An egcg-niacin co-crystal, and a preparation method and application thereof

Abstract

The application discloses an EGCG-nicotinic acid co-crystal as well as a preparation method and application thereof, and belongs to the technical field of drug co-crystals. The method comprises the following steps: EGCG and nicotinic acid are mixed according to a molar ratio of 1-5:1-5, and are ground; or, EGCG and nicotinic acid are mixed according to a molar ratio of 1-5:1-5, and are then dissolved by using water, water is removed, and grinding is performed; or, EGCG and nicotinic acid are mixed according to a molar ratio of 1-5:1-5, are then dissolved by using water, are frozen, water is removed, and grinding is performed; or, EGCG and nicotinic acid are mixed according to a molar ratio of 1-5:1-5, and an EGCG-nicotinic acid co-crystal is obtained. The EGCG-nicotinic acid co-crystal prepared by the application has significantly improved tyrosinase inhibition performance, antioxidant performance, antibacterial performance and transdermal rate; and the problem of low stability and low water solubility of EGCG in the prior art is solved.

Description

Technical Field

[0001] This invention relates to an EGCG-nicotinic acid cocrystal, its preparation method and application, belonging to the field of pharmaceutical cocrystal technology. Background Technology

[0002] Epigallocatechin gallate (EGCG) is the core active ingredient in green tea extract. As a natural polyphenol, it not only possesses excellent antioxidant, anti-inflammatory, and photoprotective effects, but also exhibits significant antibacterial activity, effectively inhibiting the growth and reproduction of common skin pathogens such as streptococci. It has broad application prospects in skin anti-aging, barrier repair, and antibacterial care. However, EGCG suffers from several drawbacks—poor stability, low water solubility, and weak transdermal absorption. These problems, combined, become key bottlenecks restricting its industrial application. Its extremely low water solubility is a fundamental weakness, making it difficult to achieve effective concentrations in aqueous formulations. This leads to precipitation and aggregation, directly compromising formulation homogeneity and creating potential problems with subsequent stability and transdermal absorption, ultimately affecting the full realization of its antibacterial and antioxidant effects.

[0003] Besides solubility and stability defects, EGCG's low transdermal absorption efficiency further exacerbates its application limitations. In practical applications, the industry typically attempts to prepare it as a complex extract or liposome-encapsulated formulation to improve the situation; however, these solutions all have significant drawbacks: the EGCG purity in complex extracts is low, and numerous impurities interfere with its efficacy, and they cannot fundamentally solve the solubility problem; liposome encapsulation is complex and costly to produce, requiring stringent equipment and process parameters, making industrial-scale production difficult. Simultaneously, the poor stability of free EGCG in formulations is related to its solubility defects. Its poor water solubility makes it more susceptible to oxidative degradation due to external factors such as light and temperature, leading to efficacy loss; furthermore, its weak lipophilicity makes it difficult to penetrate the skin's stratum corneum barrier, ultimately resulting in extremely low transdermal bioavailability and failing to fully realize its active efficacy.

[0004] Cocrystals are crystal structures formed by two or more molecules bonded together in a specific stoichiometric ratio through weak interactions such as hydrogen bonds, electrostatic attraction, and hydrophobic interactions. Hydrogen bonds, due to their high directionality, specificity, and strong stability, play a crucial role in cocrystal formation. Without altering the molecular structure of the active pharmaceutical ingredient, cocrystal technology can specifically address the solubility defects of EGCG while significantly improving its physicochemical properties, including enhanced stability and optimized transdermal performance, thereby fundamentally improving drug bioavailability. By screening for highly compatible cocrystal ligands to construct efficient cocrystal systems with EGCG, the weak interactions between the ligands and EGCG molecules can break the aggregation forces between EGCG molecules themselves, significantly improving its water solubility. This allows for stable dispersion and effective concentration in aqueous formulations, while also improving transdermal absorption efficiency. This approach holds promise for enhancing the core efficacy of EGCG, such as antioxidant, whitening, and antibacterial properties, and promoting its large-scale application in cosmetics and pharmaceuticals.

[0005] Niacin, also known as vitamin B3, is an essential water-soluble vitamin for the human body and a widely available natural active ingredient. Its main pharmacological effects include regulating skin lipid metabolism, promoting stratum corneum renewal, anti-inflammatory and soothing effects, brightening skin tone, and enhancing skin barrier function. Studies have shown that niacin can effectively inhibit excessive sebum secretion, improve the skin's water-oil imbalance, promote skin microcirculation, and reduce melanin transfer to the epidermis. In the cosmetics field, niacin is widely used in skincare products for oil control, acne treatment, brightening skin tone, and improving roughness and dullness due to its excellent oil-controlling, anti-inflammatory, and skin-brightening properties. Niacin can repair damaged skin barriers, alleviate dryness and redness, and has the potential to delay photoaging and reduce fine lines, gradually becoming one of the important representatives of natural active ingredients in skincare.

[0006] Therefore, the preparation of an EGCG eutectic system by combining EGCG and nicotinic acid using eutectic technology can achieve synergistic effects while improving its stability, water solubility and transdermal properties, and has extremely high practical and economic value. Summary of the Invention

[0007] [Technical Issues] EGCG has problems such as poor stability, low water solubility, and weak transdermal absorption. No literature has yet mentioned improving the defects of EGCG itself through eutectic methods.

[0008] [Technical Solution] To address the aforementioned problems, this invention provides an EGCG-nicotinic acid cocrystal, its preparation method, and its applications. Specifically, this invention mixes EGCG and nicotinic acid in a molar ratio of 1-5:1-5 to form an EGCG-nicotinic acid cocrystal. The EGCG-nicotinic acid cocrystal prepared by this invention exhibits significantly improved tyrosinase inhibition, antioxidant, antibacterial, and transdermal permeability; and it also solves the problems of low EGCG stability and water solubility in existing technologies.

[0009] The first objective of this invention is to provide a method for preparing EGCG-nicotinic acid eutectic, comprising the following steps: Method 1: EGCG and nicotinic acid were mixed in a molar ratio of 1~5:1~5 and ground to obtain EGCG-nicotinic acid eutectic. Method 2: EGCG and nicotinic acid were mixed in a molar ratio of 1~5:1~5; then dissolved in water, the water was removed, and the mixture was ground to obtain EGCG-nicotinic acid eutectic. Method 3: EGCG and nicotinic acid were mixed at a molar ratio of 1~5:1~5; then dissolved in water, frozen, the water was removed, and the mixture was ground to obtain EGCG-nicotinic acid eutectic. Method 4: EGCG and nicotinic acid were mixed in a molar ratio of 1~5:1~5 to obtain EGCG-nicotinic acid eutectic.

[0010] In one embodiment of the present invention, the molar ratio of EGCG to nicotinic acid in the method for preparing EGCG-nicotinic acid eutectic is 1~2:1~4, preferably 1:1~3, and specifically, 1:1, 1:2, or 1:3 can be selected.

[0011] In one embodiment of the present invention, the grinding in Method 1 is carried out at 15~35℃ (room temperature) and 80~120r / min for 1~10h; preferably, the grinding time is 1~5h; more preferably, the grinding time is 2~4h, and specifically, 2, 3, or 4h can be selected.

[0012] In one embodiment of the present invention, the ratio of the sum of the moles of EGCG and nicotinic acid to the amount of water in Method 2 is 0.01~0.1mol:500~1000g.

[0013] In one embodiment of the present invention, the water removal in Method 2 is carried out by rotary evaporation, specifically rotary evaporation at 30~70°C for 2~10 hours; preferably, the temperature is 40~60°C and the time is 2~5 hours; more preferably, the temperature is 45~55°C and the time is 2~4 hours; specifically, the temperature can be 45, 50, or 55°C and the time can be 2, 3, or 4 hours.

[0014] In one embodiment of the present invention, the grinding in Method 2 is performed at 15~35℃ (room temperature) and 80~120r / min for 5-10 minutes; specifically, the temperature can be 15℃, 25℃, or 35℃, the grinding speed can be 80r / min, 100r / min, or 120r / min, and the grinding time can be 5 minutes, 8 minutes, or 10 minutes.

[0015] In one embodiment of the present invention, the ratio of the sum of the moles of EGCG and nicotinic acid to the amount of water in Method 3 is 0.01~0.1mol:500~1000g.

[0016] In one embodiment of the present invention, freezing in method three is freezing at -80~-60°C for 40~60 hours.

[0017] In one embodiment of the present invention, the moisture removal in method three is achieved by freeze drying, specifically freeze drying at -70 to -40°C for 5 to 30 hours; preferably, freeze drying at -60 to -40°C for 5 to 20 hours; more preferably, freeze drying at -60 to -50°C for 10 to 20 hours; specifically, the temperature can be -60, -55, or -50°C, and the time can be 10, 15, or 20 hours.

[0018] In one embodiment of the present invention, the grinding in Method 3 is performed at 15~35℃ (room temperature) and 80~120r / min for 5~10min; specifically, the temperature can be 15℃, 25℃, or 35℃, the grinding speed can be 80r / min, 100r / min, or 120r / min, and the grinding time can be 5min, 8min, or 10min.

[0019] In one embodiment of the present invention, the mixing in Method 4 is carried out at 15~35℃ (room temperature) and 80~120r / min for 5~10min.

[0020] The second objective of this invention is to prepare the EGCG-nicotinic acid eutectic by the method described herein.

[0021] In one embodiment of the present invention, the particle size of the EGCG-nicotinic acid eutectic is 2~10 μm.

[0022] The third objective of this invention is the application of the EGCG-nicotinic acid eutectic described herein in the preparation of cosmetics or pharmaceuticals.

[0023] In one embodiment of the present invention, cosmetics include serums, lotions, and creams; pharmaceutical dosage forms include tinctures, liniments, lotions, oils, ointments, creams, gels, sprays, and film-forming agents.

[0024] The fourth objective of this invention is to provide a product that combines the functions of inhibiting tyrosinase, antioxidation, and antibacterial properties, which utilizes the EGCG-nicotinic acid co-crystal described in this invention.

[0025] The fifth objective of this invention is to provide a method for improving the water solubility and stability of EGCG, which employs the EGCG-nicotinic acid eutectic described in this invention.

[0026] The sixth objective of this invention is to provide a method for simultaneously improving EGCG tyrosinase inhibitory activity, antioxidant properties, antibacterial properties, and transdermal permeability, which employs the EGCG-nicotinic acid cocrystal described in this invention.

[0027] The seventh objective of this invention is to provide a daily chemical product that uses the EGCG-nicotinic acid eutectic described in this invention.

[0028] In one embodiment of the present invention, the daily chemical products include shampoo, facial cleanser, etc.

[0029] [Beneficial Effects] (1) The EGCG-nicotinic acid cocrystal of the present invention significantly improves the stability and water solubility of EGCG, and at the same time, through the synergistic effect with nicotinic acid, it significantly enhances the permeability, tyrosinase inhibitory activity, antioxidant properties, antibacterial properties, etc. of EGCG; the tyrosinase activity inhibition rate reaches more than 59.34%, and the DPPH IC 50 When the concentration reaches below 44 μg / mL, ABTS IC 50 The concentration reached below 6.17 μg / mL; the cumulative permeation of EGCG over 24 hours reached 39 μg / mL. 2 above.

[0030] (2) The EGCG-nicotinic acid co-crystal system of the present invention significantly improves the physicochemical properties of EGCG, including stability, water solubility and transdermal performance, without changing the molecular structure of the active pharmaceutical ingredient, thereby improving the bioavailability of EGCG and playing a synergistic role.

[0031] (3) The EGCG-nicotinic acid eutectic of the present invention has good stability and water solubility, is not easy to decompose, and is easy to store and transport; after being stored at 25°C for 7 days, the residual amount of EGCG reaches more than 85%; after being stored at 45°C for 7 days, the residual amount of EGCG reaches more than 81%.

[0032] (4) The preparation process of the EGCG-nicotinic acid eutectic of the present invention is green and pollution-free, and the process is simple. It is suitable for industrial promotion, and the product obtained can be used directly as a raw material for drugs or cosmetics.

[0033] (5) The EGCG-nicotinic acid cocrystal of the present invention has good antibacterial effects against Streptococcus mutans, Streptococcus suis serotype 2 and Group A Streptococcus; the MIC against Streptococcus mutans is below 0.53 mg / mL; the MIC against Streptococcus suis serotype 2 is below 2.07 mg / mL; and the MIC against Group A Streptococcus is below 3.28 mg / mL.

[0034] (6) The EGCG-nicotinic acid cocrystal of the present invention is non-cytotoxic. When the EGCG concentration does not exceed 80 μg / mL, the sample concentration does not affect the viability of B16F10 cells; it has the ability to inhibit melanin synthesis, with an inhibition rate of over 54.9%. That is, the EGCG-nicotinic acid cocrystal molecule can stably inhibit the activity of tyrosinase under safe toxicity conditions, thereby hindering melanin production. Attached Figure Description

[0035] Figure 1 The Gaussian calculation diagram is for the EGCG-nicotinic acid eutectic system of Example 1.

[0036] Figure 2 This is a water solubility monitoring graph of the EGCG-nicotinic acid eutectic system in Example 1.

[0037] Figure 3 This is a stability monitoring graph of the EGCG-nicotinic acid eutectic system in Example 1.

[0038] Figure 4 The image shows the infrared spectrum of the EGCG-nicotinic acid eutectic system of Example 1.

[0039] Figure 5 Differential scanning calorimetry (DSC) of the EGCG-nicotinic acid eutectic system in Example 1.

[0040] Figure 6 The image shows the powder X-ray diffraction pattern of the EGCG-nicotinic acid eutectic system in Example 1.

[0041] Figure 7 The effect of the EGCG-nicotinic acid cocrystal system of Example 1 on the viability of B16F10 cells.

[0042] Figure 8 The effect of the EGCG-nicotinic acid eutectic system of Example 1 on the melanin content of B16F10 cells.

[0043] Figure 9 The effect of the EGCG-nicotinic acid cocrystal system of Example 1 on the tyrosinase activity of B16F10 cells.

[0044] Figure 10 The image shows the antioxidant properties of the EGCG-nicotinic acid eutectic system in Example 1; where (a) is the DPPH radical scavenging rate and (b) is the ABTS radical scavenging rate.

[0045] Figure 11 This is a permeability test diagram of the EGCG-nicotinic acid eutectic system in Example 1. Detailed Implementation

[0046] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0047] Test method: (1) Gaussian analysis: Density functional theory (DFT) calculations were performed at the B3LYP / 6-311G(d,p) level using Gaussian 16 software. Electrostatic potentials (ESPs) were calculated, and molecular configurations of EGCG and nicotinic acid were constructed. Independent gradient models based on Hirshfeld partitioning (IGMH) and atoms-in-molecular (AIM) methods were used. Intermolecular interactions of Example 1 were analyzed using Multiwfn and VMD software. VMD 1.9.4 was used to process and graphically display the results.

[0048] (2) Water solubility test: Weigh out the eutectic and dissolve it in deionized water to prepare an aqueous solution with a mass fraction of 2% EGCG; at the same time, weigh out the EGCG and dissolve it in deionized water to prepare an aqueous solution with a mass fraction of 0.1% EGCG.

[0049] The samples were placed at 25 ℃ and 45 ℃ for a certain period of time to observe whether precipitation occurred.

[0050] (3) Stability test: The eutectic and EGCG were dissolved separately in deionized water to prepare an aqueous solution with a mass fraction of 0.1% EGCG.

[0051] The samples were placed at 25 ℃ and 45 ℃, and the residual amount of EGCG was determined at 280 nm using a UV spectrophotometer to evaluate its stability.

[0052] (4) Fourier transform infrared spectroscopy test: Machine specifications: FTS6000; Machine manufacturer: Bio-ard, USA; Experimental parameters: Scanning range 500~4000 cm -1 The resolution is 4cm. -1 .

[0053] (5) Differential scanning calorimetry: Machine specifications: DSC-8000 model; Machine manufacturer: PE Company, USA; Experimental parameters: nitrogen atmosphere, flow rate 50 mL / min, temperature range 70~300℃, heating rate 10℃ / min.

[0054] (6) Powder X-ray diffraction test: Machine specifications: D8 model; Manufacturer: Bruker AXS GmbH, Germany; Experimental parameters: degree range 5~40°, scanning speed 10° / min.

[0055] (7) The inhibitory activity of EGCG, the examples and the comparative examples on tyrosinase in mouse B16F10 melanoma cells was determined by cell experiments.

[0056] After resuscitation, B16F10 cells were cultured in 1640 medium supplemented with 10% FBS (v / v) and 1% penicillin-streptomycin (v / v). The cells were then incubated at 37°C in a humidified environment with 5% CO2. When cell confluence exceeded 80%, cell passages or subsequent experiments were performed.

[0057] The cytotoxicity of the samples to B16F10 cells was evaluated. B16F10 cells in logarithmic growth phase were seeded into 96-well plates at a density of approximately 2 × 10⁶ cells per well. 5 Cells were collected; each well was filled with 100 μL of 1640 medium supplemented with 10% FBS, and the cells were incubated in a CO2 incubator at 37 °C. After 24 h of incubation, the old medium in each well was removed using a pipette (for both cell lines), and 100 μL of fresh medium containing different concentrations of the sample (EGCG content of 20, 40, 80, 100, and 200 μg / mL) was added to each well; the cells were then incubated at 37 °C for another 24 h, with untreated cells serving as a control group. At the end of the incubation period, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and incubation was continued at 37 °C for another 4 h; afterwards, the supernatant was removed, and formazan crystals were dissolved in DMSO (gently shaken for 5 min), and the absorbance was measured at 570 nm.

[0058] After a 24-hour incubation period, B16F10 cells were seeded into 24-well plates at a density of 2 × 10⁶ cells per well. 5 Cells were collected and then treated with 100 nM α-MSH for 48 h, with kojic acid as a positive control. After incubation, the culture medium was removed, and cells were lysed at 80 °C for 1 h using 1N NaOH and 10% DMSO. The absorbance was measured at 475 nm using a spectrophotometer.

[0059] After a 24-hour incubation period, B16F10 cells were seeded into 12-well plates at a density of 2 × 10⁶ cells per well. 5 Cells were collected. Except for the blank control group, all groups were stimulated with 100 nM α-MSH for 1 h. After treatment, the culture medium was processed, and samples were added to the cells for 24 h, with kojic acid selected as a positive control. Cells were then washed with PBS, ruptured with 100 μL of PBS containing 1% Triton X-100 and 100 μM PMSF, and finally centrifuged at 4 °C and 14000 rpm for 10 min. In 96-well plates, 90 μL of supernatant was mixed with 10 μL of L-DOPA (5 mg / mL) and incubated at 37 °C for 30 min. The degree of L-DOPA oxidation to dopachrome was measured at 475 nm using a spectrophotometer.

[0060] The formula for calculating the tyrosinase activity inhibition rate is:

[0061] Among them, A sample A represents the absorbance of a sample solution containing levodopa. blank A represents the absorbance of an anhydrous ethanol solution without levodopa. control The absorbance is the value of the blank control group solution.

[0062] (8) The strength of its antioxidant properties was verified by testing the scavenging efficiency of the combination / eutectic against DPPH and ABTS free radicals: EGCG and the examples and comparative examples were diluted with methanol to 1~12 μg / mL as the ABTS test sample concentration (DPPH test sample concentration was 10~120 μg / mL). The scavenging rate of DPPH and ABTS free radicals of the sample solution was tested according to GB / T39100-2020 method.

[0063] (9) In vitro skin penetration test using Franz diffusion cell: Undamaged, clean pigskin was placed on a Franz diffusion cell, cuticle side up. EGCG was diluted with deionized water to ensure an EGCG concentration of 300 μg / mL in each system, as described in the examples and comparative studies. The receiving chamber was filled with physiological saline, and 3 mL of each drug solution was added to the diffusion cell. The mixture was continuously stirred at a constant temperature (37 ± 0.1 °C), and 1 mL of the receiving solution was collected at 1, 2, 4, 6, 12, and 24 hours, with an equal volume of fresh receiving solution added. The collected samples were filtered through a 0.45 μm microporous filter and then analyzed by HPLC.

[0064] The cumulative drug penetration is calculated using the following formula:

[0065] Among them, Q S Cumulative drug penetration per unit area (μg / cm²) 2 ), C sn V represents the drug concentration (mg / mL) in the receptor fluid measured within the sampling interval. S For the volume of the receptor pool, It is the cumulative drug concentration in the recipient fluid, S is the sampling volume, and A is the cumulative drug concentration in the recipient fluid. S It is the effective diffusion area.

[0066] (10) Antibacterial test Triple-distilled water was added to the eutectic in the example to prepare an aqueous solution of the eutectic with a mass concentration of 1%; antibacterial test was performed, with triple-distilled water as a control.

[0067] Overnight resuscitation cultures of *Streptococcus mutans*, *Streptococcus suis* serotype 2, and group A streptococci were inoculated into 5 mL of THB liquid medium at a 1% v / v inoculation rate and incubated statically at 37°C with 5% CO2 for 24 h. The bacteria were cultured until they reached mid-logarithmic growth, at which point the bacterial count reached 10⁻⁶. 9 Take it out when CFU / mL is reached and set aside for later use.

[0068] Under aseptic conditions in a biosafety cabinet, the eutectic aqueous solution was added to sterile 96-well culture plates. 100 μL of the solution was added to wells 1 to 9, and 100 μL of triple-distilled water was added to well 10 as a control.

[0069] The inoculum culture was diluted 10 times with culture medium. After dilution, 100 μL was added to the corresponding 96-well culture plate. The plate was sealed and placed in an incubator at 37°C and 5% CO2 for 24 hours to observe the antibacterial effect of the eutectic aqueous solution.

[0070] After co-incubating the sample and bacteria for 24 hours, 100 μL of the sample was plated onto a THB plate from each well and incubated statically at 37°C and 5% CO2 for 24 hours. The minimum drug concentration required for complete sterility was defined as the MIC. The assay was only meaningful when there was significant bacterial growth in the positive control wells (i.e., without the drug).

[0071] Raw materials used in the examples: EGCG was purchased from Xi'an Xinlu Biotechnology Co., Ltd., with a purity of 98%. Niacin was purchased from Shanghai Haohong Biomedical Technology Co., Ltd., with a purity of 98%.

[0072] Example 1 A method for preparing EGCG-nicotinic acid eutectic includes the following steps: Weigh out 0.01 mol EGCG and 0.02 mol nicotinic acid (NA), mix them evenly, and grind them at 25℃ (room temperature) and 80 r / min for 2 h to obtain a white solid product, namely: EGCG-nicotinic acid eutectic (particle size 2~10 μm).

[0073] The obtained EGCG-nicotinic acid eutectic was subjected to performance testing, and the test results are as follows: (1) Eutectic structure: Figure 1 The image shows the Gaussian calculation plot of the EGCG-nicotinic acid eutectic system in Example 1. From... Figure 1 It can be seen that there are obvious hydrogen bonds, van der Waals interactions and other weak interactions between EGCG and nicotinic acid components, and these interactions together promote the formation of EGCG-nicotinic acid eutectic.

[0074] Figure 4 The image shows the infrared spectrum of the EGCG-nicotinic acid eutectic system from Example 1. Figure 4 It can be seen that in the spectrum of EGCG, the main OH absorption peak is at 3451 cm⁻¹. -1 1700 cm -1 The absorption peak at 1626 cm⁻¹ is the absorption peak of the carbonyl group. -1 The absorption peak at [value] is related to benzene ring vibration. This is in contrast to the 3451 cm⁻¹ peak of EGCG. -1 And NA's 3411 cm -1 Example 1 at 3483 cm -1 Only one OH absorption peak was observed, which is due to the strong interaction between the hydroxyl groups of EGCG and NA. The wavenumber of the carbonyl absorption peak measured in Example 1 was shifted compared to both NA and EGCG, from 1640 cm⁻¹ of NA. -1 And EGCG 1700 cm -1 Offset to 1688 cm in Example 1 -1 It is speculated that the hydroxyl and carbonyl groups in EGCG and NA molecules are involved in the formation of intermolecular hydrogen bonds.

[0075] Figure 5 This is a differential scanning calorimeter of the EGCG-nicotinic acid eutectic system from Example 1. Figure 5 It can be seen that EGCG and nicotinic acid exhibit single melting peaks at 230℃ and 241℃, respectively, while the melting point of the EGCG-nicotinic acid eutectic system in Example 1 is 192℃. The appearance of a single melting point that is different from that of the raw material components indicates the formation of the eutectic system.

[0076] Figure 6 This is the powder X-ray diffraction pattern of the EGCG-nicotinic acid eutectic system in Example 1. From... Figure 6It can be seen that the EGCG-nicotinic acid eutectic in Example 1 exhibits a novel characteristic diffraction peak at 34.06°, while the intensity of some characteristic diffraction peaks of EGCG and nicotinic acid is significantly reduced or disappears. This indicates that the eutectic system is not a physical mixture, but rather forms a new crystal structure.

[0077] (2) Stability and water solubility Figure 2 This is a water solubility monitoring graph for the EGCG-nicotinic acid eutectic system in Example 1. From... Figure 2 It can be seen that after storage in aqueous solutions under different conditions for 30 days, no precipitate formed in the aqueous solution of Example 1, proving that its solubility was greatly improved; and compared with the EGCG aqueous solution, the color change of the aqueous solution of Example 1 was slower, indicating that its stability was also improved. That is, the present invention can achieve the dissolution of 2% EGCG by mass in water, greatly improving the solubility of EGCG.

[0078] Figure 3 This is a stability monitoring graph of the EGCG-nicotinic acid eutectic system in Example 1. From... Figure 3 It can be seen that the residual EGCG in the aqueous solution of Example 1 is higher than that in the aqueous solution of EGCG.

[0079] In summary, the results indicate that the formation of the EGCG-nicotinic acid eutectic system can improve the stability of EGCG.

[0080] (3) Tyrosinase inhibitory activity Figure 7 The effect of the EGCG-nicotinic acid cocrystal system of Example 1 on the viability of B16F10 cells. Figure 7 It can be seen that when the EGCG concentration does not exceed 80 μg / mL, the sample concentration does not affect the viability of B16F10 cells. Compared with EGCG and nicotinic acid, the cell viability after treatment in Example 1 was significantly improved. This is attributed to the stable hydrogen bond network formed by the combination of EGCG and nicotinic acid, creating a biocompatible microenvironment. Therefore, 80 μg / mL was chosen as the concentration for subsequent experiments.

[0081] Figure 8 The effect of the EGCG-nicotinic acid eutectic system of Example 1 on the melanin content of B16F10 cells. Figure 8 It can be seen that EGCG, nicotinic acid, and Example 1 all have the ability to inhibit melanin synthesis, producing inhibition rates of 30.9%, 21.3%, and 54.9%, respectively. These are superior to kojic acid and exhibit synergistic anti-melanin activity. This inhibition is related to tyrosinase inhibition, rather than cytotoxic effects, confirming functional melanin production interference.

[0082] Figure 9The effect of the EGCG-nicotinic acid cocrystal system of Example 1 on the tyrosinase activity of B16F10 cells. From... Figure 9 It can be seen that EGCG, nicotinic acid, and Example 1 all have the ability to inhibit intracellular tyrosinase activity. They produced inhibition rates of 43.9%, 38.1%, and 61.1%, respectively. The increased tyrosinase inhibitory activity is directly related to the inhibition of melanin production, establishing a structure-activity relationship. The therapeutic effect of the EGCG-nicotinic acid cocrystal was significantly better than that of kojic acid (p<0.01), verifying its mechanism: the EGCG-nicotinic acid cocrystal molecule can stably inhibit tyrosinase activity under safe and toxic conditions, thereby hindering melanin production.

[0083] (4) Antioxidant properties Figure 10 The image shows the antioxidant activity test results of the EGCG-nicotinic acid eutectic system in Example 1; where (a) DPPH radical scavenging rate and (b) ABTS radical scavenging rate are also shown. Figure 10 It can be seen that: IC in Example 1 50 The value was 39.24 μg / mL, and the IC50 of EGCG was... 50 The value was 43.35 μg / mL, and the IC50 of nicotinic acid was... 50 Values ​​exceeding 120 μg / mL, IC50 50 The reduction indicates that Example 1 has superior DPPH radical scavenging ability. The IC of Example 1 50 The value was 5.86 μg / mL, and the IC50 of EGCG was... 50 The value was 6.97 μg / mL, and the IC50 of nicotinic acid was... 50 Values ​​exceeding 12 μg / mL, IC50 50 The decrease in the value indicates that Example 1 has a superior ABTS free radical scavenging ability. It can be seen that both EGCG and nicotinic acid exhibit strong antioxidant properties and can effectively scavenge free radicals. After forming the EGCG-nicotinic acid eutectic system, the free radical scavenging rate is higher than that of the two component raw materials, showing a synergistic effect of "1+1>2".

[0084] (5) Permeability Figure 11 This is a permeability test diagram of the EGCG-nicotinic acid eutectic system in Example 1. From... Figure 11 It can be seen that the cumulative penetration amount of Example 1 within 24 hours was 1.4 times that of the EGCG group, reaching 46.93 μg / cm². The enhanced EGCG skin delivery by the cocrystal system can be attributed to the strong intermolecular interactions present in the system, which affect the structural integrity of the skin lipid network, thereby promoting the transdermal delivery of EGCG.

[0085] (6) Antibacterial properties The minimum complete inhibitory concentration plate experiment confirmed that the example had a significant inhibitory effect on Streptococcus mutans, Streptococcus suis serotype 2, and Group A Streptococcus.

[0086] The results are shown in Tables 1-4. The minimum inhibitory concentrations (MICs) of Examples 1-8 were lower than those of Comparative Examples 1-16. This indicates that the Examples have better antibacterial efficacy compared to the Comparative Examples.

[0087] Example 2 A method for preparing EGCG-nicotinic acid eutectic includes the following steps: Weigh 0.01 mol EGCG and 0.01 mol nicotinic acid and mix them in a round-bottom flask. Dissolve them in 1000 g of deionized water and remove the water by rotary evaporation at 45 °C for 3 h. Grind at 25 °C (room temperature) and 80 r / min for 5 min to obtain a white powder product, namely: EGCG-nicotinic acid eutectic (particle size 2~10 μm).

[0088] Example 3 A method for preparing EGCG-nicotinic acid eutectic includes the following steps: Weigh 0.01 mol EGCG and 0.03 mol nicotinic acid and mix them in a round-bottom flask. Dissolve them in 1000 g of deionized water and remove the water by rotary evaporation at 55 °C for 4 h. Grind at 35 °C (room temperature) and 100 r / min for 8 min to obtain a white powder product, namely: EGCG-nicotinic acid eutectic (particle size 2~10 μm).

[0089] Example 4 A method for preparing EGCG-nicotinic acid eutectic includes the following steps: Weigh 0.01 mol EGCG and 0.02 mol nicotinic acid and mix them in a round-bottom flask. Dissolve them in 700 g of deionized water and remove the water by rotary evaporation at 50 °C for 2 h. Grind at 120 r / min for 10 min at 15 °C (room temperature) to obtain a white powder product, namely: EGCG-nicotinic acid eutectic (particle size 2~10 μm).

[0090] Example 5 A method for preparing EGCG-nicotinic acid eutectic includes the following steps: Weigh out 0.01 mol EGCG and 0.02 mol nicotinic acid, mix them evenly, place them in a material tray, dissolve them with 700 g of deionized water and pre-freeze (freeze at -80℃ for 40 h), then treat them with a freeze dryer at -60℃ for 10 h to remove moisture, and grind them at 25℃ (room temperature) and 80 r / min for 5 min to obtain a white powder product, namely: EGCG-nicotinic acid eutectic (particle size of 2~10 μm).

[0091] Example 6 A method for preparing EGCG-nicotinic acid eutectic includes the following steps: Weigh out 0.01 mol EGCG and 0.01 mol nicotinic acid, mix them evenly, place them in a material tray, dissolve them with 1000 g of deionized water and pre-freeze (freeze at -70℃ for 50 h), then treat them with a freeze dryer at -55℃ for 15 h to remove moisture, and grind them at 25℃ (room temperature) and 80 r / min for 5 min to obtain a white powder product, namely: EGCG-nicotinic acid eutectic (particle size of 2~10 μm).

[0092] Example 7 A method for preparing EGCG-nicotinic acid eutectic includes the following steps: Weigh out 0.01 mol EGCG and 0.03 mol nicotinic acid, mix them evenly, place them in a material tray, dissolve them with 1000 g of deionized water and pre-freeze (freeze at -60℃ for 60 h), then treat them with a freeze dryer at -50℃ for 20 h to remove moisture, and grind them at 35℃ (room temperature) and 100 r / min for 10 min to obtain a white powder product, namely: EGCG-nicotinic acid eutectic (particle size of 2~10 μm).

[0093] Example 8 A method for preparing EGCG-nicotinic acid eutectic includes the following steps: Weigh out 0.01 mol EGCG and 0.02 mol nicotinic acid, mix them, and stir at 25℃ (room temperature) and 80 r / min for 5 min to obtain a white powder product, namely: EGCG-nicotinic acid eutectic (particle size 2~10 μm).

[0094] The obtained product was subjected to performance testing, and the test results are as follows: Table 1 Antibacterial effect

[0095] Table 2 Performance Testing

[0096] Comparative Example 1 Based on Example 1, the amount of nicotinic acid was changed to 0.06 mol, and the remaining steps were the same as in Example 1.

[0097] The results showed that after increasing the amount of nicotinic acid, the two components did not come into complete contact during the entire grinding process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, and there was an excess of nicotinic acid.

[0098] Comparative Example 2 Based on Example 1, the amount of EGCG was changed to 0.06 mol, the amount of nicotinic acid was changed to 0.01 mol, and the remaining steps were the same as in Example 1.

[0099] The results showed that after increasing the amount of EGCG, the two components did not come into complete contact during the entire grinding process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, indicating the presence of excessive EGCG.

[0100] Comparative Example 3 Based on Example 1, the grinding time was changed to 0.5 hours, and the remaining steps were the same as in Example 1.

[0101] The results showed that after shortening the grinding time, the two components did not come into complete contact during the entire grinding process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, and the system's self-assembly was incomplete.

[0102] Comparative Example 4 Based on Example 1, the grinding time was changed to 11 hours, and the remaining steps were the same as in Example 1.

[0103] The results show that extending the grinding time will cause the "building effect" of mechanical force to turn into a "destructive effect", ultimately leading to a deterioration of the eutectic effect and the inability to obtain a high-purity eutectic system.

[0104] Comparative Example 5 Based on Example 2, the temperature of the rotary evaporator was changed to 20°C, and the remaining steps were the same as in Example 2.

[0105] The results showed that after lowering the temperature of the rotary evaporator, the two components did not come into complete contact during the entire process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, and the system's self-assembly was incomplete.

[0106] Comparative Example 6 Based on Example 2, the temperature of the rotary evaporator was changed to 80°C, and the remaining steps were the same as in Example 2.

[0107] The results showed that increasing the temperature of the rotary evaporator resulted in excessively high temperatures throughout the process, which damaged the stability and water solubility of EGCG and nicotinic acid, leading to a decrease in the activity of the final product.

[0108] Comparative Example 7 Based on Example 2, the operating time of the rotary evaporator was changed to 11 hours, and the remaining steps were the same as in Example 2.

[0109] The results showed that extending the operating time of the rotary evaporator damaged the stability and water solubility of EGCG and nicotinic acid during the long-term rotary evaporation process, resulting in a decrease in the activity of the final product.

[0110] Comparative Example 8 Based on Example 2, the operating time of the rotary evaporator was changed to 1 hour, and the remaining steps were the same as in Example 2.

[0111] The results showed that after shortening the operating time of the rotary evaporator, EGCG and nicotinic acid did not get sufficient contact, and a stable eutectic system was not formed, resulting in incomplete self-assembly of the system.

[0112] Comparative Example 9 Based on Example 5, the temperature of the freeze dryer was changed to -80°C, and the remaining steps were the same as in Example 5.

[0113] The results showed that lowering the temperature of the freeze dryer inhibited the diffusion of eutectic molecules, affected the formation of the eutectic system, and impacted the activity of the product.

[0114] Comparative Example 10 Based on Example 5, the temperature of the freeze dryer was changed to -30°C, and the remaining steps were the same as in Example 5.

[0115] The results showed that when the temperature of the freeze dryer was increased, the adsorbed water on the eutectic surface melted rapidly throughout the process, which destroyed the eutectic system and affected the activity of the product.

[0116] Comparative Example 11 Based on Example 5, the freeze dryer usage time was changed to 4 hours, and the remaining steps were the same as in Example 5.

[0117] The results showed that after shortening the usage time of the freeze dryer, the two components did not come into complete contact during the entire process. Subsequent tests revealed that a high-purity eutectic system could not be obtained, and the system's self-assembly was incomplete.

[0118] Comparative Example 12 Based on Example 5, the freeze dryer usage time was changed to 40 hours, and the remaining steps were the same as in Example 5.

[0119] The results showed that prolonged use of the freeze dryer and long-term vacuum extraction damaged the stability and water solubility of EGCG and nicotinic acid, affecting the activity of the final product.

[0120] Comparative Example 13 Based on Example 1, nicotinamide was used instead of nicotinic acid, and the remaining steps were the same as in Example 1.

[0121] Comparative Example 14 Based on Example 2, isonicotinic acid was used to replace nicotinic acid, and the remaining steps were the same as in Example 2.

[0122] Comparative Example 15 Based on Example 5, glucose was used instead of nicotinic acid, and the remaining steps were the same as in Example 5.

[0123] Comparative Example 16 The method referenced in patent CN111601794A: 10g of green tea extract (>50% EGCG and >90% polyphenols) was dissolved in 80mL of water at room temperature to obtain a turbid solution. 3.02g of nicotinic acid was added to the solution, and the mixture was continuously slurried for 12 hours. The precipitate was filtered, the wet cake was washed with 80mL of water and blotted dry. The cake was then ground using a mortar and pestle to obtain a fine powder. The remaining steps were the same as in Example 1.

[0124] The obtained product was subjected to performance testing, and the test results are as follows: Table 3 Antibacterial effect

[0125] Table 4 Performance Testing

[0126] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing EGCG-nicotinic acid eutectic, characterized in that, Includes the following steps: Method 1: EGCG and nicotinic acid were mixed in a molar ratio of 1~5:1~5 and ground to obtain EGCG-nicotinic acid eutectic. Method 2: EGCG and nicotinic acid were mixed in a molar ratio of 1~5:1~5; then dissolved in water, the water was removed, and the mixture was ground to obtain EGCG-nicotinic acid eutectic. Method 3: EGCG and nicotinic acid were mixed at a molar ratio of 1~5:1~5; then dissolved in water, frozen, the water was removed, and the mixture was ground to obtain EGCG-nicotinic acid eutectic. Method 4: EGCG and nicotinic acid were mixed in a molar ratio of 1~5:1~5 to obtain EGCG-nicotinic acid eutectic.

2. The method according to claim 1, characterized in that, In Method 1, the grinding is carried out at 15~35℃ (room temperature) and 80~120r / min for 1~10h; preferably, the grinding time is 1~5h; and even more preferably, the grinding time is 2~4h.

3. The method according to claim 1, characterized in that, In Method 2, moisture is removed by rotary evaporation, specifically at 30-70°C for 2-10 hours; preferably, the temperature is 40-60°C and the time is 2-5 hours; more preferably, the temperature is 45-55°C and the time is 2-4 hours. Grinding is performed at 15~35℃ (room temperature) and 80~120r / min for 5~10min.

4. The method according to claim 1, characterized in that, In Method 3, moisture is removed by freeze-drying, specifically freeze-drying at -70~-40℃ for 5~30h; preferably, freeze-drying at -60~-40℃ for 5~20h; more preferably, freeze-drying at -60~-50℃ for 10~20h; grinding is performed at 15~35℃ (room temperature) and 80~120r / min for 5~10min.

5. The EGCG-nicotinic acid eutectic prepared by the method according to any one of claims 1 to 4.

6. The use of the EGCG-nicotinic acid eutectic according to claim 5 in the preparation of cosmetics or pharmaceuticals.

7. A product that combines the functions of inhibiting tyrosinase, antioxidation, and antibacterial properties, characterized in that, The EGCG-nicotinic acid eutectic as described in claim 5 was used.

8. A method for improving the water solubility and stability of EGCG, characterized in that, The EGCG-nicotinic acid eutectic as described in claim 5 was used.

9. A method for simultaneously improving EGCG tyrosinase inhibitory activity, antioxidant activity, antibacterial activity, and transdermal penetration rate, characterized in that, The EGCG-nicotinic acid eutectic as described in claim 5 was used.

10. A daily chemical product, characterized in that, The EGCG-nicotinic acid eutectic as described in claim 5 was used.

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