milling method can obtain a good mixing effect by adding a small amount of solvent, and to ensure the product yield and grinding effect, the solid-liquid ratio of the solid to the solvent should be controlled at 1 g:(0.01-0.1) g.

[0023] Furthermore, the solvent described in S1 is one or more of water, methanol, ethanol, ether, acetone, and isopropanol.

[0024] Furthermore, the ball-milling frequency described in S2 is 30 Hz, and the single grinding time is 30 s.

[0025] Preferably, setting the frequency to 30 Hz can improve the grinding efficiency. The single grinding time is set to 30-60 s, especially 30 s, the intermittent time is set to 30 s, which can effectively relieve the phenomenon of grinding heat generation, and the grinding times are set to 10-30 times, which can make the reactants be fully ground and promote their full reaction.

[0026] Furthermore, the ultrasonic frequency described in S2 is 40 KHz, and the time is 20 s.

[0027] Preferably, ultrasound and ball milling are carried out alternately, so that the powder can be fully mixed. The ultrasound frequency is selected from 25 to 45 KHz, especially 40 KHz, which can achieve the best mixing effect on substances in powder state. The ultrasound time is set to 20 s, and an interval of 10 s from ball milling is beneficial to the cooling of the material and prevents overheating.

[0028] A preparation method of adenosine dihydroxysuccinate cocrystal according to any one of the foregoing, comprising the following steps:

[0029] Step 1: Mix dihydroxysuccinic acid and adenosine, add water, and stir to form a suspension;

[0030] Step 2: Subject the suspension described in Step 1 to high-pressure homogenization at 50 - 200 bar for 2 - 5 times to obtain a mixed solution;

[0031] Step 3: Keep the mixed solution described in Step 2 under stirring and react at 60 - 80 °C for 4 - 8 h; cool the reacted solution to -10 - 10 °C, stir for crystallization, filter to obtain a solid, and dry it under vacuum to obtain the adenosine dihydroxysuccinate cocrystal.

[0032] The solvent method can rapidly reduce the particle size of solid particles through high-pressure homogenization, promote the uniform mixing of dihydroxysuccinic acid and adenosine, and at the same time, high pressure promotes the contact and reaction of dihydroxysuccinic acid and adenosine, improving the reaction efficiency; keeping stirring and raising the temperature facilitate the uniform heating of dihydroxysuccinic acid and adenosine and full reaction; cooling for crystallization can reduce impurities, thereby improving the purity of the product and consuming less energy. At the same time, stirring for crystallization can control crystal growth, and the obtained product has uniform particle size; since the reaction system is a water system, vacuum heating and drying in Step S5 can improve the drying efficiency, reduce the drying time, and avoid the growth of colonies.

[0033] Further, the molar ratio of dihydroxysuccinic acid to adenosine in Step 1 is 1:(1 - 4), and the mass ratio of the mixture of dihydroxysuccinic acid and adenosine to water is 1:(0.4 - 0.8).

[0034] Preferably, when the solid-to-water ratio of the feed liquid is 1:(0.4 - 0.8), the product yield is relatively high.

[0035] Another object of the present invention is to provide an application of adenosine dihydroxysuccinate cocrystal.

[0036] An application of the above-mentioned adenosine dihydroxysuccinate cocrystal in daily chemical products.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] 1. The adenosine dihydroxysuccinate cocrystal provided by the present invention improves the water solubility of adenosine without changing the structure of adenosine itself. The adenosine that is hardly soluble in water is prepared into the water-soluble adenosine dihydroxysuccinate cocrystal, and the water solubility of the adenosine dihydroxysuccinate cocrystal can stably reach 4%.

[0039] 2. The adenosine dihydroxysuccinate cocrystal provided by the present invention does not destroy the efficacy of dihydroxysuccinic acid and adenosine monomers, and the two synergistically enhance the effect and play a better role. It has better efficacy than monomers and simple mixtures in aspects such as antibacterial, antioxidant, anti-aging, hair care and anti-hair loss, and can be applied to different skin and hair care products.

[0040] 3. For the preparation method of the adenosine dihydroxysuccinate cocrystal provided by the present invention, a good mixing effect can be obtained by adding very little solvent in the ball milling method. The ball milling and ultrasonic act alternately, with high grinding efficiency, small reaction loss, short reaction process time, and high product purity; the solvent method uses water as the solvent, which is green and environmentally friendly, the reaction device is simple, the cost is low, and it is convenient for industrial production. Description of the Drawings

[0041] The present invention is further illustrated by the accompanying drawings, but the embodiments in the drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to the following drawings without creative efforts.

[0042] Figure 1 It is the X-ray powder diffraction pattern of the adenosine dihydroxysuccinate cocrystal in Experimental Example 1 of the present invention.

[0043] Figure 2 It is the infrared spectrum of the adenosine dihydroxysuccinate cocrystal in Experimental Example 1 of the present invention.

[0044] Figure 3 It is the nuclear magnetic resonance hydrogen spectrum (1H-NMR) of the adenosine dihydroxysuccinate cocrystal in Experimental Example 1 of the present invention.

[0045] Figure 4 It is the nuclear magnetic resonance carbon spectrum (13C-NMR) of the adenosine dihydroxysuccinate cocrystal in Experimental Example 1 of the present invention.

[0046] Figure 5 It is the X-ray powder diffraction pattern of the adenosine dihydroxysuccinate cocrystal in Experimental Example 4 of the present invention.

[0047] Figure 6 It is the nuclear magnetic resonance hydrogen spectrum (1H-NMR) of the adenosine dihydroxysuccinate cocrystal in Experimental Example 4 of the present invention.

[0048] Figure 7 It is the X-ray powder diffraction pattern of the substance in Comparative Example 2 of the present invention.

[0049] Figure 8 It is a comparison chart of the X-ray powder diffraction spectra of the adenosine dihydroxysuccinate cocrystal, dihydroxysuccinic acid, and adenosine powder of the present invention.

[0050] Figure 9 Infrared spectrum comparison chart of the adenosine dihydroxysuccinate cocrystal, dihydroxysuccinic acid, and adenosine of the present invention.

[0051] Figure 10 It is a diagram for observing the morphology of hair follicles at different days in the hair growth test of in vitro hair follicle culture of the present invention.

[0052] Figure 11 It is a result diagram of the hair cuticle repair test of the present invention.

[0053] Figure 12 It is a statistical result diagram of the product acceptance evaluation questionnaire of the present invention. Detailed implementation manners

[0054] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention is further illustrated by the following examples. Obviously, the following examples are only a part of the examples of the present invention, rather than all the examples; it should be understood that the embodiments of the present invention are only used to illustrate the technical effects of the present invention, rather than to limit the protection scope of the present invention.

[0055] The raw materials in the examples can all be obtained commercially; unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0056] Example 1: A preparation method of adenosine dihydroxysuccinate cocrystal, comprising the following steps:

[0057] Step 1: Disinfect the reaction kettle with 75% ethanol. After completely drying the reaction kettle, add 1.5009 kg of dihydroxysuccinic acid and 2.6724 kg of adenosine, and add 2.0867 kg of water, and stir to form a suspension;

[0058] Step 2: Pass the suspension described in Step 1 through a high-pressure homogenization valve, with a pressure of 150 bar and 4 circulation times, to obtain a mixed solution;

[0059] Step 3: Transfer the mixed solution described in Step 2 to a stirring crystallization kettle, keep stirring, heat up to 75 °C, and react for 6 h; cool the reacted solution to -5 °C, stir for crystallization to obtain a solid; filter, and place the obtained solid in a vacuum drying oven for drying for 16 h, with a drying temperature of 55 °C, to obtain the adenosine dihydroxysuccinate cocrystal. The molar ratio of dihydroxysuccinic acid to adenosine in the adenosine dihydroxysuccinate cocrystal is 1:1.

[0060] The weighed mass of the adenosine dihydroxysuccinate cocrystal was 3.7261 kg, and the calculated yield was 89.28%.

[0061] The obtained adenosine dihydroxysuccinate cocrystal was subjected to powder X-ray single crystal diffraction test, and the diffraction pattern is shown in Figure 1 . The results showed that in the X-ray powder diffraction pattern (XRD) of the adenosine dihydroxysuccinate cocrystal, characteristic peaks appeared at 2θ angles of approximately 7.44°±0.2°, 10.94°±0.2°, 11.79°±0.2°, 13.70°±0.2°, 14.74°±0.2°, 15.28°±0.2°, 16.39°±0.2°, and 18.84°±0.2°.

[0062] The obtained adenosine dihydroxysuccinate cocrystal was subjected to infrared spectroscopy test, and the infrared spectroscopy test results are as shown in Figure 2 . The results showed that the infrared spectrum of the adenosine dihydroxysuccinate cocrystal had characteristic broad peaks in the range of 2500 - 3500 cm -1 , and characteristic peaks at 1706 cm -1 , 1606 cm -1 , and 1063 cm -1 .

[0063] The obtained adenosine dihydroxysuccinate cocrystal was characterized by nuclear magnetic resonance hydrogen spectrum (1H-NMR) and nuclear magnetic resonance carbon spectrum ( 13 C-NMR), and the NMR spectra are shown in Figure 3 , Figure 4 respectively. Figure 3 The nuclear magnetic resonance hydrogen spectrum showed that through the characterization of nuclear magnetic resonance hydrogen spectrum ( 1 H-NMR), using D2O as the test solvent, 2 hydrogen atoms of dihydroxysuccinic acid and 8 hydrogen atoms of adenosine could be clearly found in the nuclear magnetic resonance hydrogen spectrum; the rest were deuterated reagent peaks, and no obvious impurity peaks were observed, indicating that in the adenosine dihydroxysuccinate cocrystal, dihydroxysuccinic acid and adenosine existed in a molar ratio of 1:1. Figure 4 The nuclear magnetic resonance carbon spectrum showed that through the characterization of nuclear magnetic resonance carbon spectrum ( 13 C-NMR), using D2O as the test solvent, the test results showed that dihydroxysuccinic acid and adenosine in the adenosine dihydroxysuccinate cocrystal existed in a molar ratio of 1:1.

[0064] Example 2: A preparation method of adenosine dihydroxysuccinate cocrystal, comprising the following steps:

[0065] Step 1: Disinfect the reaction kettle with 75% ethanol. After completely drying the reaction kettle, add 1.5009 kg of dihydroxysuccinic acid and 2.6724 kg of adenosine, and add 1.6693 kg of water, and stir to form a suspension;

[0066] Step 2: Pass the suspension described in Step 1 through a high-pressure homogenization valve at a pressure of 100 bar and with 4 cycles to obtain a mixed solution;

[0067] Step 3: Transfer the mixed solution described in Step 2 to a stirring crystallization kettle, keep stirring, heat up to 65 °C, and react for 8 h; cool the reacted solution to 0 °C, stir for crystallization to obtain a solid; filter, place the obtained solid in a vacuum drying oven for drying for 24 h at a drying temperature of 50 °C to obtain the adenosine dihydroxysuccinate cocrystal. The molar ratio of dihydroxysuccinic acid to adenosine in the adenosine dihydroxysuccinate cocrystal is 1:1.

[0068] Weigh the obtained adenosine dihydroxysuccinate cocrystal with a mass of 3.5826 kg, and calculate the yield to be 85.85%.

[0069] Example 3: A preparation method of adenosine dihydroxysuccinate cocrystal, comprising the following steps:

[0070] Step 1: Disinfect the reaction kettle with 75% ethanol. After completely drying the reaction kettle, put 1.5009 kg of dihydroxysuccinic acid and 2.6724 kg of adenosine, add 3.3386 kg of water, and stir to form a suspension;

[0071] Step 2: Pass the suspension described in Step 1 through a high-pressure homogenization valve at a pressure of 120 bar and with 3 cycles to obtain a mixed solution;

[0072] Step 3: Transfer the mixed solution described in Step 2 to a stirring crystallization kettle, keep stirring, heat up to 80 °C, and react for 6 h; cool the reacted solution to 4 °C, stir for crystallization to obtain a solid; filter, place the obtained solid in a vacuum drying oven for drying for 18 h at a drying temperature of 45 °C to obtain the adenosine dihydroxysuccinate cocrystal. The molar ratio of dihydroxysuccinic acid to adenosine in the adenosine dihydroxysuccinate cocrystal is 1:1.

[0073] Weigh the obtained adenosine dihydroxysuccinate cocrystal with a mass of 3.3697 kg, and calculate the yield to be 80.74%.

[0074] Example 4: A preparation method of adenosine dihydroxysuccinate cocrystal, comprising the following steps:

[0075] S1. Mix 0.0750 g of dihydroxysuccinic acid and 0.5345 g of adenosine evenly, and dropwise add 0.02 g of ethanol solution to obtain a hydroxysuccinic acid-adenosine-solvent mixture;

[0076] S2. Place the dihydroxysuccinic acid - adenosine - solvent mixture in a ball - milling jar. Set the motor frequency to 30 Hz, the single - grinding time to 30 s, the intermittent time to 30 s, and the number of grinding times to 20 times. Set that when the ball - milling jar is in the intermittent time, start the ultrasonic device with an ultrasonic frequency of 40 KHz and a time of 20 s. Take out the sample after ball - milling and dry it to obtain the dihydroxysuccinic acid - adenosine cocrystal. The molar ratio of dihydroxysuccinic acid to adenosine in the dihydroxysuccinic acid - adenosine cocrystal is 1:4.

[0077] Weigh the dihydroxysuccinic acid - adenosine cocrystal, and the mass is 0.5492 g. Calculate the yield to be 90.11%.

[0078] Perform powder X - ray single - crystal diffraction (XRD) test on the obtained dihydroxysuccinic acid - adenosine cocrystal. The diffraction pattern is shown in Figure 5 . The results show that in the X - ray powder diffraction pattern of the dihydroxysuccinic acid - adenosine cocrystal, characteristic peaks appear at 2θ angles of 7.49°±0.2°, 10.79°±0.2°, 11.89°±0.2°, 13.68°±0.2°, 14.94°±0.2°, 15.47°±0.2°, 16.22°±0.2°, 18.87°±0.2°.

[0079] Perform nuclear magnetic resonance hydrogen spectrum (1H - NMR) characterization on the obtained dihydroxysuccinic acid - adenosine cocrystal. The NMR characterization pattern is shown in Figure 6 . The results show that through 1H - NMR characterization, D2O is selected as the test solvent. Two hydrogen atoms of dihydroxysuccinic acid and 32 hydrogen atoms on adenosine can be clearly found from the 1H - NMR spectrum. The rest are deuterated reagent peaks, and no obvious impurity peaks are seen. In the dihydroxysuccinic acid - adenosine cocrystal, dihydroxysuccinic acid and adenosine exist in a molar ratio of 1:4.

[0080] Example 5: A preparation method of a dihydroxysuccinic acid - adenosine cocrystal, comprising the following steps:

[0081] S1. Mix 0.1501 g of dihydroxysuccinic acid with 0.2672 g of adenosine evenly, and dropwise add 0.02 g of isopropanol solution to obtain a hydroxysuccinic acid - adenosine - solvent mixture;

[0082] S2. Place the dihydroxysuccinic acid - adenosine - solvent mixture in a ball - milling jar. Set the motor frequency to 30 Hz, the single - grinding time to 30 s, the intermittent time to 30 s, and the number of grinding times to 25 times. Set that when the ball - milling jar is in the intermittent time, start the ultrasonic device with an ultrasonic frequency of 40 KHz and a time of 20 s. Take out the sample after ball - milling and dry it to obtain the dihydroxysuccinic acid - adenosine cocrystal. The molar ratio of dihydroxysuccinic acid to adenosine in the dihydroxysuccinic acid - adenosine cocrystal is 1:1.

[0083] The mass of the adenosine dihydroxysuccinate cocrystal obtained by weighing was 0.3815 g, and the yield was calculated to be 91.42%.

[0084] Comparative Example 1: A method for preparing a substance, comprising the following steps:

[0085] Step 1: Disinfect the reaction kettle with 75% ethanol. After completely drying the reaction kettle, add 1.5009 kg of dihydroxysuccinic acid and 2.6724 kg of adenosine, and add 1.6693 kg of absolute ethanol, and stir to form a suspension;

[0086] Step 2: Pass the suspension described in Step 1 through a high-pressure homogenization valve at a pressure of 120 bar and a circulation number of 3 times to obtain a mixed solution;

[0087] Step 3: Transfer the mixed solution described in Step 2 to a stirring crystallization kettle, keep stirring, and heat up to 65°C. It cannot be dissolved clearly, the dihydroxysuccinic acid and adenosine do not react, and the product cannot be obtained.

[0088] Comparative Example 2: A method for preparing a substance, comprising the following steps:

[0089] S1. Mix 0.1501 g of dihydroxysuccinic acid and 0.2672 g of adenosine evenly, and drop 0.02 g of isopropanol solution to obtain a dihydroxysuccinic acid-adenosine-solvent mixture;

[0090] S2. Grind for 20 minutes; recover the sample to obtain the substance.

[0091] The weighed mass was 0.3279 g, and the yield was calculated to be 78.58%.

[0092] The obtained sample was subjected to powder X-ray single crystal diffraction test, and the diffraction pattern is shown in Figure 7 . The results showed that the characteristic peaks in the X-ray powder diffraction pattern (XRD) of the sample could not correspond to those of the adenosine dihydroxysuccinate cocrystal. The product was not the adenosine dihydroxysuccinate cocrystal. It was speculated that the conditions were inappropriate and the reaction did not occur or was uneven and insufficient.

[0093] The above-mentioned examples and comparative examples were subjected to performance tests, and the experimental test methods were as follows:

[0094] The dihydroxysuccinic acid and adenosine monomers were subjected to powder X-ray single crystal diffraction test, and after obtaining the data, they were compared and analyzed with the adenosine dihydroxysuccinate cocrystal prepared in Experimental Example 1 of the present invention. Among them, the comparative diagrams of the powder XRD diffraction of the adenosine dihydroxysuccinate cocrystal, dihydroxysuccinic acid, and adenosine are as shown in Figure 8 .

[0095] Figure 8The results show that the peaks in the powder X-ray single crystal diffraction pattern of the adenosine dihydroxysuccinate cocrystal are not a simple superposition of the peaks of dihydroxysuccinic acid and adenosine monomers. This result indicates that a new cocrystal of dihydroxysuccinic acid and adenosine is formed using the preparation method of the present invention.

[0096] The infrared spectra of dihydroxysuccinic acid and adenosine monomers were tested. After obtaining the data, comparative analysis was carried out with the adenosine dihydroxysuccinate cocrystal. Among them, the infrared spectra comparison diagrams of the adenosine dihydroxysuccinate cocrystal, dihydroxysuccinic acid, and adenosine are as Figure 9 shown.

[0097] Figure 9 The results show that the peaks in the infrared spectrum of the adenosine dihydroxysuccinate cocrystal are not a simple superposition of the peaks of dihydroxysuccinic acid and adenosine monomers. Especially in the range of 2500 - 3500 cm -1 there are characteristic broad peaks, proving the formation of hydrogen bonds in the system. This result indicates that a new cocrystal of dihydroxysuccinic acid and adenosine is formed.

[0098] (1)Study on the water solubility test of adenosine dihydroxysuccinate cocrystal

[0099] The adenosine dihydroxysuccinate cocrystals obtained in Experimental Examples 1 - 5 were dissolved in water and prepared into supersaturated solutions under magnetic stirring. Samples were taken, filtered, and the content of the adenosine dihydroxysuccinate cocrystal was detected by high performance liquid chromatography. The test results are shown in Table 1.

[0100] Table 1 Results of the content determination of adenosine dihydroxysuccinate cocrystal by high performance liquid chromatography

[0101]

[0102] The test results in Table 1 show that the water solubilities of the adenosine dihydroxysuccinate cocrystals obtained in Experimental Examples 1 - 5 are all above 4%, much higher than the water solubility of adenosine, indicating that the water solubility of adenosine is greatly improved after forming a cocrystal with dihydroxysuccinic acid.

[0103] (2)Study on the antibacterial ability test of adenosine dihydroxysuccinate cocrystal against Propionibacterium acnes

[0104] The experimental materials used in this example are: the adenosine dihydroxysuccinate cocrystal obtained in Experimental Example 1, adenosine, and a physical mixture of dihydroxysuccinic acid and adenosine prepared at a molar ratio of 1:1. The antibacterial abilities of the above samples against Propionibacterium acnes were detected. According to the suspension quantitative method, after the test samples were mixed with the test bacteria for a specified time, the total number of colonies was measured and compared with the control group to calculate the antibacterial rate to evaluate the antibacterial effect. The results of the samples on the test bacteria are shown in Table 2. The specific experimental steps refer to QB / T 2738 - 2012 "Evaluation Method for Antibacterial and Bacteriostatic Effects of Daily Chemical Products".

[0105] Table 2 Results of the action of the samples on the test bacteria

[0106]

[0107] The test results in Table 2 show that after 5 minutes of action at a test concentration of 0.5%, the adenosine dihydroxysuccinate cocrystal obtained in Example 1 has an antibacterial rate of 99.75% against Propionibacterium acnes and has a strong antibacterial effect on Propionibacterium acnes. After 5 minutes of action at a test concentration of 0.5%, the physical mixture of adenosine dihydroxysuccinate has an antibacterial rate of 27.69% against Propionibacterium acnes and has no antibacterial effect on Propionibacterium acnes. After 5 minutes of action at a test concentration of 0.32%, adenosine has an antibacterial rate of 43.08% against Propionibacterium acnes and has no antibacterial effect on Propionibacterium acnes. At an equimolar test concentration, neither the adenosine dihydroxysuccinate cocrystal, adenosine, nor the physical mixture of adenosine dihydroxysuccinate has antibacterial effects, but the adenosine dihydroxysuccinate cocrystal has a strong antibacterial efficacy, demonstrating its application potential in acne removal products.

[0108] (3)Test study on the ability of adenosine dihydroxysuccinate cocrystal to scavenge free radicals (DPPH)

[0109] 1,1-Diphenyl-2-picrylhydrazyl (abbreviated as DPPH) is a stable long-lived free radical. Its ethanol solution is dark purple and has a strong absorption near 517 nm. When a free radical scavenger is present, the light absorption of the DPPH ethanol solution weakens due to the pairing of its single electron. The degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts, and thus the ability of the test sample to scavenge free radicals, that is, the magnitude of its antioxidant activity, can be evaluated.

[0110] The detection method uses T / SHRH 006-2018 "Cosmetics - Free Radical (DPPH) Scavenging Experiment Method". Set up sample tubes (T), sample backgrounds (T0), DPPH tubes (C), and solvent backgrounds (C0). For each sample tube (T) at each test concentration of each sample, 3 parallel tubes need to be set up. Add 1 mL of the sample solution with the same concentration to each of the sample tube (T) and the sample background (T0). Add the sample solvent to the sample background (T0) and the solvent background (C0) to make up 2 mL, and mix well. Add 1 mL of the DPPH ethanol solution to the sample tube (T) and the DPPH tube (C), shake gently, and let stand at room temperature for 5 minutes. Transfer the solution in each reaction tube into a 1 cm cuvette and measure the absorbance at 517 nm.

[0111]

[0112] The experimental materials used in this test example are: the adenosine dihydroxysuccinate cocrystal obtained in Experimental Example 1, adenosine, dihydroxysuccinic acid, and the adenosine dihydroxysuccinate physical mixture prepared by mixing dihydroxysuccinic acid and adenosine at a molar ratio of 1:1. The results of the scavenging rates of the adenosine dihydroxysuccinate cocrystal, adenosine, dihydroxysuccinic acid, and the adenosine dihydroxysuccinate physical mixture on DPPH radicals are shown in Tables 3 to 6.

[0113] Table 3 Scavenging Rate of Adenosine Dihydroxysuccinate Cocrystal on DPPH Radical

[0114]

[0115] Table 4 Scavenging Rate of Adenosine on DPPH Radical

[0116]

[0117] Table 5 Scavenging Rate of Dihydroxysuccinic Acid on DPPH Radical

[0118]

[0119] Table 6 Scavenging Rate of Adenosine Dihydroxysuccinate Physical Mixture on DPPH Radical

[0120]

[0121] Statistical method in Tables 3 to 6: The t-test method was used for analysis, and the test level α = 0.05; P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference. The statistical methods used in the following test examples are the same as those in this test example.

[0122] The results in Tables 3 to 6 show that due to their solubility limitations, adenosine and the adenosine dihydroxysuccinate physical mixture cannot increase the concentration and have almost no scavenging effect on DPPH radicals within the experimental concentration range, and do not have antioxidant ability. As the concentration of dihydroxysuccinic acid increases, the scavenging rate of DPPH radicals no longer increases and remains at about 15%. The adenosine dihydroxysuccinate cocrystal improves solubility, has a higher concentration than adenosine and the adenosine dihydroxysuccinate physical mixture, and the scavenging rate of DPPH radicals shows a concentration dependence. As the content increases, the scavenging rate increases, far higher than the scavenging rate of dihydroxysuccinic acid, and has the best antioxidant effect.

[0123] (4)Study on the Scavenging Ability of Adenosine Dihydroxysuccinate Cocrystal on Free Radicals (ABTS + )

[0124] In the presence of an oxidant, ABTS is oxidized to ABTS +Free radicals, the solution will turn green and have a strong absorption at a wavelength of 734 nm in the ultraviolet range. When an antioxidant is added to the system, the generation amount of ABTS + will decrease, the color of the solution will become lighter, gradually changing from dark green to light green, and the absorbance at 734 nm will become smaller. Based on this, the ABTS + free radical scavenging rate of this substance is determined.

[0125] Detection method reference: Ma Qingling. Research on the ultrasonic-assisted extraction of protein polypeptides from Uyghur medicine celery seeds [J]. Journal of Anhui Agricultural Sciences, 2011, 39(18): 3. Set appropriate mass concentration gradients according to the sample characteristics, and prepare sample solutions to be measured using PBS buffer as the solvent. Set up sample tubes (A S ), sample backgrounds (A b ), and sample blank tubes (A0). For each sample tube (A S ) at each test concentration of each sample, 3 parallel tubes need to be set up, and at the same time, 3 parallel tubes also need to be set up for the sample blank tube (A0). Add 0.2 mL of the same concentration of sample solution to the sample tubes (A S ) and sample backgrounds (A b ), and add 0.2 mL of PBS buffer to the sample blank tube (A0). Add 0.8 mL of ABTS S working solution to the sample tubes (A + ) and sample blank tubes (A0), and add 0.8 mL of PBS buffer to the sample background (A b ). React in the dark for 6 min. Transfer the solutions in each reaction tube into 1 cm cuvettes and measure the absorbance at 734 nm.

[0126]

[0127] The experimental materials used in this test example are: the adenosine dihydroxysuccinate cocrystal, adenosine, dihydroxysuccinic acid, and the physical mixture of adenosine dihydroxysuccinate prepared by mixing dihydroxysuccinic acid and adenosine at a molar ratio of 1:1 obtained in Experimental Example 1. The results of the ABTS + free radical scavenging rates of the adenosine dihydroxysuccinate cocrystal, adenosine, dihydroxysuccinic acid, and the physical mixture of adenosine dihydroxysuccinate are shown in Tables 7 to 10.

[0128] Table 7 ABTS + free radical scavenging rate of adenosine dihydroxysuccinate cocrystal

[0129]

[0130] Table 8 ABTS + free radical scavenging rate of adenosine

[0131]

[0132] Table 9 Scavenging rate of dihydroxysuccinic acid on ABTS + radical

[0133]

[0134] Table 10 Scavenging rate of physical mixture of adenosine and adenosine dihydroxysuccinate on ABTS + radical

[0135]

[0136] Statistical method in Tables 7 - 10: Analysis was performed using the t - test method, with the test level α = 0.05; P≥0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates significant difference; P < 0.01 indicates very significant difference; P < 0.001 indicates extremely significant difference.

[0137] The results of Tables 7 - 10 show that due to solubility limitations, the physical mixture of adenosine and adenosine dihydroxysuccinate cannot increase the concentration and has almost no scavenging effect on ABTS + radical within the experimental concentration range and has no antioxidant ability. Dihydroxysuccinic acid has no scavenging effect on ABTS + radical. However, the cocrystal of adenosine dihydroxysuccinate improves solubility, has a higher concentration than the physical mixture of adenosine and adenosine dihydroxysuccinate, and has a high scavenging rate for ABTS + radical and has good antioxidant effect.

[0138] (5) Study on the inhibitory ability test of adenosine dihydroxysuccinate cocrystal on elastase activity

[0139] Elastase (human leukocyte elastase, HLE) is a destructive enzyme present in the body that can hydrolyze certain connective tissue components such as elastin, proteoglycans, and certain types of collagen. Elastase can specifically hydrolyze the substrate, and the absorbance value of p - nitroaniline (NA) decomposed at 382 nm is measured by spectrophotometry to calculate the enzyme activity and inhibition rate.

[0140] Detection method reference: Chen Liping, Yin Xiangli, Fang Yuchun, Hao Lijie, Zhao Feng. Establishment of a screening method for elastase inhibitors and observation of the inhibitory activity of natural products against it [J]. Journal of Yantai University (Natural Science and Engineering Edition), 2011, 24(03): 209-213. Set up sample tubes (A1), sample background tubes (A2), enzyme activity control groups (A3), and substrate blank groups (A4). For each tested concentration of the sample, 3 parallel tubes of sample tubes (A1) need to be set up. Add 25 μL of the sample solution to the sample tubes (A1) and sample background tubes (A2) respectively, and add the same volume of the sample solvent (PBS buffer or 10% DMSO) to replace the sample in the enzyme activity control groups (A3) and substrate blank groups (A4). Add 225 μL of elastase solution to each tube in turn. After mixing, heat in a water bath at 37 °C for 5 min, then add 250 μL of the substrate solution to the sample tubes (A1) and enzyme activity control groups (A3) respectively, and add the same volume of 10% DMSO solution to the sample background tubes (A2) and substrate blank groups (A4). After mixing, heat in a water bath at 37 °C for 30 min, and measure the absorbance at a wavelength of 382 nm.

[0141]

[0142] The experimental materials used in this test example are: the adenosine dihydroxysuccinate cocrystal obtained in Experimental Example 1, adenosine, and the adenosine dihydroxysuccinate physical mixture prepared by mixing adenosine and dihydroxysuccinate at a molar ratio of 1:1. The results of the inhibition rates of the adenosine dihydroxysuccinate cocrystal, adenosine, dihydroxysuccinate, and adenosine dihydroxysuccinate physical mixture against elastase are shown in Tables 11 to 13.

[0143] Table 11 Inhibition rate of adenosine dihydroxysuccinate cocrystal against elastase

[0144]

[0145] Table 12 Inhibition rate of adenosine against elastase

[0146]

[0147] Table 13 Inhibition rate of adenosine dihydroxysuccinate physical mixture against elastase

[0148]

[0149] Statistical methods in Tables 11 to 13: Analysis was performed using the t-test method, with a test level of α = 0.05; P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference.

[0150] The results in Tables 11 to 13 show that due to its solubility limitation, the physical mixture of adenosine and adenosine dihydroxysuccinate cannot increase the concentration and has almost no inhibition of elastase within the experimental concentration range, thus not having a firming effect. However, the adenosine dihydroxysuccinate cocrystal improves solubility and can achieve an inhibition rate of more than 90% against elastase within the test range, showing a good firming and anti-aging effect.

[0151] (6)Study on the inhibitory ability of adenosine dihydroxysuccinate cocrystal against 5α-reductase

[0152] 5α-reductase is a membrane protease that depends on nicotinamide adenine dinucleotide phosphate (NADPH) and is an important androgen-metabolizing enzyme in the skin. It can irreversibly convert testosterone (T) into dihydrotestosterone (DHT). DHT is the most active androgen and can induce the sebaceous glands to secrete excessive sebum. By inhibiting the activity of 5α-reductase to reduce the level of DHT, the excessive secretion of sebum by the sebaceous glands can be effectively alleviated.

[0153] Detection method: Weigh the sample accurately, pretreat the sample for later use. Add reagents to the well plate for pre-reaction at 37°C in an incubator for 30 min. After the pre-reaction is completed, add the enzyme reagent and continue the reaction at 37°C in the incubator for 30 min. First, add the chromogenic agent to each well, develop color in the dark at 37°C in the incubator, add the termination solution to terminate the reaction. Measure the absorbance of each well by machine, record the data, and calculate the inhibition rate.

[0154] The experimental material used in this test example is the adenosine dihydroxysuccinate cocrystal obtained in Experimental Example 1. The results of the inhibition rate of the adenosine dihydroxysuccinate cocrystal against 5α-reductase are shown in Table 14.

[0155] Table 14 Inhibition rate of adenosine dihydroxysuccinate cocrystal against 5α-reductase

[0156]

[0157] The results in Table 14 show that the sample of adenosine dihydroxysuccinate cocrystal has a good inhibitory effect on 5α-reductase at a test concentration of 1%, indicating that this sample has a good oil control effect. Thus, it can be seen that the adenosine dihydroxysuccinate cocrystal of the present invention has high application potential in oil control, acne treatment, and products for seborrheic alopecia.

[0158] (7)Study on the expression level of TGF-β2 gene in human keratinocytes by adenosine dihydroxysuccinate cocrystal

[0159] Transforming growth factor β2 is a multifunctional protein that affects cell growth, differentiation, and apoptosis in the skin. Studies have shown that alopecia areata is related to the autoimmune mechanism, and the expression of TGF-β2 is closely related to the pathogenesis of alopecia areata. The anti-hair loss efficacy of a sample can be reflected by detecting the TGF-β2 gene expression.

[0160] Reference for the detection method: [1] Danielpour D, Dart L L, Flanders K C, et al. Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) secreted by cells in culture. [Journal of Cellular Physiology, 2010, 138(1): 79-86.

[0161] Zhang Ruzhi, Zhu Wenyuan, Xie Fang, et al. Effects of nicotinamide on the secretion of TGF-β1 and the expression of CXCR2 in keratinocytes after ultraviolet irradiation [J]. Chinese Journal of Dermatology and Venereology, 2008, 22(3): 3.

[0162] Wang Guangbin. Study on the effects of oleanolic acid on the expression of VEGF and TGF-β1 in the skin lesions of androgenetic alopecia mice and its therapeutic effect [D]. Luzhou Medical College, 2014.

[0163] Experimental steps: (1) Cell seeding: Inoculate the human keratinocyte suspension into a 24-well plate and incubate for 24 h.

[0164] Prepare the solution: Prepare the working solution of the test substance.

[0165] Add the drug: Divide into groups and add the drug, and culture for 24 h.

[0166] Test: Collect the RNA of each group and perform fluorescence quantitative PCR to detect the expression of the TGF-β2 gene.

[0167] The experimental materials used in this test example are: the adenosine dihydroxysuccinate co-crystal obtained in Experimental Example 1, and the adenosine dihydroxysuccinate physical mixture prepared by mixing adenosine dihydroxysuccinate and adenosine at a molar ratio of 1:1. The detection results of the TGF-β2 gene expression and the inter-group statistical results of the above samples are shown in Tables 15 to 16.

[0168] Table 15 Detection results of the TGF-β2 gene expression by different samples

[0169]

[0170] Table 16 Statistical results between groups

[0171]

[0172] Statistical methods in Tables 15 - 16: The t - test method was used for analysis, and the significance level α = 0.05; P≥0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates significant difference; P < 0.01 indicates very significant difference; P < 0.001 indicates extremely significant difference. Compared with the NC group, 0.01 < P < 0.05 is expressed as , P < 0.01 is expressed as , P < 0.001 is expressed as .

[0173] The results of Tables 15 - 16 show that at the same concentration, the adenosine dihydroxysuccinate cocrystal has the ability to inhibit TGF - β2 secretion at concentrations of 0.001% and 0.0005%, and is superior to the physical mixture of adenosine dihydroxysuccinate (P < 0.05). Then, at this concentration, the anti - hair - loss efficacy of adenosine dihydroxysuccinate cocrystal > physical mixture of adenosine dihydroxysuccinate.

[0174] (8) Study on the hair - growth promoting effect of adenosine dihydroxysuccinate cocrystal in in vitro hair follicle culture

[0175] Based on the in vitro hair follicle culture for hair - growth experiment, the promoting effects of different samples on hair growth were compared; the hair - growth cycle of mouse vibrissae is synchronized with their age, and the vibrissa hair follicles isolated in vitro can be maintained in culture for up to 23 days. Using the organ - culture method to evaluate hair follicle growth is considered to have a high correlation with the in - vivo system because the degree of hair growth can be observed as the sum of each cell's function. An in vitro model was used to study the promoting effect of samples on hair.

[0176] Reference for detection method: [1] Fan Weixin, Zhu Wenyuan, Lei Tiechi. Study on in vitro culture of mouse vibrissa hair follicles[J]. Chinese Journal of Dermatology, 1999(01):28 - 30.

[0177] [2] lida M, lhara S, Matsuzaki T. Hair cycle - dependent changes of alkaline phosphatase activity in the mesenchyme and epithelium in mouse vibrissal follices[J]. Development, growth & differentiation vol, 2007(04):185 - 195.

[0178] [3] Ohn, J., Kim, K.H.,&Kwon, O. Evaluating hair growth promoting effects of candidate substance :A review of research methods[Jl. Journal of dermatological science, 2019(03):144-149.

[0179] [4] Kang JI,Kim SC, Kim MK, Boo Hj, Jeon YJ Koh Ys, Yoo ES, Kang SM,Kang HK. Effects of Dieckola component of Ecklonia cave, on the promotion of hair growth[J]. International joumal of molecular science,2012(5):6407-6423.

[0180] [5]Yamauchi, Koichi, and Akira Kurosaka, Inhibition of glycogen synthase kinase-3 enhances the experession of alkaline phosphatase and insulin-like growth factor -1 in human primary dermaml papilla cell culture and maintains mouse hair bulbs in organ culture[J]. Archives of dermatological research2019(02):357-365.

[0181] Experimental steps: (1) Incubate the isolated hair follicles in a culture medium containing 1 mL per well at 37°C and 5% CO2 for 14 days.

[0182] Solution preparation: Prepare the working solution of the test substance.

[0183] Drug addition: Divide the groups and add the drug, changing the culture medium every 3 days.

[0184] Image analysis: Take pictures of the vibrissa follicle length every 7 days, and observe and measure the vibrissa follicle length morphologically.

[0185] Detection: After collecting the vibrissa follicles of mice on the 15th day of culture, the follicles in each group were transferred to cryotubes, PBS was added, and they were kept in a -80 °C refrigerator for standby. After the specimens were thawed, they were kept at 2 °C - 8 °C, PBS was added, the follicles were ground by hand, and the cells were broken by repeated freezing and thawing, centrifuged, and the supernatant was carefully collected. The ELISA method was used to detect the content of VEGF in the specimens.

[0186] The experimental materials used in this test example were: the adenosine dihydroxysuccinate co-crystal obtained in Experimental Example 1, and the adenosine dihydroxysuccinate physical mixture prepared by mixing adenosine dihydroxysuccinate and adenosine at a molar ratio of 1:1. Among them, the detection results of follicle growth and the statistical results between groups for different samples are shown in Tables 17 and 18, the detection results of growth rates and the statistical results between groups for different samples are shown in Tables 19 and 20, and the detection results of VEGF content and the statistical results between groups for different samples are shown in Tables 21 and 22. The observation results of follicle morphology at different days under the action of the same concentration (0.025 mg / mL) are as Figure 10 shown.

[0187] Table 17 Detection results of follicle growth for different samples

[0188]

[0189] Table 18 Statistical results between groups

[0190]

[0191] Table 19 Detection results of growth rates for different samples

[0192]

[0193] Table 20 Statistical results between groups

[0194]

[0195] Table 21 Detection results of VEGF content for different samples

[0196]

[0197] Table 22 Statistical results between groups

[0198]

[0199] Statistical method in Tables 17 - 22: The t-test method was used for analysis, and the test level α = 0.05; P ≥ 0.05 indicates no statistical difference; 0.01 < P < 0.05 indicates a significant difference; P < 0.01 indicates a very significant difference; P < 0.001 indicates an extremely significant difference. Compared with the NC group, 0.01 < P < 0.05 is expressed as , P<0.01 is indicated as , P<0.00l is indicated as .

[0200] Table 17~Table 22 and Figure 10 The results showed that in the in vitro hair follicle culture hair growth test, at the same concentration (0.025 mg / mL), the length growth, growth rate and VEGF content of the dihydroxyadenosine eutectic group were better than those of the dihydroxyadenosine physical mixture (P<0.05). At this concentration, the hair growth promoting effect of the dihydroxyadenosine eutectic was greater than that of the dihydroxyadenosine physical mixture.

[0201] (9) In vitro test on combing power, tensile strength and hair scale repair of real hair bundles

[0202] The scales of normal hair are arranged from the root to the end like fish scales from the head to the tail. When the hair is damaged, the tail end of the scales will lift up and detach from the hair. The greater the lift, the greater the opening and closing of the scales. In severe cases, the scales may fall off. When the scales lift up and open or fall off, the hair becomes coarse and loses its luster. The factors that affect the combability of a single hair include the combing coefficient between hair fibers and between hair fibers and combs, hair diameter, hair rigidity, and the presence of static charges. Hair products can make hair easier to comb by changing the combing coefficient, oil, and adhesion of the hair.

[0203] Hair Care Test Method:

[0204] Baseline dry hair test: The hair to be tested is fully moistened with running constant temperature water, treated with cleaning solution, and placed in a constant temperature and humidity chamber (20~25℃, 40%~60%) for more than 4 hours. It is then taken out and fixed in the test position for a combing cycle test.

[0205] Sample dry hair test: The hair to be tested is fully moistened with running constant temperature clean water, and the sample is evenly applied on the surface of the hair at a rate of 0.2g per gram of hair. The application time is about 30s, and it is allowed to stand for about 3-5min. After rinsing, it is placed in a constant temperature and humidity chamber (20~25℃, 40%~60%) for more than 4 hours, and then taken out and fixed in the test position for combing cycle test.

[0206] Record the combing work values ​​of the hair bundle tested by CTX texture analyzer before and after using the sample and perform data processing.

[0207] Anti-hair breakage test method:

[0208] (1) Test before using the sample: The hair strand to be tested is fully moistened with running constant temperature clean water, treated with cleaning solution, placed in a constant temperature and humidity chamber [(26±2)℃, (60±10)%RH] to dry naturally for 24 hours, and then a single-strand tensile test is performed, and the test data of each time is recorded.

[0209] (2) Post - use testing of the sample: The hair strands to be tested are rinsed with flowing constant - temperature clear water or not rinsed according to the sample usage method. The sample is uniformly applied on the hair strand surface at a dosage of 0.2 g of the sample per gram of hair strand or in accordance with the sample usage method. After staying for a period of time, then rinse and blot the moisture on the hair strand surface with a soft and clean facial tissue or do not rinse. Place the ex - vivo hair in a constant - temperature and humidity chamber [(26 ± 2)°C, (60 ± 10)%RH] and let it air - dry naturally for 24 hours, then conduct a single - strand tensile test and record the data of each test.

[0210]

[0211] Where: C0: Combing work (tensile strength) of the baseline test;

[0212] Cs: Combing work (tensile strength) of the sample - added treatment test

[0213] Using the adenosine dihydroxysuccinate cocrystal obtained in Experimental Example 1 as the active ingredient, an adenosine dihydroxysuccinate cocrystal shampoo is prepared. This method measures the combability of the hairpiece before and after using the sample through equipment, and uses the degree of reduction in combability to measure the efficacy of the sample in improving combability. The descriptive statistical results of the changes in combing work and tensile strength before and after using the sample are shown in Tables 23 - 24. The result graph of the hair cuticle repair test is as Figure 11 shown.

[0214] Table 23 Descriptive statistical table of the change rate of combing work before and after using the sample

[0215]

[0216] Table 24 Descriptive statistical table of the change rate of tensile strength before and after using the sample

[0217]

[0218] Tables 23 - 24 and Figure 11 The results show that: compared with before use, the combing work of the hair strands decreases, the tensile strength increases, the hair strands are more compliant, tough, the degree of hair cuticle opening and closing decreases, and there is an obvious improvement in the degree of hair cuticle opening and closing.

[0219] (10)Research on the evaluation test of adverse reactions of cosmetic skin irritation

[0220] Using the adenosine dihydroxysuccinate cocrystal obtained in Experimental Example 1 as the active ingredient, an adenosine dihydroxysuccinate cocrystal shampoo was prepared. Qualified patch test equipment was selected. By the closed patch test method, 0.020 g to 0.025 g of the test substance was placed in the patch test equipment and externally applied to the flexor side of the forearm of the subject with a low-sensitization tape. After 24 hours, the test substance was removed, and the skin reactions were observed at 0.5, 24, and 48 hours after removal respectively. The results were recorded according to the skin reaction grading standard in the "Technical Specifications for Cosmetics Safety" (2015 Edition). The results of the human skin closed patch test are shown in Table 25.

[0221] Table 25 Summary of the Results of the Human Skin Patch Test

[0222]

[0223] The results of the human skin closed patch test of the adenosine dihydroxysuccinate cocrystal shampoo showed that 0 out of 30 people had skin adverse reactions.

[0224] (11) Consumer Product Acceptance Evaluation Test Research

[0225] Using the adenosine dihydroxysuccinate cocrystal obtained in Experimental Example 1 as the active ingredient, an adenosine dihydroxysuccinate cocrystal shampoo was prepared. After 14 days and 28 days of using the test product, the subjects evaluated the acceptance of the product-related indicators. The higher the percentage, the more people agreed with the indicator. The statistical results of the product acceptance evaluation questionnaire are as Figure 12 shown.

[0226] Figure 12 The results showed that after 14 days and 28 days of using the test product, the subjects had a high acceptance in terms of skin feel, oil control, and anti-hair loss. The overall evaluation was that they were satisfied with the product as a whole, and there were significant differences.

[0227] In summary, without changing the structure of adenosine itself, the present invention improves the water solubility of adenosine, prepares the poorly water-soluble adenosine into the water-soluble adenosine dihydroxysuccinate cocrystal, and the water solubility of the adenosine dihydroxysuccinate cocrystal can reach 4%. It not only does not destroy the efficacy of dihydroxysuccinic acid and adenosine monomers, but also the two work synergistically to achieve better effects. It has better effects than the monomers and simple mixtures in many aspects such as antibacterial, antioxidant, anti-aging, hair care and anti-hair loss, and has good application prospects in skin care and hair care products.

[0228] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A dihydroxysuccinate adenosine cocrystal, characterized in that: The molar ratio of dihydroxysuccinic acid to adenosine in the dihydroxysuccinic acid adenosine cocrystal is 1:1; the cocrystal has an X-ray powder diffraction pattern represented by 2θ angle values, comprising the following 2θ angle values: 7.44°±0.2°, 10.94°±0.2°, 11.79°±0.2°, 13.70°±0.2°, 14.74°±0.2°, 15.28°±0.2°, 16.39°±0.2°, 18.84°±0.2°; in the infrared spectrum of the cocrystal, at 2500 cm -1 -3500 cm -1 There is a characteristic broad peak at 1706 cm -1 , 1606 cm -1 , 1063 cm -1 The invention relates to a method for preparing an aqueous solution of at least one hydroxyadenosine dihydroxysuccinate cocrystal and a cocrystal comprising: providing a characteristic peak at a position; or the molar ratio of dihydroxysuccinic acid to adenosine in the dihydroxysuccinic acid adenosine cocrystal is 1:4; the cocrystal has an X-ray powder diffraction pattern expressed as 2θ angle values, comprising the following 2θ angle values: 7.49°±0.2°, 10.79°±0.2°, 11.89°±0.2°, 13.68°±0.2°, 14.94°±0.2°, 15.47°±0.2°, 16.22°±0.2°, 18.87°±0.2°.

2. A method for preparing adenosine dihydroxysuccinate cocrystal according to claim 1, characterized in that: The following steps are involved: S1. mixing dihydroxysuccinic acid and adenosine uniformly, and adding a solvent dropwise to obtain a dihydroxysuccinic acid-adenosine-solvent mixture; S2. The dihydroxysuccinic acid-adenosine-solvent mixture of S1 is ball-milled at 25-35 Hz for 10-30 times, with a single grinding time of 30-60 s and an interval time of 30 s; during the interval time, ultrasound is started at a frequency of 25-45 KHz; after the ball milling is completed, the sample is taken out and dried to obtain the dihydroxysuccinic acid adenosine eutectic; The molar ratio of dihydroxysuccinic acid to adenosine in S1 is 1:1 or 1:4; the mass ratio of the mixture of dihydroxysuccinic acid and adenosine in S1 to the solvent is 1:(0.01-0.1); S1 The solvent is one or more of water, methanol, ethanol, ether, acetone, and isopropanol.

3. The method for preparing the dihydroxysuccinate adenosine cocrystal according to claim 1, characterized in that: The following steps are involved: Step 1, mixing dihydroxysuccinic acid and adenosine, adding water, and stirring to form a suspension; Step 2: homogenize the suspension in step 1 at 50-200 bar high pressure for 2-5 times to obtain a mixed solution; Step 3, keep stirring the mixed solution in step 2, and react at 60-80°C for 4-8h; cool the solution after the reaction to -10-10°C, stir and crystallize, filter to obtain a solid, and vacuum dry to obtain the dihydroxysuccinic acid adenosine cocrystal; In step 1, the molar ratio of dihydroxysuccinic acid to adenosine is 1:1 or 1:4, and the mass ratio of the mixture of dihydroxysuccinic acid and adenosine to water is 1:(0.4-0.8).

4. The adenosine dihydroxysuccinate cocrystal according to claim 1, characterized in that The water solubility of the co-crystal is 4%.

5. Use of the dihydroxyadenosine succinate cocrystal according to claim 1 in daily chemical products.

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