Glycyrrhetinic acid-tromethamine eutectic crystal, preparation method thereof and application of glycyrrhetinic acid-tromethamine eutectic crystal in skin care

Abstract

The invention discloses a glycyrrhetinic acid-tromethamine eutectic crystal as well as a preparation method and application thereof in skin care, and belongs to the technical field of biological medicines. The molecular formula of the glycyrrhetinic acid-tromethamine eutectic crystal is [C4H11NO3. 2C30H46O4], the accurate structure of the eutectic crystal is characterized by single crystal X-ray diffraction, and the eutectic crystal forms a basic structural unit through two glycyrrhetinic acid molecules and one tromethamine molecule. The preparation method of the eutectic comprises the following steps: mixing glycyrrhetinic acid and tromethamine according to a molar ratio of (3: 1)-(1: 3), adding an organic solvent such as methanol, ethanol, acetonitrile or isopropanol, stirring and dissolving at 40-60 DEG C, then evaporating to remove the solvent at 40-50 DEG C under reduced pressure, and carrying out vacuum drying to obtain a white eutectic solid. The method is simple in process and suitable for large-scale production. According to the prepared eutectic crystal, the water solubility of glycyrrhetinic acid is remarkably improved, the core problem that glycyrrhetinic acid is prone to layering and precipitation in a water-based system is solved, and the application range and effect of glycyrrhetinic acid are widened.

Description

Technical Field

[0001] This invention relates to a glycyrrhetinic acid-aminobutyric acid eutectic, its preparation method, and its application in skin care, belonging to the field of biomedical technology. Background Technology

[0002] Glycyrrhetinic acid (GA) is one of the core active ingredients of licorice. Belonging to the pentacyclic triterpenoid class of compounds, it possesses excellent anti-inflammatory and soothing properties, reduces melanin production, repairs the skin barrier, and has antioxidant effects, making it a highly valuable natural active ingredient in the cosmetics industry. However, the inherent physicochemical defects of glycyrrhetinic acid create numerous bottlenecks in its cosmetic applications, severely limiting its widespread use and efficacy. Specific problems include: 1. Poor water solubility and limited compatibility: Glycyrrhetinic acid is a typical fat-soluble compound with extremely low solubility in water. Modern cosmetics are primarily water-based (such as serums, lotions, creams, and masks), and the fat-soluble glycyrrhetinic acid is difficult to disperse evenly in water-based systems, easily leading to problems such as layering, precipitation, and a grainy texture. This not only affects the appearance, texture, and user experience of the cosmetics but also results in uneven distribution of active ingredients, preventing them from fully exerting their efficacy.

[0003] 2. Low skin absorption efficiency and poor efficacy conversion: The stratum corneum of the skin's surface is a lipophilic barrier, but the aqueous environment inside the skin has a weak ability to conduct lipid-soluble components. Due to its insufficient water solubility, glycyrrhetinic acid has difficulty penetrating the stratum corneum to reach the deeper layers of the skin, remaining mostly on the skin surface. This prevents it from effectively exerting its anti-inflammatory, soothing, and barrier-repairing effects, requiring higher doses to achieve the desired results. This increases raw material costs and may also lead to skin irritation risks due to excessively high local concentrations.

[0004] 3. Poor formulation stability, affecting product shelf life: In conventional cosmetic production processes (such as emulsification, homogenization, and sterilization), glycyrrhetinic acid is prone to instability due to crystal form changes and particle aggregation. This not only leads to a rougher product texture and reduced efficacy, but may also cause system instability (such as stratification and precipitation), shortening the shelf life of cosmetics.

[0005] To address the shortcomings of glycyrrhetinic acid in cosmetic applications, existing technologies have significant limitations: ① While preparing sodium and potassium glycyrrhetinic acid salts slightly improves water solubility, these salts are pH-sensitive in cosmetic systems, easily precipitating free glycyrrhetinic acid in acidic formulations. Furthermore, the salt components increase product stickiness, negatively impacting the user experience. ② Cyclodextrin inclusion technology, while improving dispersibility, results in low drug loading, increasing redundant components in the formulation. The inclusion process is also complex and costly. ③ Nanoparticle dispersion technology, while improving solubility, leads to nanoparticle aggregation in cosmetic systems, poor storage stability, and significant challenges in industrial production, making it unsuitable for large-scale application in the cosmetic industry.

[0006] Co-crystallization is a novel technique developed in recent years to improve the physicochemical properties of poorly soluble active ingredients. It involves the formation of a co-crystall with a specific crystal structure through non-covalent interactions (hydrogen bonds, π-π stacking, van der Waals forces, etc.) between active ingredient molecules and co-crystallizer molecules. This technique does not alter the covalent structure of the active ingredient, and can significantly improve properties such as water solubility, dispersibility, and stability while preserving its original efficacy. Furthermore, the preparation process is simple and suitable for industrial production in cosmetics.

[0007] Tris(hydroxymethyl)aminomethane (Tris for short) is an organic compound containing multiple hydroxyl and amino groups. It exhibits excellent biocompatibility and skin tolerance, and its molecular structure contains multiple hydrogen-bonding sites (hydroxyl and amino groups), allowing it to form stable co-crystals with glycyrrhetinic acid. Furthermore, tromethamine itself has a certain pH-regulating ability, adapting to the common pH range of cosmetics (4.5–7.5) without negatively impacting the stability of cosmetic formulations. Currently, there are no reports on the preparation of co-crystals from glycyrrhetinic acid and tromethamine and their application in the field of skin care. Summary of the Invention

[0008] The purpose of this invention is to provide a glycyrrhetinic acid-tromethamine cocrystal and its preparation method. By screening suitable cocrystal formations and preparation processes, this invention successfully prepares glycyrrhetinic acid-tromethamine cocrystal, effectively solving the technical problems of poor water solubility, uneven dispersion, and low skin absorption efficiency of glycyrrhetinic acid in skincare applications.

[0009] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: A glycyrrhetinic acid-tributanol eutectic has the molecular formula [C4H] 11 NO3·2C 30 H 46[O4] is composed of two glycyrrhetinic acid molecules and one tromethamine molecule as the basic structural unit; the eutectic space group is C121, and the cell parameters are: a = 30.891(11) Å, b = 30.891(11) Å, c = 30.891(11) Å, α = 90.00°, β = 92.910(11)°, γ = 90.00°.

[0010] Preferably, the characteristic X-ray diffraction peaks of the eutectic powder are at 6.6°, 9.06°, 12.08°, 13.12°, 14.72°, and 16.08°.

[0011] The present invention also provides a method for preparing the above-mentioned glycyrrhetinic acid-tributanol eutectic, comprising the following steps: 1) Weigh out glycyrrhetinic acid and tromethamine in molar ratio and place them in a reaction vessel; 2) Add organic solvent to the above container and stir to dissolve at 40~60℃ for 0.5~2 h to form a clear solution; 3) Place the above solution in a rotary evaporator and evaporate the solvent at 40~50℃ under reduced pressure to obtain a white crude solid. Then place the white crude solid in a vacuum drying oven and dry at 40℃ for 12~24 h to obtain the final product.

[0012] Preferably, the molar ratio of glycyrrhetinic acid and tromethamine in step (1) is 3:1 to 1:3.

[0013] Preferably, the organic solvent is one or a mixture of two or more of methanol, ethanol, acetonitrile, and isopropanol.

[0014] Preferably, the solid-liquid ratio of the mixture powder to the organic solvent is 100 mg:(8~15) mL.

[0015] The application of the glycyrrhetinic acid-tributanol cocrystal prepared in this invention in skin care, wherein the skin care includes moisturizing, anti-aging, anti-oxidation, whitening, anti-wrinkle or repair.

[0016] The beneficial effects of this invention are: (1) For the first time, glycyrrhetinic acid was co-crystallized with tromethamine, which significantly improved the water solubility of glycyrrhetinic acid and greatly enhanced its application effect in water-based systems.

[0017] (2) The preparation process of the present invention is simple, the production process does not require complex equipment, the cost is low, it can realize large-scale industrial production, the application process is simple, and the obtained eutectic product has strong process adaptability in subsequent applications, which is convenient for further processing and use.

[0018] (3) The eutectic morphology prepared by the present invention is stable and does not easily undergo phase separation or precipitation, which is conducive to maintaining performance consistency; it performs well in aqueous formulation systems such as cosmetics, and has high safety and wide applicability.

[0019] (4) This study provides a practical new scheme for the co-crystallization modification of fat-soluble natural active ingredients, which not only expands the application forms of glycyrrhetinic acid, but also provides a reference for improving the solubility and optimizing the performance of other poorly soluble functional ingredients. Attached Figure Description

[0020] Figure 1 This is a molecular structure diagram of the glycyrrhetinic acid-aminobutyric acid eutectic prepared in this invention; Figure 2 This is a powder X-ray diffraction pattern of the glycyrrhetinic acid-aminobutyric acid eutectic prepared in this invention. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0022] Example 1 A glycyrrhetinic acid-tributanol eutectic is prepared by the following method: (1) Accurately weigh 3.0 mmol (1.161 g) of glycyrrhetinic acid and 1.0 mmol (0.121 g) of tromethorphanol, and place them together in a 50 mL round-bottom flask to ensure that the two raw materials are fully mixed.

[0023] (2) Add methanol as an organic solvent to the reaction vessel. Based on the solid-liquid ratio of 100 mg: 10 mL, the total solid mass is 1.282 g. 128.2 mL of methanol needs to be added. Stir to make the powder evenly dispersed in the solvent. Place the round-bottom flask in a constant temperature water bath and set the temperature to 45℃. Turn on the magnetic stirrer and continue stirring for 1.0 h. It is observed that the system gradually forms a uniform and transparent clear solution with no obvious solid residue.

[0024] (3) Close the water bath, connect the round-bottom flask to the rotary evaporator, set the water bath temperature to 42℃ and the vacuum degree to 0.08 MPa, start the rotary evaporator to remove methanol. During the process, a white solid can be seen gradually precipitating on the inner wall of the flask. After the solvent is completely removed, stop the equipment to obtain a white solid crude product. Transfer the white solid crude product to a vacuum drying oven, set the drying temperature to 40℃ and the vacuum degree to 0.09 MPa, and dry for 18 h. After drying, take it out to obtain pure glycyrrhetinic acid-aminobutyric acid eutectic.

[0025] Example 2 A glycyrrhetinic acid-tributanol eutectic is prepared by the following method: (1) Accurately weigh 1.5 mmol (0.5805 g) of glycyrrhetinic acid and 1.5 mmol (0.1815 g) of tromethorphanol, put them into a 100 mL three-necked flask, and gently grind and mix them with a glass stirring rod to ensure that the raw materials are in full contact.

[0026] (2) A mixed solvent of ethanol and acetonitrile (volume ratio 1:1) was selected as the dissolving medium. Based on a solid-liquid ratio of 100 mg: 12 mL, the total solid mass was 0.762 g. 91.44 mL of mixed solvent was added. The solvent was slowly poured into a round-bottom flask. The round-bottom flask was placed in a constant temperature water bath. The heating temperature was set to 50 °C and the stirring rate was 300 r / min. The mixture was stirred continuously for 1.5 h to finally form a clear and transparent solution.

[0027] (3) Connect the reaction solution to a rotary evaporator, set the water bath temperature to 45℃ and the vacuum degree to 0.075 MPa, and perform vacuum distillation to remove the mixed solvent. As the solvent evaporates, the white solid gradually precipitates and aggregates. After the solvent is completely removed, a white solid crude product is obtained. Place the white solid crude product in a vacuum drying oven and maintain a vacuum condition of 40℃ and 0.095 MPa for 20 h. After drying, grind it through an 80-mesh sieve to obtain a powdered glycyrrhetinic acid-aminobutyric acid eutectic.

[0028] Example 3 A glycyrrhetinic acid-tributanol eutectic is prepared by the following method: (1) Accurately weigh 1.0 mmol (0.387 g) of glycyrrhetinic acid and 3.0 mmol (0.363 g) of tromethorphanol, place them in a 250 mL flat-bottomed flask, mix them evenly and set aside.

[0029] (2) Isopropanol was selected as the organic solvent. Based on the solid-liquid ratio of 100 mg: 14 mL, the total solid mass was calculated to be 0.75 g. 105 mL of isopropanol was added and slowly poured into the flask to cover all the solid raw materials. The flat-bottomed flask was placed in a constant temperature water bath shaker, and the shaking temperature was set to 55 °C and the shaking frequency was set to 200 times / min. The shaking was continued for 0.8 h to fully dissolve the raw materials and finally obtain a clear solution without suspended particles.

[0030] (3) Remove the flask and connect it to a rotary evaporator. Set the water bath temperature to 48℃ and the vacuum degree to 0.085 MPa. Start the equipment to remove isopropanol. During the process, control the rotation speed to 60 r / min to ensure uniform precipitation of the solid. After the solvent is completely removed, a loose white crude solid product is obtained. Transfer the white crude solid product to a vacuum drying oven and dry it at 40℃ and 0.09 MPa for 22 h to remove residual solvent. After drying, a glycyrrhetinic acid-aminobutyric acid eutectic product with good crystallinity is obtained.

[0031] Performance testing 1. Structural characterization The pure glycyrrhetinic acid-tromethamine eutectic prepared in Example 1 was used as the sample to be tested. The precise structure of the eutectic was characterized by single-crystal X-ray diffraction. The eutectic consists of two glycyrrhetinic acid molecules and one tromethamine molecule as the basic structural unit. The molecular structure diagram is shown below. Figure 1 As shown. The space group of the glycyrrhetinic acid-aminobutyric acid eutectic is C121, and the cell parameters are: a = 30.891(11) Å, b = 30.891(11) Å, c = 30.891(11) Å, α = 90.00°, β = 92.910(11)°, γ = 90.00°. Its powder X-ray diffraction (PXRD) pattern, Figure 2 Characteristic diffraction peaks appear at 6.6°, 9.06°, 12.08°, 13.12°, 14.72°, and 16.08°.

[0032] 2. Stability test The glycyrrhetinic acid-tromethamine eutectic (T1), pure glycyrrhetinic acid (T2), pure tromethamine (T3), and a physical mixture of glycyrrhetinic acid and tromethamine in a 2:1 molar ratio (T4) prepared in Example 1 were each ground through an 80-mesh sieve to ensure uniform particle size. 2 g samples were accurately weighed, sealed, and labeled as initial conditions. The samples were then placed in a constant temperature and humidity chamber at 40°C and 75% relative humidity for 6 months. The chamber was calibrated beforehand to ensure temperature fluctuations of ±1°C and humidity fluctuations of ±3%. The actual temperature and humidity inside the chamber were recorded monthly after sample placement. Samples were taken after 6 months, and the crystal form, solubility, dispersibility, and appearance were observed and tested.

[0033] The specific testing method is as follows: Crystal form detection: X-ray powder diffraction (XRD) is used to compare the characteristic diffraction peaks of samples at different time points to determine whether peak shape shift, new peak generation, or disappearance of characteristic peaks occurs.

[0034] Solubility detection: Take 0.1 g of sample at each time point, add 10 mL of deionized water, stir magnetically at 25℃ for 30 min, filter with a 0.45 μm filter membrane, determine the concentration of active ingredient in the filtrate by high performance liquid chromatography (HPLC), and calculate the solubility (mg / mL).

[0035] Dispersibility test: Take 0.5 g of sample, add 20 mL of deionized water, stir at 25℃ and 300 r / min for 10 min, and use a laser particle size analyzer to measure the particle size distribution (D50, D90) and observe whether agglomeration occurs (an increase of more than 20% in D90 is considered a decrease in dispersibility).

[0036] Appearance observation: Record the sample color (whether it turns yellow or darkens) and state (whether it clumps or absorbs moisture).

[0037] Table 1 Results of 6-month stability test As can be seen from the results in Table 1 above, the co-crystal obtained in this invention, after being placed under accelerated conditions of 40℃ and 75% relative humidity for 6 months, showed no change in crystal form, and no significant decrease in solubility and dispersibility. This indicates that when used in the conventional production of cosmetics, it exhibits good stability after emulsification (80℃, 30 min) and sterilization (60℃, 30 min) processes, ensuring the consistency of efficacy throughout the product's shelf life. The co-crystal was prepared by combining glycyrrhetinic acid and tromethamine through intermolecular hydrogen bonds, with a molar ratio limited to 3:1 to 1:3. This ratio range is the core parameter for ensuring the stability and property improvement effect of the co-crystal; any result exceeding this ratio will not achieve the effects described in this invention.

[0038] 3. Solubility test Measure 30 ml of water into 50 mL Erlenmeyer flasks with glass stoppers, and add excess of the test sample (glycyrrhetinic acid-tromethamine eutectic prepared in Examples 1-3, pure glycyrrhetinic acid, and a physical mixture of glycyrrhetinic acid and tromethamine in a 2:1 molar ratio). Seal the flasks and place them in a 25°C constant temperature water bath and stir for 2 hours to ensure solid-liquid equilibrium is reached. After standing for 30 min, filter through a 0.45 μm filter membrane and collect the filtrate. Measure the absorbance at a wavelength of 250 nm. Calculate the solubility by measuring the absorbance of the standard reference standard using HPLC. Each sample is tested in triplicate, and the results are expressed as the mean. The results are shown in Table 2.

[0039] Table 2. Solubility of different test samples in aqueous solution (mg / mL) As can be seen from the results in Table 2, the solubility of the glycyrrhetinic acid-tromethamine eutectic of the present invention is significantly better than that of pure glycyrrhetinic acid, increasing by 20 to 30 times compared to the solubility of the glycyrrhetinic acid raw material. Examples 1-3 of the present invention show similar solubility test results. This indicates that when this product is used in water-based systems commonly used in cosmetics, it can be uniformly dispersed without stratification or precipitation.

[0040] 4. Skin absorption rate The transdermal delivery efficiency of the active ingredient was evaluated using a Franz diffusion cell to simulate skin penetration. The specific method is as follows: Fresh pigskin (approximately 0.8 mm thick) was used as a transdermal barrier and fixed between the supply and receiving pools. The receiving pool was filled with phosphate-buffered saline (PBS) at pH 7.4 and maintained at a constant temperature of 37°C with continuous magnetic stirring. Test samples containing equal amounts of glycyrrhetinic acid (0.5%, w / v) (the eutectic suspension from Example 1, pure glycyrrhetinic acid suspension, and a physical mixture suspension, all prepared with buffer solution at pH 5.5 to simulate the skin surface environment) were added to the supply pool. At predetermined time points (2, 4, 6, 8, 12, 24 h), 2 mL samples were taken from the receiving pool (and immediately replenished with an equal volume of fresh receiving solution at the same temperature). The cumulative permeation of glycyrrhetinic acid in the receiving solution was determined using high-performance liquid chromatography (HPLC). The cumulative permeation over 24 hours (Q) was calculated. 24 μg / cm 2 ) and transdermal rate (Jss, μg / cm 2 / h). The results are shown in Table 3.

[0041] Table 3. Results of skin penetration test (mean ± SD, n=6) As shown in Table 3, the 24-hour cumulative skin penetration and steady-state transdermal rate of the glycyrrhetinic acid-tromethamine cocrystal prepared in this invention are significantly higher than those of pure glycyrrhetinic acid and its physical mixtures. This demonstrates that the cocrystal technology of this invention effectively improves the transdermal absorption performance of glycyrrhetinic acid, which is conducive to the more efficient delivery of its active ingredients to the deep layers of the skin, thereby enhancing its conversion efficiency in anti-inflammatory, repairing and other effects.

[0042] 5. Melanin content The inhibitory effect of the samples on melanin production was evaluated using a B16F10 mouse melanoma cell model. Cells were seeded in 96-well plates, and after cell attachment, the medium was replaced with fresh medium containing different concentrations of the samples (cocrystal and pure glycyrrhetinic acid, both dissolved in DMSO and diluted with culture medium, final DMSO concentration <0.1%) and α-melanocyte-stimulating hormone (α-MSH, 200 nM). After 72 hours of culture, cells were collected, lysed, and treated with NaOH solution. Absorbance was measured at 405 nm, and melanin content was calculated (with the normal cell group without α-MSH as the baseline, set as 100%). Cell viability was simultaneously determined using the CCK-8 assay to ensure that the tested concentrations were not cytotoxic. Safe and effective concentrations (10 μM, based on glycyrrhetinic acid) were selected for comparison, and the results are shown in Table 4.

[0043] Table 4. Effects of α-MSH on melanin production in B16F10 cells (mean ± SD, n=6) As can be seen from the results in Table 4 above, at the same safe concentration, the cocrystals prepared in Examples 1-3 of this invention showed significantly better inhibition rates against α-MSH-induced melanin overproduction than pure glycyrrhetinic acid. This indicates that improving solubility through cocrystalization enhances the bioavailability of glycyrrhetinic acid in cell models, thereby allowing its whitening and spot-fading potential to be more fully realized.

[0044] 6. Patch test skin reaction Human occlusive patch tests were conducted according to the *Cosmetic Safety Technical Specifications* to assess the safety of the samples. A test cream base containing 1.0% glycyrrhetinic acid (in eutectic or pure form) was prepared, with a blank cream base serving as a control. The test substance was evenly applied to a standard patch applicator and then applied to the flexor surface of the upper arm of 30 healthy volunteers (half male and half female). The patch applicator was removed at 24 and 48 hours after application. Dermatologists observed and recorded skin reactions (erythema, edema, papules, etc.) at 30 minutes, 24 hours, and 48 hours after removal, scoring them on a 0-4 scale. The test results are shown in Table 5.

[0045] Table 5 Results of human closed patch test The above-mentioned human patch test showed that the test sample containing 1% of the cocrystal of this invention did not induce any skin irritation in any volunteers, and the safety evaluation was non-irritating. In contrast, the same concentration of pure glycyrrhetinic acid caused mild, transient, and reversible erythema in a few volunteers. This indicates that preparing glycyrrhetinic acid into a tromethamine cocrystal not only does not introduce new safety risks, but may also reduce the risk of high-concentration crystal formation of active ingredients that irritate the skin due to better dispersibility and solubility, resulting in better skin tolerance, especially suitable for cosmetics repairing sensitive skin.

[0046] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A glycyrrhetinic acid-tributanol eutectic, characterized in that, Its molecular formula is [C4H] 11 NO3·2C 30 H 46 [O4] is composed of two glycyrrhetinic acid molecules and one tromethamine molecule as the basic structural unit; the eutectic space group is C121, and the cell parameters are: a = 30.891(11) Å, b = 30.891(11) Å, c = 30.891(11) Å, α = 90.00°, β = 92.910(11)°, γ = 90.00°.

2. The glycyrrhetinic acid-tributanol eutectic according to claim 1, characterized in that, The characteristic X-ray diffraction peaks of the eutectic powder are at 6.6°, 9.06°, 12.08°, 13.12°, 14.72°, and 16.08°.

3. A method for preparing the glycyrrhetinic acid-tributanol eutectic according to claim 1 or 2, characterized in that, Includes the following steps: 1) Weigh out glycyrrhetinic acid and tromethamine in molar ratio and place them in a reaction vessel; 2) Add organic solvent to the above container and stir to dissolve at 40~60℃ for 0.5~2 h to form a clear solution; 3) Place the above solution in a rotary evaporator and evaporate the solvent at 40~50℃ under reduced pressure to obtain a white crude solid. Then place the white crude solid in a vacuum drying oven and dry at 40℃ for 12~24 h to obtain the final product.

4. The method for preparing glycyrrhetinic acid-tributanol eutectic according to claim 3, characterized in that, In step (1), the molar ratio of glycyrrhetinic acid and tromethamine is 3:1 to 1:

3.

5. The method for preparing glycyrrhetinic acid-tributanol eutectic according to claim 3, characterized in that, The organic solvent is one or a mixture of two or more of methanol, ethanol, acetonitrile, and isopropanol.

6. The method for preparing glycyrrhetinic acid-tributanol eutectic according to claim 3, characterized in that, The solid-liquid ratio of the mixture powder to the organic solvent is 100 mg:(8~15) mL.

7. The application of the glycyrrhetinic acid-tributanol eutectic as described in claim 1 or 2 in skin care.

8. The application according to claim 7, characterized in that, The skin care includes moisturizing, anti-aging, anti-oxidation, whitening, anti-wrinkle, or repair.

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