Rutin-l-proline eutectic hydrate, preparation method therefor and use thereof

Abstract

The present invention relates to a rutin-L-proline eutectic hydrate, a preparation method therefor and a use thereof. In the rutin-L-proline eutectic hydrate of the present invention, the molar ratio of rutin to L-proline to water molecules is 1:1:3, and cell parameters are (I), α=90°, β=110.327°, and γ=90°. The rutin-L-proline eutectic hydrate obtained in the present invention has good dissolution performance and remarkably improved bioavailability, thereby better achieving the medicinal value of rutin.

Description

Rutin-L-proline eutectic hydrate, preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and more specifically, relates to a rutin-L-proline cocrystal hydrate, a method for preparing the same, and its use as a medicine or dietary supplement. The present invention also relates to a composition comprising the cocrystal. Background Art

[0002] Rutin (vitamin P) is a natural flavonoid glycoside that is widely found in plants such as buckwheat and sophora japonica. Its structural formula is shown in Formula I:

[0003] Rutin is a glycoside composed of the flavonoid aglycone quercetin and a disaccharide. Numerous studies have demonstrated its diverse physiological activities, including antioxidant, vascular protection, anticancer, neuroprotective, and antidiabetic properties. Rutin is widely used in pharmaceuticals, health foods, and cosmetics. However, its low solubility in water (only 125 μg / mL) and low bioavailability (4.9%) limit its application.

[0004] To address the problems of low solubility and low bioavailability of rutin, the inventors of the present application designed and synthesized rutin-L-proline cocrystal hydrate through cocrystal technology, which significantly improved the solubility and bioavailability of rutin and exhibited better hypoglycemic physiological activity. Summary of the Invention

[0005] One object of the present invention is to provide a rutin-L-proline cocrystal hydrate.

[0006] Another object of the present invention is to provide a method for preparing rutin-L-proline cocrystal hydrate.

[0007] Another object of the present invention is to provide the beneficial effects of rutin-L-proline cocrystal hydrate on blood sugar control.

[0008] Another object of the present invention is to provide an amorphous material obtained by dehydrating the rutin-L-proline eutectic hydrate.

[0009] Another object of the present invention is to provide a composition comprising rutin-L-proline cocrystal hydrate.

[0010] Another object of the present invention is to provide the use of rutin-L-proline cocrystal hydrate or a composition comprising rutin-L-proline cocrystal hydrate in the preparation of foods, cosmetics, medicines, and health products for blood sugar control.

[0011] To achieve the purpose of the present invention, according to one aspect of the present invention, a rutin-L-proline eutectic hydrate is provided, wherein the molar ratio of rutin, L-proline and water molecules is 1:1:3.

[0012] Wherein, the structural formula of L-proline is shown in the following formula II:

[0013] According to one embodiment of the present invention, the unit cell parameters of the rutin-L-proline eutectic hydrate are α=90°, β=110.327°, γ=90°.

[0014] In some embodiments, compared to rutin, the rutin-L-proline cocrystal hydrate prepared by the present invention has significantly improved dissolution performance.

[0015] In some embodiments, the rutin-L-proline cocrystal hydrate prepared by the present invention has higher bioavailability than rutin.

[0016] In some embodiments, compared with rutin, the rutin-L-proline cocrystal hydrate prepared by the present invention has a better effect on controlling blood sugar.

[0017] According to one embodiment of the present invention, the X-ray powder diffraction of the rutin-L-proline cocrystal hydrate expressed in 2θ angles has characteristic peaks at least at diffraction angles of 4.3°±0.2°, 5.8°±0.2°, 8.2°±0.2°, 8.7°±0.2°, 12.7°±0.2°, 17.5°±0.2°, 18.6°±0.2°, and 26.6°±0.2°.

[0018] According to one embodiment of the present invention, the X-ray powder diffraction of the rutin-L-proline cocrystal hydrate expressed in 2θ angles has characteristic peaks at least at diffraction angles of 4.3°±0.2°, 5.8°±0.2°, 6.1°±0.2°, 8.2°±0.2°, 8.7°±0.2°, 11.1°±0.2°, 11.8°±0.2°, 12.7°±0.2°, 16.5°±0.2°, 17.5°±0.2°, 18.6°±0.2°, 19.9°±0.2°, 22.1°±0.2°, 24.4°±0.2°, 24.9°±0.2°, and 26.6°±0.2°.

[0019] According to one embodiment of the present invention, the rutin-L-proline cocrystal hydrate has an X-ray powder diffraction pattern substantially as shown in FIG1 .

[0020] According to one embodiment of the present invention, the rutin-L-proline cocrystal hydrate has two weight loss steps at 25-60° C. and 60-150° C. according to thermogravimetric analysis.

[0021] Among them, the weight loss at 25-60°C is about 2.4%, the weight loss at 60-150°C is about 3.3%, and the total weight loss is 5.7%, which corresponds to the three crystal waters in the crystal structure.

[0022] According to one embodiment of the present invention, the rutin-L-proline cocrystal hydrate has a thermogravimetric analysis spectrum substantially as shown in FIG2 .

[0023] According to one embodiment of the present invention, the rutin-L-proline cocrystal hydrate has an endothermic peak at 153±2° C. and 170±2° C., respectively, as determined by differential scanning calorimetry.

[0024] According to one embodiment of the present invention, the rutin-L-proline cocrystal hydrate has a differential scanning calorimetry diagram substantially as shown in FIG3 .

[0025] According to one embodiment of the present invention, the infrared absorption spectrum of the rutin-L-proline cocrystal hydrate is at least at 3754 cm -1 、2913cm -1 、1655cm -1 、1572cm -1 、1507cm -1 、1350cm -1 、1295cm -1 、1198cm -1 , 1070cm -1 、1027cm -1 、978cm -1 There is an absorption peak at.

[0026] According to one embodiment of the present invention, the infrared absorption spectrum of the rutin-L-proline cocrystal hydrate is at least at 3754 cm -1 、2913cm -1 、2897cm -1 、1655cm -1 、1572cm -1 、1507cm -1 、1476cm -1 、1350cm -1 、1295cm -1 、1198cm -1 、1167cm -1 , 1070cm -1 、1027cm -1 、978cm -1, 933cm -1 、805cm -1 , 775cm -1 , 730cm -1 、687cm -1 、609cm -1 , 560cm -1 、467cm -1 There is an absorption peak at.

[0027] According to one embodiment of the present invention, the rutin-L-proline cocrystal hydrate has an infrared absorption spectrum substantially as shown in FIG4 .

[0028] According to another aspect of the present invention, the present invention provides a method for preparing rutin-L-proline cocrystal hydrate, comprising:

[0029] (1) contacting rutin or its hydrate with L-proline in a molecular state by a solution method;

[0030] (2) Crystallization to obtain rutin-L-proline cocrystal hydrate.

[0031] The method allows rutin or its hydrate and L-proline to contact in a solution in a molecular state, and then crystallizes through suspension crystallization, slow evaporation crystallization, cooling crystallization and the like to obtain rutin-L-proline eutectic hydrate.

[0032] According to one embodiment of the present invention, the method for preparing rutin-L-proline is one of the following methods:

[0033] Method 1:

[0034] suspending rutin or its hydrate and L-proline in an organic solvent to obtain a rutin-L-proline cocrystal hydrate;

[0035] Method 2:

[0036] The rutin or its hydrate and L-proline are recrystallized in an organic solvent to obtain a rutin-L-proline eutectic hydrate.

[0037] According to one embodiment of the present invention, the method 1 comprises the following steps:

[0038] S11, fully dissolving excess L-proline in an organic solvent and stirring at room temperature;

[0039] S12, filtering the suspension obtained in S11 and collecting the filtrate;

[0040] S13, adding rutin or its hydrate to the filtrate obtained in S12, and stirring at room temperature overnight;

[0041] S14, separating the solid, and slowly volatilizing and drying at room temperature to obtain the rutin-L-proline eutectic hydrate.

[0042] According to one embodiment of the present invention, in S11, when preparing rutin-L-proline eutectic hydrate, the solvent is selected from one or more of methanol, ethanol, n-propanol, n-butanol, n-pentanol, acetone, methyl ethyl ketone, acetonitrile, tetrahydrofuran, nitromethane, ethyl acetate, and water. Preferably, the organic solvent can be selected from a mixed solvent of methanol and water, a mixed solvent of methanol and n-propanol, a mixed solvent of methanol and n-butanol, a mixed solvent of methanol and n-pentanol, a mixed solvent of methanol and acetone, a mixed solvent of methanol and acetonitrile, a mixed solvent of ethanol and water, a mixed solvent of ethanol and n-propanol, a mixed solvent of ethanol and n-butanol, a mixed solvent of ethanol and n-pentanol, a mixed solvent of ethanol and acetone, a mixed solvent of ethanol and acetonitrile, and a mixed solvent of methanol, ethanol, and water. The volume ratio of the solvent in the mixed solvent may be 1:0.1 to 10, preferably 1:1 to 5, and more preferably 1:1.

[0043] The solvent itself still contains a certain amount of water without drying treatment, and the low humidity environment during the crystallization process is not strictly controlled, so that crystallization water can be obtained from the environment or the solvent.

[0044] The hydrate of rutin is preferably rutin trihydrate, so that water molecules can be provided even when the solvent does not contain water.

[0045] According to one embodiment of the present invention, the second method comprises at least the following steps:

[0046] S21, dissolving rutin or its hydrate and L-proline in an organic solvent, and stirring at a first temperature until completely dissolved;

[0047] S22, cooling the solution obtained in S21 at a second temperature lower than the first temperature to crystallize;

[0048] S23, separating the solid and drying it to obtain the rutin-L-proline eutectic hydrate.

[0049] Specifically, in S21, the first temperature is 25-80°C, preferably 50-70°C.

[0050] Specifically, in S22, the second temperature is -20 to 25°C, preferably -20 to 0°C.

[0051] Specifically, in S21, when preparing rutin-L-proline eutectic hydrate, the solvent is selected from one or more of methanol, ethanol, n-propanol, n-butanol, n-pentanol, acetone, methyl ethyl ketone, acetonitrile, tetrahydrofuran, nitromethane, ethyl acetate, and water. Preferably, the organic solvent can be selected from a mixed solvent of methanol and water, a mixed solvent of methanol and n-propanol, a mixed solvent of methanol and n-butanol, a mixed solvent of methanol and n-pentanol, a mixed solvent of methanol and acetone, a mixed solvent of methanol and acetonitrile, a mixed solvent of ethanol and water, a mixed solvent of ethanol and n-propanol, a mixed solvent of ethanol and n-butanol, a mixed solvent of ethanol and n-pentanol, a mixed solvent of ethanol and acetone, a mixed solvent of ethanol and acetonitrile, and a mixed solvent of methanol, ethanol, and water. The volume ratio of the solvent in the mixed solvent can be 1:0.1 to 10, preferably 1:1 to 5, and more preferably 1:1.

[0052] Specifically, in S22, seed crystals and an appropriate amount of water may be added during the crystallization process to accelerate the formation of crystals.

[0053] Specifically, in S23, solid-liquid separation can be performed by filtration, centrifugation, etc., and filtration is preferred; drying can be performed by normal pressure drying, vacuum drying, spray drying, etc., and vacuum drying at room temperature is preferred.

[0054] The hydrate of rutin is preferably rutin trihydrate, so that water molecules can be provided even when the solvent does not contain water.

[0055] According to another aspect of the present invention, there is provided an amorphous material obtained by dehydrating the above-mentioned rutin-L-proline cocrystal hydrate, wherein the amorphous material has an X-ray powder diffraction pattern substantially as shown in FIG5 .

[0056] According to another aspect of the present invention, the present invention provides a composition comprising the above-mentioned rutin-L-proline cocrystal hydrate.

[0057] According to one embodiment of the present invention, the composition may contain, in addition to the rutin-L-proline eutectic hydrate of the present invention, an excess of L-proline, an excess of rutin or its hydrate, and other excipients acceptable to food, cosmetics, medicines, health products or feed.

[0058] In other words, there's no specific molar ratio between rutin and L-proline in the composition, as long as the raw materials can produce the aforementioned rutin-L-proline eutectic hydrate. For example, the molar ratio of rutin to L-proline in the composition can range from 10:1 to 1:10, with some of the raw materials existing in the form of the aforementioned rutin-L-proline eutectic hydrate and some existing in a free form. To fully address rutin's low water solubility, it's preferred that all of the rutin be converted to the rutin-L-proline eutectic hydrate.

[0059] According to another aspect of the present invention, the present invention also provides the beneficial effects of the rutin-L-proline cocrystal hydrate on blood sugar control.

[0060] According to another aspect of the present invention, the present invention also provides the use of the rutin-L-proline cocrystal hydrate or a composition comprising the rutin-L-proline cocrystal hydrate in the preparation of food, cosmetics, medicines, health products or feed for blood sugar control.

[0061] The beneficial effects of the present invention include at least:

[0062] The rutin-L-proline cocrystal hydrate of the present invention exhibits non-covalent interactions between L-proline and rutin at the molecular level, and the cocrystal has good water solubility, thereby improving the water solubility of rutin at the molecular level. Therefore, compared to rutin itself, the rutin-L-proline cocrystal hydrate of the present invention exhibits significantly improved dissolution properties, higher bioavailability, and improved blood sugar control.

[0063] The present invention prepares rutin-L-proline eutectic hydrate by a cooling crystallization method, which has the technical advantages of high yield, low cost and suitability for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG1 is an X-ray powder diffraction (PXRD) pattern of the rutin-L-proline cocrystal hydrate of Example 1 of the present invention;

[0065] FIG2 is a thermogravimetric analysis (TGA) diagram of the rutin-L-proline cocrystal hydrate of Example 1 of the present invention;

[0066] FIG3 is a differential scanning calorimetry (DSC) diagram of the rutin-L-proline cocrystal hydrate of Example 1 of the present invention;

[0067] FIG4 is an infrared spectrum (IR) of the rutin-L-proline cocrystal hydrate of Example 1 of the present invention;

[0068] FIG5 is an X-ray powder diffraction (PXRD) pattern of an amorphous material obtained by dehydration of the rutin-L-proline cocrystal hydrate described in Example 14 of the present invention;

[0069] FIG6 is a dissolution curve of the rutin-L-proline cocrystal hydrate and the rutin raw material in pH 2.0 buffer (containing 1% Tween 80) according to Example 1 of the present invention;

[0070] 7 is a dissolution curve of the rutin-L-proline cocrystal hydrate and the rutin raw material in pH 4.5 buffer (containing 1% Tween 80) according to Example 1 of the present invention;

[0071] FIG8 is a dissolution curve of the rutin-L-proline cocrystal hydrate and the rutin raw material in pH 6.8 buffer (containing 1% Tween 80) according to Example 1 of the present invention;

[0072] FIG9 is a plasma drug-dose-time curve of the rutin-L-proline cocrystal hydrate and the rutin raw material in Example 1 of the present invention in SD rats. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0074] Reagents and instruments

[0075] The X-ray powder diffraction patterns in the embodiments of the present invention were obtained using a Bruker D8 Advanced model X-ray powder diffractometer, which uses Cu-Kα irradiation. The scanning range was from 3° to 40° in the 2θ interval, and the scanning speed was 2° / min.

[0076] Differential scanning calorimetry was performed using a TA DSC Q2000 device with a heating rate of 10 K / min;

[0077] Thermogravimetric analysis was performed using a TGA-55 thermogravimetric analyzer from TA Instruments, USA;

[0078] The Fourier transform infrared spectrometer used was Thermo Scientific Nicolet 6700;

[0079] The micro-dissolution apparatus uses Mini-IDR micro-dissolution apparatus;

[0080] High performance liquid chromatography was performed using an Agilent Technologies 1260 high performance liquid chromatograph;

[0081] Mass spectrometry analysis was performed using Triple QuadTM 4500LC-MS from Aibocaisi Analytical Instrument Trading Co., Ltd.

[0082] Rutin trihydrate was purchased from Beijing Yinuokai Technology Co., Ltd. with a purity of ≥96%;

[0083] L-Proline was purchased from Aladdin Reagent with a purity of ≥99%.

[0084] Example 1

[0085] L-proline (about 2 mmol) was weighed and added to 20 mL of 95% ethanol aqueous solution. The mixture was ultrasonically dissolved for 20 min, and the undissolved powder was removed by centrifugation. The supernatant was collected and rutin trihydrate (about 2 mmol) was weighed and added to the supernatant in a molar ratio of 1:1. The mixture was stirred overnight, and the precipitate was filtered through a Buchner funnel. The solid was slowly evaporated and dried in the air to obtain 1.2 g of rutin-L-proline cocrystal hydrate with a yield of about 77.02%.

[0086] The mother liquor evaporates and crystallizes to obtain a single crystal with the unit cell parameters being: α=90°, β=110.327°, γ=90°.

[0087] The above experimental data verified that the molar ratio of rutin, L-proline and water molecules in the cocrystal was 1:1:3.

[0088] The cocrystal was characterized by X-ray powder diffraction (PXRD), differential scanning calorimetry, thermogravimetric analysis, and infrared spectroscopy. The experimental results are shown in Figures 1 to 4.

[0089] Example 2

[0090] 94.1 g of rutin trihydrate and 16.3 g of L-proline (molar ratio of 1:1) were added to 500 mL of methanol, stirred at 100 rpm / min, heated to 60°C at 3°C / min and maintained for 10 minutes to dissolve, then cooled to 25°C at -1°C / min. When the temperature dropped to 30°C, 0.1% seed crystals were added, and the temperature was then maintained at a constant temperature for 12 hours. A yellow precipitate gradually precipitated, and the precipitate was filtered through a Buchner funnel. The solid was placed in a vacuum drying oven and dried at room temperature for 18 hours to obtain 86.3 g of rutin-L-proline cocrystal hydrate with a yield of 78.2%.

[0091] Example 3

[0092] 332 g of rutin trihydrate and 57.5 g of L-proline (molar ratio 1:1) were weighed and added to 800 mL of methanol. The mixture was stirred at 100 rpm / min, heated to 60 °C at 3 °C / min and maintained for 10 min to dissolve the mixture. The mixture was cooled to 20 °C at -1 °C / min. 0.1% seed crystals were added when the temperature dropped to 30 °C, and the temperature was then kept constant for 2 h. The mixture was cooled to 5 °C at -1 °C / min and maintained at a constant temperature for 12 h. A yellow precipitate gradually precipitated. The precipitate was filtered through a Buchner funnel, and the solid was placed in a vacuum drying oven and dried at room temperature for 18 h to obtain 313.98 g of rutin-L-proline eutectic hydrate with a yield of 80.6%.

[0093] Example 4

[0094] 94.1 g of rutin trihydrate and 16.3 g of L-proline (molar ratio 1:1) were weighed and added to 500 mL of methanol. The mixture was stirred at 100 rpm / min, heated to 60°C at 3°C / min and maintained for 10 min to dissolve the mixture. The mixture was cooled to -20°C at -1°C / min (0.1% seed crystals were added when the temperature dropped to 30°C). The temperature was then maintained at a constant temperature for 12 h. A yellow precipitate gradually precipitated. The precipitate was filtered through a Buchner funnel, and the solid was placed in a vacuum drying oven and dried at room temperature for 18 h to obtain 89.2 g of rutin-L-proline cocrystal hydrate with a yield of 80.8%.

[0095] Example 5

[0096] Weigh 86.7 g of anhydrous rutin and 16.3 g of L-proline (molar ratio 1:1) and add them to 500 mL of methanol. Stir at 100 rpm / min, heat to 60 ° C at 3 ° C / min and keep for 10 min to dissolve. Add 50 mL of pure water, cool to 20 ° C at -1 ° C / min (add 0.1% seed crystals when cooling to 30 ° C), keep constant temperature for 2 h, cool to 5 ° C at -1 ° C / min and keep constant temperature for 6 h. A yellow precipitate gradually precipitates. Filter the precipitate through a Buchner funnel, and place the solid in a vacuum drying oven at room temperature and dry it for 18 h to obtain 86 g of rutin-L-proline eutectic hydrate with a yield of 77.9%.

[0097] Example 6

[0098] Weigh 86.7 g of anhydrous rutin and 16.3 g of L-proline (molar ratio 1:1) and add 500 mL of the remaining mother liquor of the methanol process obtained in Example 5 (the filtrate obtained by filtering in Example 5), stir at 100 rpm / min, heat to 70°C at 3°C / min and hold for 20 min to dissolve, cool to 20°C at -1°C / min (add 0.1% seed crystals when cooling to 30°C), keep constant temperature for 2 h, cool to 5°C at -1°C / min and keep constant temperature for 6 h, gradually precipitate a yellow precipitate, filter the precipitate through a Buchner funnel, and place the solid in a vacuum drying oven and dry at room temperature for 18 h to obtain 101 g of rutin-L-proline eutectic hydrate with a yield of 91.5%.

[0099] Example 7

[0100] 461.9 mg of rutin trihydrate and 80 mg of L-proline (molar ratio 1:1) were weighed and added to 3 mL of methanol. The mixture was heated to 75 °C to completely dissolve the mixture. The temperature was then cooled to 25 °C at -0.5 °C / min. A yellow precipitate gradually precipitated. The precipitate was filtered through a Buchner funnel and the solid was placed in a vacuum drying oven and dried at room temperature for 18 h to obtain 155 mg of rutin-L-proline cocrystal hydrate with a yield of 28.6%.

[0101] Example 8

[0102] Weigh 461.9 mg of rutin trihydrate and 80 mg of L-proline (molar ratio 1:1) into 2 mL of methanol, heat to 75 ° C to completely dissolve, cool to 4 ° C at -0.5 ° C / min, and gradually precipitate a yellow precipitate. Filter the precipitate through a Buchner funnel, and place the solid in a vacuum drying oven at room temperature for 18 h to obtain 320 mg of rutin-L-proline cocrystal hydrate with a yield of 59%.

[0103] Example 9

[0104] Weigh 461.9 mg of rutin trihydrate and 80 mg of L-proline (molar ratio 1:1) into 1.25 mL of methanol, heat to 75 ° C to completely dissolve, cool to -0.5 ° C / min to -20 ° C, gradually precipitate a yellow precipitate, filter the precipitate through a Buchner funnel, and place the solid in a vacuum drying oven at room temperature for 18 h to obtain 400 mg of rutin-L-proline cocrystal hydrate with a yield of 73.8%.

[0105] Example 10

[0106] Weigh 45.6 g of rutin trihydrate and 7.9 g of L-proline (molar ratio 1:1) and add 500 mL of a mixed solvent of methanol, ethanol and water (ratio 50:20:2, v / v), stir at 100 rpm / min, heat to 70 ° C. at 3 ° C. / min and maintain for 20 minutes to dissolve, then cool and crystallize for 24 hours, and a yellow precipitate gradually precipitates. The precipitate is filtered through a Buchner funnel, and the solid is placed in a vacuum drying oven and dried at room temperature for 18 hours to obtain rutin-L-proline eutectic hydrate.

[0107] Example 11

[0108] Weigh 45.6 g of rutin trihydrate and 7.9 g of L-proline (molar ratio 1:1) and add 500 mL of a mixed solvent of methanol, ethanol and water (ratio 50:20:4), stir at 100 rpm / min, heat to 70 ° C. at 3 ° C. / min and maintain for 20 minutes to dissolve, then cool and crystallize for 24 hours, gradually precipitate a yellow precipitate, filter the precipitate through a Buchner funnel, and place the solid in a vacuum drying oven at room temperature for 18 hours to obtain rutin-L-proline eutectic hydrate.

[0109] Example 12

[0110] Weigh 45.6 g of rutin trihydrate and 7.9 g of L-proline (molar ratio 1:1) and add 500 mL of a mixed solvent of methanol, ethanol and water (ratio 50:30:4), stir at 100 rpm / min, heat to 70 ° C. at 3 ° C. / min and maintain for 20 minutes to dissolve, then cool and crystallize for 24 hours, gradually precipitate a yellow precipitate, filter the precipitate through a Buchner funnel, and place the solid in a vacuum drying oven at room temperature for 18 hours to obtain rutin-L-proline eutectic hydrate.

[0111] Example 13

[0112] Weigh 45.6 g of rutin trihydrate and 7.9 g of L-proline (molar ratio 1:1) and add 500 mL of a mixed solvent of methanol, ethanol and water (ratio 50:40:4), stir at 100 rpm / min, heat to 70 ° C. at 3 ° C. / min and maintain for 20 minutes to dissolve, then cool and crystallize for 24 hours, gradually precipitate a yellow precipitate, filter the precipitate through a Buchner funnel, and place the solid in a vacuum drying oven at room temperature for 18 hours to obtain rutin-L-proline eutectic hydrate.

[0113] Example 14

[0114] The rutin-L-proline eutectic hydrate prepared in Example 1 was dehydrated at 150±2° C. to obtain an amorphous substance obtained by dehydration of the rutin-L-proline eutectic hydrate.

[0115] The amorphous material was characterized by X-ray powder diffraction (PXRD). The experimental results are shown in Figure 5.

[0116] Test Example 1

[0117] The dissolution properties of rutin (trihydrate) and rutin-L-proline obtained in Example 2 in different buffer media were compared.

[0118] About 50 mg of rutin (trihydrate) and rutin-L-proline cocrystal hydrate (non-sedimentation conditions) were weighed and added to 15 mL of buffer solutions containing 1% Tween 80 at pH 2.0, 4.5, and 6.8. Powder dissolution experiments were performed at 37°C and 50 rpm / min. 500 μL of liquid was collected at 2, 5, 10, 15, 30, 45, 60, 90, and 120 min, respectively. After centrifugation, 400 μL of the supernatant was transferred to a liquid phase bottle, diluted with 400 μL of methanol, and the concentration was detected by HPLC.

[0119] The test conditions are as follows:

[0120] Wavelength: 256 nm; chromatographic column: Agilent Zorbax Eclipse Plus C18 column (4.6×150 mm, 5 μm); column temperature: 30°C; injection volume: 10 μL; flow rate: 1 mL / min; mobile phase: A: 1% trifluoroacetic acid in water; B: methanol, gradient elution. Specific methods are shown in the table below.

[0121] Table 1 Mobile phase gradient

[0122] The experimental results are shown in Figures 6 to 8.

[0123] As shown in Figures 6-8, the dissolution of rutin-L-proline cocrystal hydrate in pH 2.0, pH 4.5, and pH 6.8 buffers (containing 1% Tween 80) is significantly improved. Rutin-L-proline can quickly reach a high concentration (about 2 mg / mL), which is about 20-30 times that of the raw material (about 100 μg / mL), and can maintain a dissolution advantage of about 3-4 times within 1 hour, and a dissolution advantage of about 2 times within 2 hours. And according to PXRD characterization, rutin-L-proline cocrystal hydrate quickly dissolves in the dissolution medium to a high degree of supersaturation, and then the cocrystal dissociates and precipitates in the form of rutin. This shows that rutin-L-proline cocrystal hydrate can significantly improve the in vitro dissolution performance of rutin.

[0124] Experimental Example 1

[0125] The pharmacokinetic properties of rutin (trihydrate) and the rutin-L-proline cocrystal hydrate obtained in Example 2 were compared in SD rats.

[0126] 7 Sprague-Dawley rats of 200-250g are divided into one group, give the rutin of 105mg / kg (in rutin) and the rutin-L-proline eutectic hydrate that equivalent embodiment 2 obtains respectively, sample is suspended by 0.5%CMC-Na aqueous solution, by gavage, sample homogeneity detects through HPLC.Administration is fasted for 12 hours before, can drink water at will, and after administration, recovery feeds in 4 hours.According to the pharmacokinetic property of rutin, after administration, 20,40,60,90,120,240,360,480,600min, from rat orbital venous plexus, about 700 μ L of blood is drawn in the centrifuge tube that contains 20 μ L1% heparin sodium, after 14000rpm / min centrifugal 10min, get supernatant 500 μ L and be-80 ℃ of freezing preservations.

[0127] Plasma sample processing: Take 100 μL of plasma sample and place it in a centrifuge tube, add 20 μL of a mixed solution of glucuronidase (500 units / mL) and sulfatase (100 units / mL), heat in a 37°C water bath for 1 hour, then add 600 μL of methanol, shake for 30 minutes to precipitate the protein, then centrifuge at 14000 rpm / min for 10 minutes, and take 400 μL of the supernatant for content analysis by mass spectrometry.

[0128] HPLC-MS analysis method: mobile phase: A: 1 mmol / L ammonium formate aqueous solution; B: 1 mmol / L ammonium formate methanol solution, gradient elution, specific conditions are shown in the table below; acquisition time: 6 min; flow rate: 0.6 mL / min; column temperature: 30°C; injection volume: 10 μL; scanning mode: MRM, m / z 609.2→300.0.

[0129] The experimental results are shown in FIG9 , which show that the rutin-L-proline cocrystal hydrate obtained in Example 2 has a higher bioavailability than the rutin prototype at the same dosage. max It is 3.8 times that of the original data, and its AUC is 5.6 times that of the original data.

[0130] Table 2 Mobile phase gradient

[0131] Experimental Example 2

[0132] The blood sugar lowering effects of rutin (trihydrate) and the rutin-L-proline cocrystal hydrate obtained in Example 2 on diabetic model rats were compared.

[0133] To prepare an animal model of pancreatic islet injury and hyperglycemia, 150 Sprague-Dawley rats weighing approximately 200 g were fed adaptively for one week. Afterward, they were weighed and fasted with free access to water. Four hours later, tail tip blood was drawn for fasting blood glucose measurement. Eight rats were selected as blank controls. The experimental group received an intraperitoneal injection of STZ solution at a dose of 60 mg / kg, while the blank control group received an equal volume of blank solvent. The rats were housed normally after the injections. Fasting blood glucose was measured by tail tip blood sampling on days 2 and 7 after the injections. Rats with fasting blood glucose values ​​greater than 11.1 mmol / L on two consecutive measurements were considered successful models and could be used for subsequent experiments.

[0134] Animal grouping and continuous administration: 56 rats with successful modeling were grouped according to the last measured fasting blood glucose value. Rats with a difference of 1.1 mmol / L or less in fasting blood glucose values ​​were divided into 7 groups, namely high-, medium-, and low-dose rutin groups, high-, medium-, and low-dose rutin-L-proline cocrystal hydrate groups, and a model control group. The high, medium, and low doses of 50 mg / kg, 100 mg / kg, and 300 mg / kg (calculated as rutin) were administered by gavage, respectively. The samples were suspended in soybean oil. The model control group was given an equal amount of blank solvent, and the blank control group was given 100 mg / kg (calculated as rutin) of rutin-L-proline cocrystal hydrate once a day for 40 consecutive days.

[0135] Related index test: Fasting blood glucose test. 40 days after administration, rats were fasted and allowed to drink water freely. 4 hours later, fasting blood glucose values ​​were measured by blood sampling from the tail tip. The measurements were performed 3-5 times in parallel. The average value was taken to compare the percentage of fasting blood glucose reduction in each group.

[0136] Glucose tolerance test: The rats were fasted for 40 days after administration and were allowed to drink water freely. Fasting blood glucose was measured after 4 hours. Then different experimental groups were administered drugs. The model control group was given an equal amount of blank solvent, and the blank control group was given rutin-L-proline cocrystal hydrate at a dose of 100 mg / kg. Then, 50% glucose solution was administered by gavage at a dose of 2 g / kg. The blood glucose levels of the rats were measured by blood sampling from the tail tip 0.5 hours and 2 hours after the administration of glucose solution, and the area under the blood glucose response curve was calculated based on the fasting blood glucose, 0.5 hour and 2 hour blood glucose.

[0137] The experimental results are shown in the table below. The results show that both rutin and rutin-L-proline cocrystal hydrate have a certain effect on lowering fasting blood sugar, but the effect of rutin-L-proline cocrystal hydrate is more obvious. In addition, both rutin and rutin-L-proline cocrystal hydrate have a significant effect on reducing glucose tolerance, and the effect of rutin-L-proline cocrystal hydrate on reducing glucose tolerance is better than rutin itself.

[0138] Table 3 Effects of rutin and its cocrystal on fasting blood glucose in diabetic rats

[0139] Table 4 Effects of rutin and its cocrystals on glucose tolerance in diabetic rats

[0140] The superscripts with different letters indicate significant differences (p<0.05).

[0141] This application is only a preferred embodiment of the present invention, but is not intended to limit the claims. Any modifications, equivalent substitutions, and improvements made by any person skilled in the art without departing from the concept of this application should be within the scope of protection of the present invention.

Claims

1. A rutin-L-proline co-crystal hydrate, characterized in that, In the rutin-L-proline cocrystal hydrate, the molar ratio of rutin, L-proline, and water is 1:1:

3.

2. The rutin-L-proline cocrystal hydrate according to claim 1, wherein The unit cell parameters of the rutin-L-proline cocrystal hydrate are as follows: α = 90°, β = 110.327°, γ = 90°.

3. The rutin-L-proline cocrystal hydrate according to claim 1, characterized in that, The X-ray powder diffraction of the rutin-L-proline cocrystal hydrate expressed in 2θ angles has characteristic peaks at least at diffraction angles of 4.3°±0.2°, 5.8°±0.2°, 8.2°±0.2°, 8.7°±0.2°, 12.7°±0.2°, 17.5°±0.2°, 18.6°±0.2°, and 26.6°±0.2°.

4. The rutin-L-proline cocrystal hydrate according to claim 1, wherein The rutin-L-proline cocrystal hydrate has two weight loss steps by thermogravimetric analysis at 25-60 °C and 60-150 °C.

5. The rutin-L-proline cocrystal hydrate according to claim 1, wherein, The rutin-L-proline cocrystal hydrate has an endothermic peak at 153±2 °C and 170±2 °C respectively by differential scanning calorimetry.

6. The rutin-L-proline cocrystal hydrate according to claim 1, wherein, The infrared absorption spectrum of the rutin-L-proline cocrystal hydrate has absorption peaks at least at 3754 cm -1 , 2913 cm -1 , 1655 cm -1 , 1572 cm -1 , 1507 cm -1 , 1350 cm -1 , 1295 cm -1 , 1198 cm -1 , 1070 cm -1 , 1027 cm -1 , 978 cm -1 .

7. A preparation method of the rutin-L-proline cocrystal hydrate according to any one of claims 1 to 6, characterized in that, At least includes the following steps: S11. Dissolve an excess of L-proline fully in an organic solvent and stir at room temperature. S12. Filter the suspension obtained in S11 and collect the filtrate. S13. Add rutin or its hydrate to the filtrate obtained in S12 and stir overnight at room temperature. S14. Separate the solid, slowly volatilize and dry at room temperature to obtain the rutin-L-proline cocrystal hydrate.

8. The preparation method according to claim 7, characterized in that, The selected organic solvent is one or more selected from methanol, ethanol, n-propanol, n-butanol, n-pentanol, acetone, methyl ethyl ketone, acetonitrile, tetrahydrofuran, nitromethane, ethyl acetate, and water.

9. A method for preparing the rutin-L-proline cocrystal hydrate according to any one of claims 1 to 6, characterized in that, At least includes the following steps: S21. Dissolve rutin or its hydrate and L-proline in an organic solvent and stir at a first temperature until completely dissolved. S22. Cool the solution obtained in S21 at a second temperature lower than the first temperature to crystallize. S23. Separate the solid and dry to obtain the rutin-L-proline cocrystal hydrate. Preferably, the first temperature is 25-80 °C, preferably 50-70 °C. Preferably, the second temperature is -20-25 °C, preferably -20-0 °C. Preferably, in S22, seeds and water are added during the crystallization process to accelerate crystal formation.

10. The preparation method according to claim 9, characterized in that, The selected organic solvent is one or more selected from methanol, ethanol, n-propanol, n-butanol, n-pentanol, acetone, methyl ethyl ketone, acetonitrile, tetrahydrofuran, nitromethane, ethyl acetate, and water.

11. An amorphous substance obtained by dehydrating the rutin-L-proline cocrystal hydrate according to any one of claims 1 to 6, characterized in that, The amorphous substance has an X-ray powder diffraction pattern substantially as shown in Figure 5.

12. A composition comprising the rutin-L-proline cocrystal hydrate according to any one of claims 1-6, and optionally rutin or its hydrate, L-proline, and / or excipients acceptable in food, cosmetics, pharmaceuticals, health products.

13. Use of the rutin-L-proline cocrystal hydrate according to any one of claims 1-6 or the composition according to claim 12 in the preparation of food, cosmetics, pharmaceuticals, health products, or feeds for blood glucose control.

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