Co-crystal of silybin and l-proline, and preparation method therefor and use thereof

Abstract

The present invention relates to a co-crystal of silybin and L-proline, and a preparation method therefor and the use thereof. The stoichiometric ratio of silybin to L-proline in the co-crystal of silybin and L-proline of the present invention is 1:2. The present invention further provides a composition comprising the co-crystal of silybin and L-proline as described above, and optionally a pharmaceutically, bromatologically or cosmetically acceptable excipient. In the composition, the stoichiometric ratio of silybin to L-proline is 5:1-1:9. Compared with silybin, the co-crystal of silybin and L-proline and the composition comprising same described in the present invention have better dissolution performance and significantly improved bioavailability.

Description

A eutectic of silymarin and L-proline, its preparation method and uses Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, and particularly relates to pharmaceutical co-crystal technology. Specifically, this invention relates to the co-crystal of silymarin and L-proline and its preparation method, as well as products containing the co-crystal and their uses. Background Technology

[0002] Silybin is a natural flavonoid and the main active ingredient in milk thistle extract. It consists of two diastereomers, silybin A and silybin B, in a stoichiometric ratio of 1:1. Their structural formulas are shown below:

[0003] Silymarin is a flavonoid compound extracted and isolated from the fruit of the milk thistle plant. It has significant protective and stabilizing effects on hepatocyte membranes, can improve liver function, and produce enzyme-lowering effects. Currently, it is used clinically for the recovery of abnormal liver function in acute and chronic hepatitis and fatty liver. However, due to its extremely poor water solubility (only 51 ng / mL in pure water at 37°C), silymarin has low bioavailability and requires relatively high dosages. Summary of the Invention

[0004] To improve the bioavailability of silybin, this invention improves the solubility and dissolution properties of silybin by forming a eutectic with L-proline, thereby enhancing its bioavailability.

[0005] One of the objectives of this invention is to provide a silymarin-L-proline co-crystal.

[0006] The second objective of this invention is to provide a method for preparing the silymarin-L-proline cocrystal.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] In one aspect, the present invention provides a eutectic of silybin and L-proline, wherein the stoichiometric ratio of silybin to L-proline in the eutectic is 1:2.

[0009] In some embodiments, the silybin is silybin A, or silybin B, or a racemic mixture of both.

[0010] In some embodiments, the X-ray powder diffraction pattern of the silymarin-L-proline cocrystal exhibits characteristic peaks at at least the 2θ angles of 8.9°±0.2°, 11.0°±0.2°, 14.9°±0.2°, 16.7°±0.2°, 18.0°±0.2°, 19.0°±0.2°, 19.6°±0.2°, and 23.9°±0.2°. Specifically, the X-ray powder diffraction pattern of the cocrystal also exhibits characteristic peaks at the 2θ angles of 17.3°±0.2°, 18.7°±0.2°, 20.9°±0.2°, and 22.1°±0.2°.

[0011] Preferably, the silymarin-L-proline eutectic has an X-ray powder diffraction pattern as shown in Figure 1.

[0012] In some embodiments, the eutectic of silymarin and L-proline, as determined by differential scanning calorimetry, exhibits an endothermic peak at 206±2℃; preferably, it has a differential scanning calorimetric spectrum as shown in Figure 2.

[0013] In some embodiments, the infrared absorption spectrum of the silymarin-L-proline cocrystal is at least at 3473 cm⁻¹. -1 3283cm -1 3129cm -1 2961cm -1 1609cm -1 1510cm -1 1461cm -1 1384cm -1 1272cm -1 1171cm -1 1131cm -1 1085cm -1 1029cm -1 996cm -1 829cm -1 It has an absorption peak at a certain point; preferably, it has an infrared spectrum as shown in Figure 3.

[0014] Secondly, the present invention provides a method for preparing the silymarin-L-proline cocrystal, the method comprising: recrystallizing silymarin and L-proline in a solvent at a stoichiometric ratio of 1:2-1:2.8, for example 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, and obtaining the cocrystal after separation and drying of the precipitate.

[0015] In some embodiments, the solvent is one or more selected from water, alcohols, ketones, esters, alkanes, aromatic hydrocarbons and haloalkanes; preferably, the solvent is ethanol or a solvent containing ethanol.

[0016] In some embodiments, the recrystallization temperature is 10-70°C, for example 15°C, 25°C, 35°C, 45°C, 50°C, 60°C.

[0017] In some embodiments, the recrystallization time is 1-36 hours, preferably 10-24 hours.

[0018] In some embodiments, the drying method employs vacuum drying, boiling drying, or forced-air drying.

[0019] In some specific embodiments, silymarin and L-proline in the above stoichiometric ratio are dissolved / suspended in a solvent at 55-65°C, then cooled to 20-50°C to crystallize, separated, and dried to obtain a eutectic.

[0020] According to another aspect of the invention, a composition is provided comprising the silymarin-L-proline cocrystal as described above, and optionally pharmaceutically, food-, or cosmetically acceptable excipients.

[0021] In some specific embodiments, the stoichiometric ratio of silymarin to L-proline in the composition is 5:1 to 1:9, for example 5:1, 4:1, 3:1, 2:1, 1:1, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8.

[0022] When the silybin to L-proline stoichiometric ratio in the silybin composition is 1:1, the X-ray powder diffraction pattern of the composition exhibits characteristic peaks at 2θ angles of approximately 8.9°±0.2°, 11.1°±0.2°, 12.4°±0.2°, 13.8°±0.2°, 14.8°±0.2°, 15.0°±0.2°, 16.8°±0.2°, and 18.0°±0.2°. These peaks are characteristic of the silybin-L-proline cocrystal of the present invention, thus confirming that the composition contains the cocrystal of the present invention.

[0023] The silybin-L-proline stoichiometric ratio of 1:1 silybin composition has an X-ray powder diffraction pattern substantially as shown in Figure 5.

[0024] When the stoichiometric ratio of silybin to L-proline in the silybin composition is 1:3, the X-ray powder diffraction pattern of the composition exhibits characteristic peaks at 2θ angles of approximately 9.0°±0.2°, 11.1°±0.2°, 15.1°±0.2°, 16.8°±0.2°, 17.5°±0.2°, and 18.1°±0.2°. These peaks are characteristic of the silybin-L-proline co-crystal of the present invention, thus confirming that the composition contains the co-crystal of the present invention.

[0025] The silybin-L-proline stoichiometric ratio of 1:3 silybin composition has an X-ray powder diffraction pattern substantially as shown in Figure 6.

[0026] When the stoichiometric ratio of silybin to L-proline in the silybin composition is 1:5, the X-ray powder diffraction pattern of the composition exhibits characteristic peaks at 2θ angles of approximately 8.9°±0.2°, 11.1°±0.2°, 15.1°±0.2°, 16.8°±0.2°, 17.4°±0.2°, and 18.0°±0.2°. These peaks are characteristic of the silybin-L-proline co-crystal of the present invention, thus confirming that the composition contains the co-crystal of the present invention.

[0027] The silybin-L-proline stoichiometric ratio of 1:7 silybin composition has an X-ray powder diffraction pattern substantially as shown in Figure 7.

[0028] When the stoichiometric ratio of silybin to L-proline in the silybin composition is 1:7, the X-ray powder diffraction pattern of the composition exhibits characteristic peaks at 2θ angles of approximately 8.6°±0.2°, 8.9°±0.2°, 11.1°±0.2°, 15.1°±0.2°, 16.8°±0.2°, 17.5°±0.2°, and 18.0°±0.2°. These peaks are characteristic of the silybin-L-proline co-crystal of the present invention, thus confirming that the composition contains the co-crystal of the present invention.

[0029] The silybin-L-proline stoichiometric ratio of 1:7 silybin composition has an X-ray powder diffraction pattern substantially as shown in Figure 8.

[0030] When the stoichiometric ratio of silybin to L-proline in the silybin composition is 1:9, the X-ray powder diffraction pattern of the composition exhibits characteristic peaks at 2θ angles of approximately 8.6°±0.2°, 8.9°±0.2°, 11.1°±0.2°, 15.1°±0.2°, 16.8°±0.2°, 17.4°±0.2°, and 18.0°±0.2°. These peaks are characteristic of the silybin-L-proline co-crystal of the present invention, thus confirming that the composition contains the co-crystal of the present invention.

[0031] The silybin-L-proline stoichiometric ratio of 1:9 silybin composition has an X-ray powder diffraction pattern substantially as shown in Figure 9.

[0032] When the silybin to L-proline stoichiometric ratio in the silybin composition is 3:1, the X-ray powder diffraction pattern of the composition exhibits characteristic peaks at 2θ angles of approximately 9.0°±0.2°, 11.1°±0.2°, 12.4°±0.2°, 13.7°±0.2°, 14.7°±0.2°, 16.7°±0.2°, 17.6°±0.2°, and 17.9°±0.2°. These peaks are characteristic of the silybin-L-proline co-crystal of the present invention, thus confirming that the composition contains the co-crystal of the present invention.

[0033] The silybin-L-proline stoichiometric ratio of 3:1 has an X-ray powder diffraction pattern that is substantially as shown in Figure 10.

[0034] When the silybin to L-proline stoichiometric ratio in the silybin composition is 5:1, the X-ray powder diffraction pattern of the composition exhibits characteristic peaks at 2θ angles of approximately 9.0°±0.2°, 11.2°±0.2°, 12.4°±0.2°, 13.8°±0.2°, 14.7°±0.2°, 16.8°±0.2°, 17.6°±0.2°, and 18.0°±0.2°. These peaks are characteristic of the silybin-L-proline cocrystal of the present invention, thus confirming that the composition contains the cocrystal of the present invention.

[0035] The silybin-L-proline stoichiometric ratio of 5:1 has an X-ray powder diffraction pattern substantially as shown in Figure 11.

[0036] According to another aspect of the present invention, the use of the silymarin-L-proline cocrystal or the composition thereof in health products, food, cosmetics, pharmaceuticals, and feed is provided.

[0037] The beneficial effects of the present invention are as follows: The present invention provides a silybin-L-proline cocrystal and a composition containing the same. Compared with silybin crystals themselves, the cocrystal and the composition improve the dissolution performance of silybin and significantly improve the bioavailability of silybin. Attached Figure Description

[0038] Figure 1 is the X-ray powder diffraction (PXRD) pattern of the silymarin-L-proline cocrystal of Example 1 of the present invention;

[0039] Figure 2 is the 1H NMR spectrum of the silymarin-L-proline cocrystal of Example 1 of the present invention;

[0040] Figure 3 is a differential scanning calorimetry (DSC) diagram of the silymarin-L-proline cocrystal of Example 1 of the present invention;

[0041] Figure 4 is the infrared (IR) spectrum of the silymarin-L-proline cocrystal of Example 1 of the present invention;

[0042] Figure 5 is the X-ray powder diffraction (PXRD) pattern of the silymarin:L-proline = 1:1 composition of Example 6 of the present invention;

[0043] Figure 6 is the X-ray powder diffraction (PXRD) pattern of the silymarin:L-proline = 1:3 composition of Example 7 of the present invention;

[0044] Figure 7 is the X-ray powder diffraction (PXRD) pattern of the silymarin:L-proline = 1:5 composition of Example 8 of the present invention;

[0045] Figure 8 is the X-ray powder diffraction (PXRD) pattern of the silymarin:L-proline = 1:7 composition of Example 9 of the present invention;

[0046] Figure 9 is the X-ray powder diffraction (PXRD) pattern of the silymarin:L-proline = 1:9 composition of Example 10 of the present invention;

[0047] Figure 10 is the X-ray powder diffraction (PXRD) pattern of the silymarin:L-proline = 3:1 composition of Example 11 of the present invention;

[0048] Figure 11 is an X-ray powder diffraction (PXRD) pattern of the silymarin:L-proline = 5:1 composition of Example 12 of the present invention;

[0049] Figure 12 shows the dissolution curves of silymarin-L-proline cocrystal and silymarin raw material in pH 2.0 buffer solution in Example 1 of the present invention.

[0050] Figure 13 shows the dissolution curves of silymarin-L-proline cocrystal and silymarin raw material in pH 4.5 buffer solution in Example 1 of the present invention.

[0051] Figure 14 shows the dissolution curves of silymarin-L-proline cocrystal and silymarin raw material in pH 6.8 buffer solution in Example 1 of the present invention.

[0052] Figure 15 shows the dissolution curves of silymarin-L-proline cocrystal and silymarin raw material in pH 2.0 + 0.5% PVP K30 buffer solution in Example 1 of the present invention;

[0053] Figure 16 shows the dissolution curves of the silymarin:L-proline = 1:1 composition and silymarin raw material in pH 2.0 + 0.5% PVP K30 buffer solution of Example 6 of the present invention;

[0054] Figure 17 shows the dissolution curves of the silymarin:L-proline = 1:3 composition and silymarin raw material in pH 2.0 + 0.5% PVP K30 buffer solution of Example 7 of the present invention;

[0055] Figure 18 shows the dissolution curves of the silymarin:L-proline = 1:5 composition and silymarin raw material in pH 2.0 + 0.5% PVP K30 buffer solution of Example 8 of the present invention;

[0056] Figure 19 shows the dissolution curves of the silymarin:L-proline = 1:7 composition and silymarin raw material in pH 2.0 + 0.5% PVP K30 buffer solution of Example 9 of the present invention;

[0057] Figure 20 shows the dissolution curves of the silymarin:L-proline = 1:9 composition and silymarin raw material in pH 2.0 + 0.5% PVP K30 buffer solution of Example 10 of the present invention;

[0058] Figure 21 shows the dissolution curves of the silymarin:L-proline = 3:1 composition and silymarin raw material in pH 2.0 + 0.5% PVP K30 buffer solution of Example 11 of the present invention;

[0059] Figure 22 shows the dissolution curves of the silymarin:L-proline = 5:1 composition and silymarin raw material in pH 2.0 + 0.5% PVP K30 buffer solution of Example 12 of the present invention;

[0060] Figure 23 shows the plasma drug-time curves of silymarin-L-proline cocrystal and silymarin extract in SD rats in Example 1 of the present invention. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0062] Reagents and Instruments

[0063] In the embodiments of the present invention

[0064] The X-ray powder diffraction pattern was obtained using a Bruker D8 Advanced X-ray powder diffractometer, which employs Cu-Kα irradiation. The scanning range is from 3° to 40° in the 2θ interval, and the scanning speed is 5° / minute;

[0065] Differential scanning calorimetry was performed using a TA DSC Q2000 instrument at a heating rate of 10 K / min.

[0066] The Fourier transform infrared spectrometer used is a Thermo Scientific Nicolet™ iS50.

[0067] The micro-dissolution apparatus used is the Mini-IDR micro-dissolution apparatus;

[0068] High-performance liquid chromatography was performed using an Agilent Technologies 1260 high-performance liquid chromatograph.

[0069] Mass spectrometry analysis was performed using a Triple Quad™ 4500 LC-MS from Iberia Analytical Instruments Trading Co., Ltd.

[0070] The raw material for silybin is a racemic mixture of silybin, purchased from Xi'an Qianyecao Biotechnology Co., Ltd., with a purity of ≥96%.

[0071] The silymarin extract was purchased from Liaoning Fengrui Tiancheng Biotechnology Co., Ltd., and the silymarin content in the extract was 30%.

[0072] Anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0073] L-proline was purchased from Shanghai Haohong Biomedical Technology Co., Ltd., with a purity of ≥99%.

[0074] Example 1

[0075] 100 g of silybin (0.2 mol) was weighed into a reaction vessel, and 1.43 L of ethanol was added. The mixture was heated to 60 °C to completely dissolve the silybin. After the solid was completely dissolved, 47.7 g of L-proline (0.4 mol) was added, and the mixture was heated to 60 °C to completely dissolve both silybin and L-proline. The temperature was then lowered to 50 °C, and a white solid precipitated. The temperature was further lowered to 25 °C and stirred for 2 hours. The precipitated white powder was filtered and dried under vacuum to obtain 130.7 g of silybin-L-proline eutectic, with a yield of 88.5%.

[0076] Example 2

[0077] 100 g of silybin (0.2 mol) was weighed into a reaction vessel, and 1.43 L of ethanol was added. The mixture was heated to 60 °C until the silybin was completely dissolved. After the solid was completely dissolved, 47.7 g of L-proline (0.4 mol) was added, and the mixture was heated to 60 °C until both silybin and L-proline were completely dissolved. The mixture was then cooled to 25 °C, and a white solid precipitated. The mixture was stirred overnight. The precipitated eutectic was filtered and dried under vacuum to obtain 136.4 g of silybin-L-proline eutectic, with a yield of 92.3%.

[0078] Example 3

[0079] 100 g of silybin (0.2 mol) was weighed into a reaction vessel, and 1.43 L of ethanol was added. The mixture was heated to 60 °C until the silybin was completely dissolved. After the solid was completely dissolved, 62 g of L-proline (0.54 mol) was added, and the mixture was heated to 60 °C until both silybin and L-proline were completely dissolved. The mixture was then cooled to 25 °C, and a white solid precipitated. The mixture was stirred overnight. The precipitated eutectic was filtered and dried under vacuum to obtain 140 g of silybin-L-proline eutectic, with a yield of 94.8%.

[0080] Example 4

[0081] 100 g of silybin (0.2 mol) and 47.7 g of L-proline (0.4 mol) were weighed into a reaction vessel, and 0.88 L of ethanol was added. The mixture was heated to 60 °C and stirred for 1 h. The temperature was then lowered to 25 °C, and stirring was continued for 12 h. The solution was filtered and dried under vacuum to obtain 137.1 g of silybin-L-proline eutectic, with a yield of 92.8%.

[0082] Example 5

[0083] 100 g of silybin (0.2 mol) and 52.5 g of L-proline (0.44 mol) were weighed into a reaction vessel, and 0.88 L of ethanol was added. The mixture was heated to 60 °C and stirred for 1 h. The temperature was then lowered to 25 °C, and stirring was continued for 12 h. The eutectic was filtered and dried under vacuum to obtain 145.6 g of silybin-proline eutectic, with a yield of 98.6%.

[0084] Example 6

[0085] 115 mg of L-proline (1 mmol) was weighed into a 15 mL single-necked glass bottle, and 3.0 mL of solvent (V:V90% ethanol = 7:3) was added. The mixture was heated to 65 °C and stirred for 30 minutes. Then, 482 mg of silybin (1 mmol) was added, and the mixture was stirred at 65 °C for 4 hours. The temperature was lowered to 40 °C, and the mixture was evaporated to dryness under reduced pressure. After vacuum drying for 24 hours, a silybin composition was obtained, wherein the stoichiometric ratio of silybin to L-proline was 1:1. This silybin composition was characterized by X-ray powder diffraction (XRPD). The results are shown in Figure 5 and Table 4. The composition is a mixture of eutectic and silybin.

[0086] Example 7

[0087] 310 mg of L-proline (2.7 mmol) was weighed into a 15 mL single-necked glass bottle, and 3.8 mL of solvent (V:V90% ethanol = 7:3) was added. The mixture was heated to 65 °C and stirred for 30 minutes. Then, 434 mg of silybin (0.9 mmol) was added, and the mixture was stirred at 65 °C for 4 hours. The temperature was lowered to 40 °C, and the mixture was evaporated to dryness under reduced pressure. The mixture was then vacuum dried for 24 hours to obtain a silybin composition, wherein the stoichiometric ratio of silybin to L-proline was 1:3. This silybin composition was characterized by X-ray powder diffraction (XRPD). The results are shown in Figure 6 and Table 5. The composition is a mixture of eutectic and L-proline.

[0088] Example 8

[0089] 345 mg (3 mmol) of L-proline was weighed into a 15 mL single-necked glass bottle, and 3.0 mL of solvent (V:V90% ethanol = 7:3) was added. The mixture was heated to 65 °C and stirred for 30 minutes. Then, 289 mg (0.6 mmol) of silybin was added, and the mixture was stirred at 65 °C for 4 hours. The temperature was lowered to 40 °C, and the mixture was evaporated to dryness under reduced pressure. The mixture was then vacuum dried for 24 hours to obtain a silybin composition, wherein the stoichiometric ratio of silybin to L-proline was 1:5. This silybin composition was characterized by X-ray powder diffraction (XRPD). The results are shown in Figure 7 and Table 6. The composition is a mixture of eutectic and L-proline.

[0090] Example 9

[0091] 402 mg of L-proline (3.5 mmol) was weighed into a 15 mL single-necked glass bottle, and 3.2 mL of solvent (V:V:90% ethanol = 7:3) was added. The mixture was heated to 65 °C and stirred for 30 minutes. Then, 241 mg of silybin (0.5 mmol) was added, and the mixture was stirred at 65 °C for 4 hours. The temperature was lowered to 40 °C, and the mixture was evaporated to dryness under reduced pressure. The mixture was then vacuum dried for 24 hours to obtain a silybin composition, wherein the stoichiometric ratio of silybin to L-proline was 1:7. This silybin composition was characterized by X-ray powder diffraction (XRPD). The results are shown in Figure 8 and Table 7. The composition is a mixture of eutectic and L-proline.

[0092] Example 10

[0093] 414 mg of L-proline (3.6 mmol) was weighed into a 15 mL single-necked glass bottle, and 3.2 mL of solvent (V:V90% ethanol = 7:3) was added. The mixture was heated to 65 °C and stirred for 30 minutes. Then, 193 mg of silybin (0.4 mmol) was added, and the mixture was stirred at 65 °C for 4 hours. The temperature was lowered to 40 °C, and the mixture was evaporated to dryness under reduced pressure. The mixture was then vacuum dried for 24 hours to obtain a silybin composition, wherein the stoichiometric ratio of silybin to L-proline was 1:9. This silybin composition was characterized by X-ray powder diffraction (XRPD). The results are shown in Figure 9 and Table 8. The composition is a mixture of eutectic and L-proline.

[0094] Example 11

[0095] 46 mg of L-proline (0.4 mmol) was weighed into a 15 mL single-necked glass bottle, and 3.2 mL of solvent (V:V:90% ethanol = 7:3) was added. The mixture was heated to 65 °C and stirred for 30 minutes. Then, 578 mg of silybin (1.2 mmol) was added, and the mixture was stirred at 65 °C for 4 hours. The temperature was lowered to 40 °C, and the mixture was evaporated to dryness under reduced pressure. The mixture was then vacuum dried for 24 hours to obtain a silybin composition, wherein the stoichiometric ratio of silybin to L-proline was 3:1. This silybin composition was characterized by X-ray powder diffraction (XRPD). The results are shown in Figure 10 and Table 9. The composition is a mixture of eutectic and silybin.

[0096] Example 12

[0097] 35 mg of L-proline (0.3 mmol) was weighed into a 15 mL single-necked glass bottle, and 3.2 mL of solvent (V:V:90% ethanol = 7:3) was added. The mixture was heated to 65 °C and stirred for 30 minutes. Then, 723 mg of silybin (1.5 mmol) was added, and the mixture was stirred at 65 °C for 4 hours. The temperature was lowered to 40 °C, and the mixture was evaporated to dryness under reduced pressure. The mixture was then vacuum dried for 24 hours to obtain a silybin composition, wherein the stoichiometric ratio of silybin to L-proline was 5:1. This silybin composition was characterized by X-ray powder diffraction (XRPD). The results are shown in Figure 11 and Table 10. The composition is a mixture of eutectic and silybin.

[0098] Test Example 1

[0099] The eutectic products in the examples were characterized by X-ray powder diffraction (PXRD), differential scanning calorimetry, infrared spectroscopy, and nuclear magnetic resonance. The experimental results of the eutectic obtained in Example 1 are shown in Figures 1-5. The PXRD diffraction patterns of the eutectic obtained in Examples 2-5 are similar to those in Figure 1.

[0100] The PXRD data of the cocrystal product obtained in Example 1 and the silymarin and L-proline raw materials are shown in Tables 1-3. The NMR spectrum of the cocrystal is shown in Figure 2. As can be seen from Figure 2, δ = 3.9 ppm (s, 3H) represents the methyl hydrogen on the methoxy group of the silymarin benzene ring, and δ = 2.0 ppm (m, 4H) represents the hydrogen on the methylene group of L-proline. The integral ratio of the two indicates that the stoichiometric ratio of silymarin to L-proline in the cocrystal product is 1:2.

[0101] Table 1. PXRD diffraction data of silymarin-L-proline cocrystal

[0102] Table 2. PXRD diffraction pattern data of silymarin raw material

[0103] Table 3. PXRD diffraction data of L-proline

[0104] Table 4. PXRD diffraction data of the 1:1 composition of silymarin and L-proline.

[0105] Table 5. PXRD diffraction data of the 1:3 composition of silymarin and L-proline.

[0106] Table 6. PXRD diffraction data of the 1:5 composition of silymarin and L-proline.

[0107] Table 7. PXRD diffraction data of the 1:7 composition of silymarin and L-proline.

[0108] Table 8. PXRD diffraction data of the 1:9 composition of silymarin and L-proline.

[0109] Table 9. PXRD diffraction data of the 3:1 composition of silymarin and L-proline.

[0110] Table 10. PXRD diffraction data of the silymarin and L-proline 5:1 composition.

[0111] Test Example 2

[0112] The dissolution properties of silybin raw material and silybin-L-proline cocrystal obtained in Example 1 were compared in different buffer media.

[0113] The experimental method is as follows:

[0114] Experimental apparatus: Mini IDR

[0115] Experimental Procedure: Weigh 60 mg (calculated as silymarin) of sample powder that has passed through a 100-mesh sieve and place it in a dissolution vessel containing 15 mL of dissolution medium. The dissolution medium consisted of buffer solutions with pH 2.0 (glycine + hydrochloric acid), 4.5 (disodium hydrogen phosphate + citric acid), 6.8 (disodium hydrogen phosphate + citric acid), and pH 2.0 (glycine + hydrochloric acid) + 0.5% polyvinylpyrrolidone (PVP K30). The powder dissolution experiment was conducted at 37 °C with a rotation speed of 50 rpm. Sampling time points were 2, 5, 10, 15, 20, 30, 40, 60, 90, and 120 minutes. The sample volume was 0.6 mL. After filtration through a 0.45 μm aqueous filter membrane, 300 μL of the filtrate was taken and diluted by half with the same volume of methanol. Liquid chromatography analysis was then performed.

[0116] The test conditions are as follows:

[0117] Wavelength: 288 nm; Column: Agilent Zorbax Eclipse Plus C18 column (4.6 × 150 mm, 5 μm); Column temperature: 25℃; Injection volume: 10 μL; Flow rate: 1 mL / min; Mobile phase: A: 0.5% acetic acid aqueous solution; B: methanol, isocratic elution. Mobile phase ratio A:B = 48:52.

[0118] The experimental results are shown in Figures 12-23.

[0119] As shown in Figures 12-14, the dissolution performance of silymarin-L-proline cocrystal in buffer solutions at pH 2.0, pH 4.5, and pH 6.8 was significantly improved. In the pH 2.0 buffer solution, the apparent solubility of the cocrystal at 2 min was approximately 87.4 μg / mL, which is 35-40 times that of the silymarin raw material (approximately 2.0 μg / mL). It maintained a solubility advantage of approximately 3-4 times within 1 h and approximately 2 times within 2 h.

[0120] Figures 15-22 show the dissolution curves of silybin cocrystals from Example 1, compositions of silybin and L-proline in different ratios (Examples 6-12), and silybin raw materials in a buffer solution of pH 2.0 + 0.5% PVP K30. The results show that the silybin cocrystals and all silybin compositions exhibited significantly improved solubility compared to silybin itself. After 120 minutes, the solubility of the silybin cocrystals and silybin compositions (silybin:L-proline (molar ratio) = 5:1, 3:1, 1:1, 1:3, 1:5, 1:7, 1:9) increased by 19.7 times, 5.6 times, 9.3 times, 19.7 times, 19.7 times, 19.6 times, 18.6 times, and 17.5 times, respectively. The results show that adding a small amount of proline to form a composition with silybin can significantly improve the solubility of silybin.

[0121] Test Example 3

[0122] The pharmacokinetic properties of silymarin extract (containing approximately 30% silymarin) and the silymarin-L-proline cocrystal obtained in Example 1 were compared in SD rats.

[0123] Male SD rats (weighing 200-300 g) were used in the experiment under well-fed conditions. Fourteen rats were randomly divided into two groups of seven each. Silymarin extract and cocrystals were uniformly dispersed in soybean oil and administered as a suspension via gavage at a dose of 50 mg / kg (calculated as silymarin). Rats were fasted for 12 hours prior to administration but allowed free access to water. Feeding was resumed 4 hours after administration. Based on the pharmacokinetic properties of silymarin, approximately 500 μL of blood was collected from the infraorbital venous plexus at 10, 25, 40, 60, 120, 180, 240, 360, and 480 min after administration. The blood was collected in centrifuge tubes containing 20 μL of 1% heparin sodium, centrifuged at 14000 rpm for 10 min, and 300 μL of the supernatant was stored at -80°C.

[0124] Plasma sample processing: After thawing the frozen plasma, take 200 μL of plasma and add 600 μL of internal standard solution. Vortex for 15 min, then centrifuge at low temperature (10 min, 14000 rpm, 4℃). Take the supernatant and detect it by mass spectrometry.

[0125] Preparation of internal standard solution: Naringenin was used as the internal standard and prepared into a methanol solution of approximately 20 ng / mL.

[0126] HPLC-MS analysis method: Ion source: Electrospray ionization (ESI source); Detection mode: Negative ion mode; Column: Agilent Eclipse Plus C18 column (4.6×150mm, 5μm); Mobile phase: A: 0.5% acetic acid solution, B: methanol; Elution program: Isocratic elution, mobile phase A: mobile phase B = 44:56; Acquisition time: 6 minutes; Flow rate: 1.2 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Absorption wavelength: 288 nm; Scan mode: MRM, m / z 481.00→m / z 300.90 (silymarin), m / z 271.00→m / z 151.00 (naringenin).

[0127] The experimental results are shown in Table 11 and Figure 23. The results indicate that the silymarin-L-proline cocrystal has higher bioavailability than the silymarin extract. max It is 16 times that of the extract, and its AUC is 16 times that of the extract.

[0128] Table 11

Claims

1. A co-crystal of silymarin and L-proline, characterized in that, The stoichiometric ratio of silymarin to L-proline in the eutectic is 1:

2.

2. The eutectic as described in claim 1 or 2, characterized in that, The silybin is silybin A, or silybin B, or a racemic mixture of the two.

3. The eutectic of silymarin and L-proline according to claim 1 or 2, characterized in that, The X-ray powder diffraction pattern of the eutectic has characteristic peaks at least at 2θ angles of 8.9°±0.2°, 11.0°±0.2°, 14.9°±0.2°, 16.7°±0.2°, 18.0°±0.2°, 19.0°±0.2°, 19.6°±0.2°, and 23.9°±0.2°. In particular, the X-ray powder diffraction pattern of the eutectic also has characteristic peaks at 2θ angles of 17.3°±0.2°, 18.7°±0.2°, 20.9°±0.2°, and 22.1°±0.2°. More particularly, the eutectic has an X-ray powder diffraction pattern essentially as shown in Figure 1.

4. The eutectic of silymarin and L-proline according to claim 1 or 2, characterized in that, The eutectic was determined by differential scanning calorimetry and showed an endothermic peak at 206±2℃.

5. The eutectic of silymarin and L-proline according to claim 1 or 2, characterized in that, The infrared absorption spectrum of the eutectic is at least at 3473 cm⁻¹. -1 3283cm -1 3129cm -1 2961cm -1 1609cm -1 1510cm -1 1461cm -1 1384cm -1 1272cm -1 1171cm -1 1131cm -1 1085cm -1 1029cm -1 996cm -1 829cm -1 It has an absorption peak.

6. The method for preparing the eutectic of silymarin and L-proline as described in any one of claims 1-5, characterized in that, The method includes: recrystallizing silymarin and L-proline in a solvent at a stoichiometric ratio of 1:2 to 1:2.8, and obtaining a eutectic after separation and drying of the precipitate.

7. The preparation method according to claim 6, characterized in that, The solvent is selected from one or more of water, alcohols, ketones, esters, alkanes, aromatic hydrocarbons, and haloalkanes; preferably, the solvent is ethanol or a mixed solvent containing ethanol; and / or The recrystallization temperature is 10-70℃; and / or The recrystallization time is 1-36 hours; and / or The drying method employs vacuum drying, boiling drying, or forced-air drying.

8. The preparation method according to claim 6, characterized in that, Silymarin and L-proline in a stoichiometric ratio of 1:2-1:2.8 are dissolved or suspended in a solvent at 55-65℃, then cooled to 20-50℃ to crystallize, separated, and dried to obtain a eutectic.

9. A composition comprising a cocrystal of silymarin as described in any one of claims 1-5 and L-proline, and optionally an excipient acceptable for use in health products, pharmaceuticals, food, cosmetics, or animal feed.

10. The composition according to claim 9, characterized in that, In the composition, the stoichiometric ratio of silymarin to L-proline is 5:1 to 1:

9.

11. Use of the eutectic of silymarin and L-proline as described in any one of claims 1-5 or the composition according to claim 9 in health products, food, cosmetics, pharmaceuticals, or animal feed.

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