WO2023109855A1 - Curcumin cocrystal, preparation method therefor, and use thereof as drug or in pharmaceutical preparation - Google Patents

Abstract

The present invention relates to a pharmaceutical cocrystal of curcumin, a preparation method therefor, and the use thereof as a drug or in a pharmaceutical preparation. The pharmaceutical cocrystal of curcumin has a good solubility, a good stability, a process developability, etc., and the preparation method therefor is simple and low in cost, which hold an important value for the optimization and development of the drug in the future.

Description

Curcumin co-crystal, preparation method and application thereof as medicine or in pharmaceutical preparations 【Technical field】

The invention belongs to the technical field of drug co-crystals, and in particular relates to curcumin co-crystals, a preparation method and their application as medicine or in pharmaceutical preparations.

【technical background】

French scientist M. Lehn et al. proposed the complete concept of "supramolecular chemistry" in 1978. Supramolecular chemistry is a kind of science used to study complex and ordered molecular aggregates formed by intermolecular interactions. These molecules Aggregates contain specific structures and functions. Crystal engineering was first proposed by Pepinsky and other scientists in 1955. Crystal engineering applies the principles and methods of supramolecular chemistry to the design and growth of crystals. Through the joint action of molecular recognition and self-assembly process, the structure can be adjusted. , a new crystal with specific physicochemical properties.

Supramolecular chemistry and crystal engineering provide a good platform for the rapid development of drug co-crystals. Pharmaceutical co-crystal is a new crystalline form with specific physical and chemical properties formed by active pharmaceutical ingredient (API) and cocrystal former (CCF) under the action of hydrogen bond or other non-covalent bonds. Co-crystals can change many physical and chemical properties of drugs, such as drug solubility, stability, melting point, etc. Drug co-crystal is a kind of solid state of drug, similar to salt, polymorph, solvent and hydrate of drug, all of which can change the physical and chemical properties of drug, thereby affecting the curative effect and pharmacological action of drug. The greatest application value of drug co-crystals in pharmacy is that it can achieve the purpose of modifying the physical and chemical properties of drugs without changing the covalent structure of drug molecules, which also provides a basis for improving the solubility and bioavailability of insoluble active ingredients in Chinese medicine. new solution.

Curcumin is a fat-soluble compound, insoluble in water, easily oxidized in vitro, less absorbed in vivo, metabolized too fast, and poor in stability, which restricts the wide application of curcumin in clinical practice. In recent years, the dosage forms of curcumin mainly include microemulsions, microspheres, solid dispersions, liposomes, phospholipid complexes, micelles, nanoparticles, and cyclodextrin inclusion complexes. Although the above methods have certain improvements in dissolution or oral absorption, there are still deficiencies, such as low drug loading, potential toxicity, and possible degradation of curcumin during the preparation process.

Compared with the above-mentioned methods for improving the properties of active pharmaceutical ingredients, the preparation of co-crystals is more operable, that is, there are no ionizable groups in the structure of active pharmaceutical molecules, and co-crystals can also be produced through process improvement, so that To achieve the purpose of improving the properties of the drug. Therefore, from the perspective of improving the efficacy of curcumin, increasing solubility, increasing dissolution rate, enhancing stability, and improving bioavailability, it is of great significance to design and synthesize new crystalline forms of curcumin.

【Content of invention】

The application of co-crystal technology to curcumin and other Chinese herbal medicines and natural pharmaceutical chemical components can greatly improve their poor water solubility and low oral absorption and utilization.

There are many types of curcumin co-crystal formers, which can be divided into: (1) amides: such as nicotinamide; (2) phenols: such as hydroquinone, resorcinol, pyrogallol, resorcinol Triphenols, pyroglucinol, 4-aminophenol; (3) carboxylic acids: such as p-hydroxybenzoic acid, L-tartaric acid, lysine, salicylic acid; (4) heterocycles: such as BPNO, 2- Aminopyridine, 4-hydroxypyridine, 4,6-dihydroxy-5-nitropyrimidine. The drug co-crystal structure can be obtained by solvent method, grinding method or solvent-assisted grinding method, which has the characteristics of improving the solubility and dissolution rate of curcumin, improving drug stability and bioavailability (Research progress of curcumin co-crystal, Pharmaceutical Chemical Industry, 2017 , 48(12):12-17).

However, in previous studies, researchers mostly chose non-natural compounds such as nicotinamide, hydroquinone, resorcinol, and pyrogallol as CCFs. Due to the toxic and side effects of these chemically synthesized compounds, curcumin co-crystals There are certain limitations in clinical application research. Traditional Chinese medicine has a long history, and its drug safety has been tested for a long time. Most of the active ingredients of traditional Chinese medicine can be used as potential CCFs to form co-crystals with therapeutic drugs. While improving the physical and chemical properties and bioavailability of API, they can also play a role in drug synergy. effect.

Ferulic acid is an aromatic acid commonly found in the plant kingdom. It has a variety of physiological activities, such as scavenging free radicals, antithrombotic, antibacterial and anti-inflammatory, inhibiting tumors, preventing and treating hypertension, heart disease, and enhancing sperm motility. Ferulic acid has low toxicity and is easy to be metabolized by the human body. It can be used as a food preservative and has a wide range of uses in food and medicine. N.Rajesh Goud et al. reported the preparation of curcumin-ferulic acid co-melt by grinding method. It was proved by PXRD, IR and 13C NMR tests that curcumin and ferulic acid did not form eutectic compounds (Fast dissolving eutectic compositions of curcumin, International Journal of Pharmaceuticals, 2012, 439:63-72).

In this eutectic system, although the two components are also combined into a "one body" through weak interactions, the microstructure of the eutectic and the eutectic compound is not completely consistent, that is, the eutectic forms a secondary component (CCF ) in the supersaturated state of the main component (API) lattice, usually in a layered arrangement, is a "heterogeneous system"; in contrast, eutectic compounds are formed by mutual "permeation" between the two components "Homogeneous system".

In this co-melting system, due to the high solubility of the secondary components in the solvent (40% ethanol aqueous solution), curcumin was separated out in the form of precipitation, which indicated that the inconsistency of the solubility of curcumin and ferulic acid caused each component in the Dissociation occurs during dissolution, so ferulic acid has limited effect on improving the solubility of curcumin. However, the eutectic compound is different. Due to the homogeneous system formed by the mutual "permeation" between the two phases, the eutectic form always maintains "connection" with curcumin when the system dissolves, thereby improving the solubility of curcumin to a certain extent. effect.

Gallic acid is a polyphenolic organic compound, which is widely found in plants such as Rhubarb palmatum, Eucalyptus eucalyptus, dogwood, etc. It has significant antioxidant effects, and it also has anti-tumor, trypanocidal, liver protection and anti-hepatitis B properties. It has many functions such as viruses, and is widely used in food, biology, medicine, chemical industry and other fields. Wenzhe Pang et al. reported the preparation of curcumin-gallic acid co-crystal powder by grinding method. Although the preparation of drug co-crystal by grinding method is easy to operate, the experimental reproducibility is not good, and there is still the interference of raw materials when the grinding method is insufficient. The obtained The crystal structure is unstable (Cocrystals of curcumin-isonicotinamide and curcumin-gallic acid: Does the weak forces in cocrystals effect on binding profiles with BSA and cell cytotoxicity, European Journal of Pharmaceutics and Biopharmaceutics, 2 019,140:78–90).

In order to solve the deficiencies in the prior art, the present invention provides curcumin co-crystals and a preparation method of the curcumin co-crystals, aiming at stably obtaining the drug co-crystals.

The second object of the present invention is to provide the application of the curcumin co-crystal as medicine or in pharmaceutical preparations.

Through a lot of research, the present inventors have found originality that curcumin is used as the co-crystal pharmaceutical active ingredient (API), and ferulic acid and gallic acid are used as the co-crystal precursor (CCF), which can be obtained by intermolecular hydrogen bonding. The curcumin co-crystal described above.

Preferably, the curcumin-ferulic acid cocrystal has main characteristic peaks at diffraction angles 2θ of 13.82°±0.2°, 17.91°±0.2° and 27°±0.2° under powder X-ray diffraction. Described curcumin-gallic acid eutectic is under powder X-ray diffraction, is 8.5 ° ± 0.2 °, 9.2 ° ± 0.2 °, 9.9 ° ± 0.2 °, 12.1 ° ± 0.2 °, 13.9 ° ± 0.2 ° at diffraction angle 2θ °, 28°±0.2° have main characteristic peaks.

In the curcumin-ferulic acid eutectic, the ratio of curcumin to ferulic acid is 1/1 molar ratio.

In the described curcumin-gallic acid co-crystal, the ratio of curcumin to gallic acid is 1/2 molar ratio.

As a preference, the melting point of the curcumin-ferulic acid co-crystal is 153°C±0.2°C as tested by a differential scanning calorimeter. The melting point of the curcumin-gallic acid co-crystal is 178°C±0.2°C.

The present invention also provides a kind of preparation method of described curcumin-ferulic acid cocrystal, comprises the following steps:

Step (1): Self-assemble curcumin and eutectic precursor ferulic acid with a mass ratio of 5:1-1:5 in a crystallization solvent; the temperature of the self-assembly process is 25-70°C; the time is 2 ~12 hours. Described crystallization solvent is the mixed solvent of one or at least two compositions in toluene, acetonitrile, methylene chloride, methyl alcohol, dehydrated alcohol, Virahol and ether; The weight of curcumin and eutectic precursor and the weight of crystallization solvent The volume ratio is 5-30 mg/mL;

Step (2): The solution obtained in step (1) is volatilized and crystallized at room temperature, washed and dried to obtain the curcumin co-crystal.

The present invention also provides a kind of preparation method of described curcumin-gallic acid co-crystal, comprises the following steps:

Step (1): Put curcumin and co-crystal precursor gallic acid with a mass ratio of 5:1 to 1:5 in an agate mortar, add 5-10 drops of absolute ethanol to the above mixture, and wet grind 5 minutes; after the mixed powder is dried, gradually add a crystallization solvent for self-assembly; the temperature of the self-assembly process is 25-80° C.; the time is 2-12 hours. The crystallization solvent is a mixed solvent of one or at least two of methanol, absolute ethanol, isopropanol, tetrahydrofuran and ether; the volume ratio of the weight of curcumin and the co-crystal precursor to the crystallization solvent is 5- 30mg/mL;

Step (2): The solution obtained in step (1) is volatilized and crystallized at room temperature, washed and dried to obtain the curcumin co-crystal.

Compared with curcumin, the curcumin co-crystal prepared by the invention has better solubility and stability, is convenient for storage and use, can be directly used in the preparation of solid preparations, and has good powder properties. At the same time, the present invention selects ferulic acid and gallic acid as co-crystal precursors from natural drug active ingredients, and the prepared drug co-crystal and its pharmaceutical composition can play a synergistic effect.

【Description of drawings】

Fig. 1 is the PXRD pattern of curcumin, ferulic acid, curcumin-ferulic acid co-crystal and curcumin-ferulic acid blend prepared according to literature, wherein: CU is curcumin, FA is ferulic acid, CU -FA is curcumin-ferulic acid co-crystal, CU-FA_eutectic is curcumin-ferulic acid co-fusion.

Fig. 2 is the DSC figure that curcumin and ferulic acid and its cocrystal test under nitrogen atmosphere, wherein: Fig. 2 (a) is curcumin (CU); Fig. 2 (b) is ferulic acid (FA); Fig. 2(c) is curcumin-ferulic acid co-crystal (CU-FA).

Fig. 3 is the ¹ H NMR figure of curcumin-ferulic acid co-crystal (CU-FA), curcumin and ferulic acid equiproportion mixture (CU-FA Blend), wherein: Fig. 3a is curcumin-ferulic acid co-crystal crystal (CU-FA); Figure 3b is a mixture of curcumin and ferulic acid in equal proportions (CU-FA Blend).

Fig. 4 is the equilibrium solubility of curcumin (CU), curcumin-ferulic acid eutectic (CU-FA) and curcumin-ferulic acid blend (CU-FA_eutectic) prepared according to literature in 40% ethanol aqueous solution Curve (25°C).

Fig. 5 is a relation diagram of curcumin content (CU%) in curcumin solid powder (CU) and curcumin-ferulic acid eutectic compound (CU-FA) with light time.

Fig. 6 is a graph showing the relationship between curcumin content (CU%) in curcumin solid powder (CU) and curcumin-ferulic acid eutectic compound (CU-FA) as the time of the accelerated experiment changes.

Fig. 7 is the PXRD pattern of curcumin-ferulic acid co-crystal compound (CU-FA) and its product after light experiment and accelerated experiment. Among them, CU-FA_illumination experiment is the PXRD pattern of the illumination test sample of CU-FA; CU-FA_accelerated experiment is the PXRD pattern of the accelerated test sample of CU-FA.

Fig. 8 is the PXRD figure of curcumin, gallic acid, curcumin-gallic acid eutectic and the curcumin-gallic acid powder prepared by grinding method according to literature, wherein: CU is curcumin, GA is gallic acid, and CU-GA is Curcumin-gallic acid co-crystal, CU-GA-grinding powder is curcumin-gallic acid powder prepared by grinding method.

Fig. 9 is the DSC figure that curcumin and gallic acid and its cocrystal test under nitrogen atmosphere, wherein: Fig. 9 (a) is curcumin (CU); Fig. 9 (b) is gallic acid (GA); Fig. 9 ( c) is curcumin-gallic acid co-crystal (CU-GA).

Fig. 10 is the ¹ H NMR figure of curcumin-gallic acid cocrystal (CU-GA), curcumin and gallic acid equiproportion mixture (CU-GA Blend), wherein: Fig. 10a is curcumin-gallic acid cocrystal (CU -GA); Figure 10b is an equal proportion mixture of curcumin and gallic acid (CU-GA physical blend).

Fig. 11 is curcumin (CU), curcumin-gallic acid eutectic (CU-GA) and (CU-GA-grinding powder) of curcumin-gallic acid powder prepared by grinding method according to literature in 40% ethanol aqueous solution Dissolution rate graph.

Fig. 12 is a relation diagram of curcumin content (CU%) in curcumin solid powder (CU) and curcumin-gallic acid eutectic compound (CU-GA) as a function of light time.

Fig. 13 is a graph showing the relationship between curcumin content (CU%) in curcumin solid powder (CU) and curcumin-gallic acid eutectic compound (CU-GA) as a function of accelerated test time.

Fig. 14 is the PXRD pattern of curcumin-gallic acid co-crystal compound (CU-GA) and its product after light experiment and accelerated experiment investigation. Among them, CU-GA_illumination experiment is the PXRD pattern of the illumination test sample of CU-GA; CU-GA_accelerated experiment is the PXRD pattern of the accelerated test sample of CU-GA.

【Detailed ways】

The instrument that detects drug eutectic structure among the present invention is as follows:

1. X-ray powder diffractometer, produced by Shimadzu Corporation, model XRD-6000X, Cu-K(α), tube voltage 40kV, tube current 40mA, scanning speed 2°/min.

2. Differential scanning calorimeter, American TA company, model Q20, the present invention adopts nitrogen atmosphere, heating rate 10 ℃/min.

3. Varian Unity INOVA 400 nuclear magnetic resonance instrument, using DMSO-d6 as the deuterated reagent and TMS as the internal standard to measure ¹ H NMR. The observation frequency of the ¹ H NMR spectrum is 400 MHz, the observation spectrum width of the ¹ H NMR spectrum is 10 KHz, the pulse angle is 30°, and the pulse repetition time is 10 s. The test temperature is 40°C.

4. Agilent 1260Infinity high performance liquid chromatography, C18 column (4.6mm×250mm, 5μm). Among them, the liquid phase conditions of curcumin-ferulic acid cocrystal: mobile phase is acetonitrile-2% glacial acetic acid aqueous solution (55:45); detection wavelength is 425nm; flow rate is 1mL/min, column temperature is 30 ℃, 20 μL. The liquid phase conditions of curcumin-gallic acid cocrystal: mobile phase is water-2% glacial acetic acid aqueous solution (90:10); detection wavelength is 300nm; flow rate is 1mL/min, column temperature is 30 ℃, and injection volume is 20 μ L.

5. The excess sample was added to 6 mL of 40% ethanol-water, and the obtained supersaturated solution was placed at 25° C. to test its equilibrium solubility.

6. Sample light stability test method:

Place the samples in a weighing bottle with a thickness of no more than 2mm, spread them evenly, place them in a D65/ID65 emission standard light source, and expose them to cool white fluorescent lamps and near-ultraviolet lamps (spectral range: 320-400nm, maximum emission Energy 350~370nm). Under the condition that the total illuminance is not lower than 1.2×106Lux·hr, and the near-ultraviolet energy is not lower than 200w·hr/m ² ), the investigation is carried out for 8 weeks, and on the 1st day, 3rd day, 5th day, 2nd week, 3rd week, 4th day Samples were taken at 1 week, 5 weeks, 6 weeks, 7 weeks, and 8 weeks to detect changes in curcumin content.

7. Sample acceleration test method:

Place the samples in weighing bottles with a thickness of no more than 2mm, and spread them evenly. Under the conditions of 40°C and RH75%, inspect them for 8 weeks. At 4 weeks, 5 weeks, 6 weeks, 7 weeks, and 8 weeks, samples were taken to detect changes in curcumin content.

Example

Co-crystals were synthesized by solvent evaporation at room temperature using curcumin and co-crystal precursor ferulic acid:

The reactants are fed according to the mass ratio of curcumin:ferulic acid=1:1.

Use a pipette gun to accurately pipette 2mL of ethanol, 3mL of dichloromethane and 2mL of acetone solvent into a glass vial, stir on a magnetic stirrer for 4 hours at a temperature of 25°C, take out the stirring bar, and place it between the bottle cap and the bottle body Add a layer of tin foil to increase the airtightness and tighten.

Place the glass vial at room temperature, seal it slightly with tin foil to prevent foreign matter from entering the culture solution, let it stand for 5 days, the solution reaches saturation, and crystals begin to precipitate. With the continuous volatilization of the solvent, orange-yellow needle-like transparent crystals are obtained, namely It is curcumin-ferulic acid co-crystal.

As can be seen from Fig. 1, curcumin-ferulic acid co-crystal (CU-FA) has obvious differences from raw materials (CU and The characteristic diffraction peaks of FA) and the main characteristic peaks of CU and FA disappeared in the co-crystal compound, indicating that CU and FA formed a co-crystal compound with a new crystal structure through hydrogen bonding. On the other hand, the characteristic diffraction peaks of the curcumin-ferulic acid co-fusion (CU-FA_eutectic) obtained according to the preparation method in the literature are completely consistent with the raw materials curcumin and ferulic acid, and no new characteristic diffraction peaks appear. It is consistent with the results reported in the literature.

The ratio of curcumin to ferulic acid in the curcumin-ferulic acid co-crystal is determined to be 1/1 molar ratio by high performance liquid chromatography (HPLC).

It can be seen from Figure 2 that the melting point of curcumin (CU) is 186°C, the melting point of ferulic acid (FA) is 175°C, and the melting point of curcumin-ferulic acid cocrystal (CU-FA) is 153°C, which are significantly different. The melting point (145°C) of the curcumin-ferulic acid co-melt reported in the literature is also different from that of the raw material drug, which indicates that CU and FA form a co-crystal compound through self-assembly.

From the comparison of Figure 3a and Figure 3b, it can be seen that the H NMR spectrum of curcumin-ferulic acid co-crystal (CU-FA), curcumin and ferulic acid equal proportion mixture (CU-FA Blend) is between 9.5-9.75ppm and 12 There is a clear difference in the ~12.25ppm region. Because both CU-FA and CU-FA Blend are dissolved with DMSO-d6 deuterated reagent and tested for ¹ H NMR, it will not affect the nuclear magnetic resonance effect of active hydrogen in the sample. After the active hydrogen on the carboxyl group of ferulic acid forms a cocrystal with curcumin through hydrogen bonding, its active hydrogen completely disappears in the 12-12.25ppm region in Figure 3a (see Figure 3a); meanwhile, the curcumin and ferulic acid The phenolic hydroxyl group interacts through hydrogen bonds, and its peak shape in the 9.5-9.75ppm region (see Figure 3a) is also different from the physical mixture of raw materials (see Figure 3b). These differences all indicate that curcumin and ferulic acid pass hydrogen Bonding forms the eutectic compound.

It can be seen from Figure 4 that the equilibrium solubility of curcumin-ferulic acid co-crystal (CU-FA) and curcumin-ferulic acid co-fusion (CU-FA_eutectic) prepared according to the literature is higher than that of curcuma longa in 40% ethanol aqueous solution Element (CU). In view of the difference in microstructure between eutectic and eutectic systems, the solubility of CU-FA was significantly higher than that of CU-FA_eutectic at different times (1h, 3h, 5h, 7h, 12h).

As can be seen from Figure 5, after 3 consecutive days of indoor light experiments, the content of curcumin in the curcumin solid powder decreased significantly. In contrast, the content of curcumin in the curcumin-ferulic acid cocrystal compound remained basically unchanged after 8 consecutive weeks of indoor light experiments, indicating that it had better photostability.

It can be seen from Figure 6 that under the accelerated test conditions, the content of curcumin in the curcumin solid powder and the curcumin-ferulic acid eutectic compound basically remained unchanged, indicating that the two have better heat and humidity stability.

It can be seen from Figure 7 that the crystal form of the curcumin-ferulic acid co-crystal compound is consistent with that before the test after the light test and accelerated test, indicating that the crystal structure of the curcumin-ferulic acid co-crystal compound is very stable.

Co-crystals were synthesized by solvent evaporation at room temperature using curcumin and co-crystal precursor gallic acid:

The reactant is fed by curcumin: gallic acid=1:1 mass ratio. Add 5-10 drops of absolute ethanol dropwise to the above mixture, and wet grind for 5 minutes; after the mixed powder is dry, transfer the mixture powder to a glass vial, and gradually add a mixed solvent of 2mL of absolute ethanol and 4mL of tetrahydrofuran, at 60°C Stir for 4 hours on a magnetic stirrer under the same conditions, take out the stirring bar, add a layer of tin foil between the bottle cap and the bottle body to increase the sealing, and tighten it.

Place the glass vial at room temperature, seal it slightly with tin foil to prevent foreign matter from entering the culture solution, let it stand for 7 days, the solution reaches saturation, and crystals begin to precipitate. With the continuous volatilization of the solvent, orange-yellow needle-like transparent crystals are obtained, namely It is curcumin-gallic acid co-crystal.

It can be seen from Figure 8 that curcumin-gallic acid eutectic (CU-GA) is 8.5°±0.2°, 9.2°±0.2°, 9.9°±0.2°, 12.1°±0.2°, 13.9°±0.2° at diffraction angle 2θ °, 28°±0.2°, there are characteristic diffraction peaks that are obviously different from those of the raw materials (CU and GA). At the same time, the main characteristic peaks in CU and GA disappear in the eutectic compound, indicating that CU and GA form a A co-crystal compound with a new crystal structure. On the other hand, the CU-GA prepared by the present invention and the eutectic powder reported in the literature have obvious differences in crystal structure, that is, the eutectic powder reported in the literature has a new characteristic peak at 6.5°±0.2°, while the present invention The prepared CU-GA does not appear; at the same time, the eutectic powder reported in the literature is the main characteristic peak at 9.9°±0.2°, while the characteristic peak intensity of the CU-GA prepared by the present invention is extremely weak at the position of 13.9° Obvious characteristic peaks appear and are the main characteristic peaks.

By means of high performance liquid chromatography (HPLC), it is determined that in the curcumin-gallic acid co-crystal, the ratio of curcumin to gallic acid is 1/2 molar ratio.

The melting point of gallic acid is between 235°C and 240°C (unstable at this temperature, when gallic acid is heated above 200°C, it loses carbon dioxide and produces pyrogallic acid, namely pyrogallol). It can be seen from Figure 9 that gallic acid (GA) does not have a melting endothermic peak within the test temperature range (that is, no melting point is measured), and only a solid-solid phase transition occurs in the range of 60°C to 90°C. Figure 9 shows that the melting point of curcumin (CU) is 186 ° C, while the melting point of curcumin-gallic acid cocrystal (CU-GA) is 178 ° C, which is obviously different from the raw material drug, and is different from that of curcumin-gallic acid reported in the literature. The melting point (174.8°C) of the eutectic powder is also different, which indicates that CU and GA form a eutectic compound through self-assembly.

From the comparison of Figure 10a and Figure 10b, it can be seen that due to the weak interaction between the phenolic hydroxyl group and the carboxyl group in the molecular structure of curcumin and gallic acid, the curcumin-gallic acid co-crystal (CU-GA), a mixture of curcumin and gallic acid in equal proportions (CU-GA physical blend) The peak shape and intensity of the hydrogen nuclear magnetic resonance spectrum are significantly different in the 8.75-9.75ppm and 12-12.5ppm regions, which shows that curcumin and gallic acid form a co-crystal compound through hydrogen bonding.

As can be seen from Figure 11, the equilibrium solubility of curcumin-gallic acid eutectic (CU-GA) in 40% ethanol aqueous solution is obviously higher than curcumin (CU) and curcumin-gallic acid powder (CU-GA- grinding powder).

It can be seen from Figure 12 that after 3 consecutive days of indoor lighting experiments, the content of curcumin in the curcumin solid powder decreased significantly. In contrast, the content of curcumin in the curcumin-gallic acid co-crystal compound remained basically unchanged after 8 consecutive weeks of indoor light experiments, indicating that it had better photostability.

It can be seen from Figure 13 that under the accelerated test conditions, the content of curcumin in the curcumin solid powder and the curcumin-gallic acid eutectic compound basically remained unchanged, indicating that the two have better heat and humidity stability.

It can be seen from Figure 14 that after the light test and accelerated test of the curcumin-gallic acid co-crystal compound, the crystal form of the sample is consistent with that before the test, indicating that the crystal structure of the curcumin-gallic acid co-crystal compound is very stable.

Curcumin and Curcumin Co-crystal Inhibitory Rate Determination of Cancer Cells

Dispersing curcumin into DMEM medium to prepare curcumin suspension is recorded as curcumin group; dissolving curcumin-ferulic acid co-crystal in DMEM medium is recorded as curcumin-ferulic acid co-crystal group; The curcumin-ferulic acid blend prepared by grinding method according to the literature (Fast dissolving eutectic compositions of curcumin, International Journal of Pharmaceuticals, 2012,439:63-72) was dissolved in DMEM medium , recorded as the curcumin-ferulic acid co-crystal group; the curcumin-gallic acid co-crystal was dissolved in DMEM medium, and recorded as the curcumin-gallic acid co-crystal group; the concentrations of the above four groups of samples were calculated by curcumin , both 100 μmol/L. At the same time, ferulic acid and gallic acid were dissolved in DMEM medium respectively, and the corresponding ligand mass concentration samples in the curcumin-ferulic acid co-crystal group and curcumin-gallic acid co-crystal group were prepared, respectively recorded as Ferulic acid group, gallic acid group.

MTT method was used to study the inhibitory effect of samples on the proliferation of MCF-7 cells and A549 cells. MCF-7 and A549 cells were cultured with DMEM medium containing 10% fetal bovine serum and 100 U/mL double antibody, and cultured in a constant temperature incubator containing 5% _CO2 gas at 37°C until the cells covered 80% of the bottom area of the bottle. %; After digested with trypsin, inoculated to 96-well plate at a concentration of 1×10 ⁵ cells/mL, 200 μL per well, and cultured for 6 hours. Discard the old medium, add 200 μL of fresh medium or sample solution, and continue culturing for 24 h. The old medium was discarded, washed twice with phosphate buffered saline, 200 μL of fresh medium and 10 μL of MTT (5 mg/mL) were added to each well, and the culture was continued for 4 h. Pour off the liquid in the culture medium, suck off the foam, add 150 μL DMSO to react for 10 minutes, measure the absorbance of the sample well at a wavelength of 550 nm with a microplate reader, record the absorbance of the sample well with MTT as A2, and record the absorbance of the well with an equal volume of medium as A1, The well without MTT was recorded as A0, and the inhibition rate was calculated according to formula (1). The inhibition rate of curcumin co-crystal group is the data after deducting the corresponding inhibition rate of ferulic acid group or gallic acid group.

Inhibition rate/%＝[(A1-A0)-(A2-A0)]/(A1-A0)×100 - formula (1)

Table 1 The inhibitory effect of compounds on A549 and MCF-7 cells

Compound group	Inhibition rate of MCF-7 cells/%	Inhibition rate of A549 cells/%
Curcumin group	27±0.93	19±0.93
ferulic acid group	31±1.88	22±2.8
gallic acid group	29±2.48	18±1.7
Curcumin-Ferulic Acid Blend Group	43±0.36	51±2.4
Curcumin-ferulic acid co-crystal group	82±0.9	92±0.5
Curcumin-Gallic Acid Co-Crystal Group	76±0.33	85±1.9

As shown in Table 1, curcumin group, ferulic acid group and gallic acid group have little inhibitory effect on the above two cancer cells, and the curcumin-ferulic acid blend group has slightly less inhibitory effect on the two cancer cells. increased, but the inhibition rate remained at a low level.

The inhibition rates of the two curcumin cocrystals on cancer cells were significantly higher than those of the control group above under most concentration conditions. Among them, the curcumin-ferulic acid co-crystal group had a slightly better inhibitory effect on the two cancer cells than the curcumin-gallic acid co-crystal group.

Curcumin has low water solubility. After reaching the saturated concentration, continue to increase the amount of curcumin in water, and the number of curcumin molecules dissolved in water will not increase, so the inhibitory effect on cancer cells will no longer be enhanced. In the same way, ferulic acid and gallic acid are insoluble compounds in water, and their inhibitory effects on the two cancer cells are relatively low.

The prepared curcumin co-crystal can increase the solubility of curcumin, and can provide more free curcumin molecules to contact with cancer cells, thus improving the inhibitory effect on cancer cells.

As mentioned earlier, in the curcumin-ferulic acid fusion group, because ferulic acid has a limited effect on improving the solubility of curcumin, the curcumin-ferulic acid fusion group has no effect on the two cancers. The inhibition of cells is not ideal.

Claims (10)

1. The curcumin co-crystal is characterized in that: curcumin, as an active substance, forms a co-crystal with other compounds as co-crystal precursors, wherein the co-crystal precursor is selected from one or more of the following compounds: ferulic acid , Gallic acid.
2. The curcumin co-crystal according to claim 1, characterized in that: the curcumin co-crystal is self-assembled from curcumin and a co-crystal precursor with a mass ratio of 5:1 to 1:5.
3. Curcumin co-crystal as claimed in claim 2 is characterized in that: curcumin-ferulic acid co-crystal is one or at least two in toluene, acetonitrile, methylene chloride, methyl alcohol, dehydrated alcohol, Virahol and ether self-assembly, volatilization and crystallization in a mixed solvent; curcumin-gallic acid co-crystal self-assembly, volatilization obtained by crystallization.
4. The curcumin eutectic as claimed in any one of claims 1 to 3, wherein the curcumin-ferulic acid eutectic is 13.82° ± 0.2° at the diffraction angle 2θ under powder X-ray diffraction. There are main characteristic peaks at 17.91°±0.2° and 27°±0.2°; preferably, in the curcumin-ferulic acid co-crystal, the ratio of curcumin to ferulic acid is 1/1 molar ratio.
5. The curcumin eutectic according to any one of claims 1 to 3, characterized in that: the curcumin-gallic acid eutectic is 8.5°±0.2°, 9.2° at the diffraction angle 2θ under powder X-ray diffraction. °±0.2°, 9.9°±0.2°, 12.1°±0.2°, 13.9°±0.2°, 28°±0.2° have main characteristic peaks; preferably, in curcumin-gallic acid cocrystal, curcumin and gall The ratio of acid is 1/2 molar ratio.
6. The curcumin eutectic as claimed in any one of claims 1 to 3, wherein the fusing point of the curcumin-ferulic acid eutectic is 153°C ± 0.2°C, and the fusing point of the curcumin-gallic acid eutectic is 178°C °C±0.2°C.
7. The preparation method of the curcumin eutectic as described in any one of claim 1～6, it is characterized in that:

   The preparation method of the curcumin-ferulic acid co-crystal includes step (1): self-assembling curcumin and co-crystal precursor ferulic acid with a mass ratio of 5:1 to 1:5 in a crystallization solvent; The temperature of the self-assembly process is 25-70°C; the time is 2-12 hours; the crystallization solvent is one or at least one of toluene, acetonitrile, methylene chloride, methanol, absolute ethanol, isopropanol and ether A mixed solvent of two compositions; the volume ratio of the weight of curcumin and the co-crystal precursor to the crystallization solvent is 5 to 30 mg/mL; step (2): the solution obtained in step (1) is volatilized and crystallized at room temperature, washed, drying to obtain the curcumin co-crystal;

   The preparation method of described curcumin-gallic acid co-crystal comprises, step (1): mix curcumin and co-crystal precursor gallic acid that mass ratio is 5:1～1:5, drop 5 to above-mentioned mixture - 10 drops of absolute ethanol, wet grinding for 5 minutes; after the mixed powder is dried, gradually add in the crystallization solvent for self-assembly; the temperature of the self-assembly process is 25-80°C; the time is 2-12 hours; The crystallization solvent is a mixed solvent composed of one or at least two of methanol, absolute ethanol, isopropanol, tetrahydrofuran and ether; the weight ratio of curcumin and co-crystal precursor to the crystallization solvent is 5-30mg/mL Step (2): The solution obtained in step (1) is volatilized and crystallized at room temperature, washed and dried to obtain the curcumin co-crystal.
8. A pharmaceutical composition comprising the curcumin co-crystal compound according to any one of claims 1 to 7, optionally further comprising a pharmaceutically acceptable carrier or excipient.
9. The pharmaceutical composition of claim 8, wherein the solid state of the compound is any crystalline polymorph or amorphous form or a mixture thereof.
10. Curcumin cocrystal as described in any one of claims 1 to 6 or pharmaceutical composition as described in claim 8 is used in the preparation of anticancer and antitumor, anti-Alzheimer's disease and the medicine for treating diabetes application.

Images