Phytosterol-cholic acid composition as well as preparation method and application thereof

Abstract

The invention relates to a phytosterol-cholic acid composition as well as a preparation method and application thereof. In the phytosterol-cholic acid composition, the molar ratio of phytosterol to cholic acid ranges from 10: 1 to 1: 10. The obtained phytosterol-cholic acid composition is better in dissolution performance, the bioavailability is remarkably improved, and the medicinal value of phytosterol is better embodied.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine. More specifically, the present invention relates to a phytosterol-cholic acid composition, a preparation method thereof, and uses thereof as a drug or a dietary supplement. The present invention also relates to a preparation comprising the phytosterol-cholic acid composition. Background Art

[0002] Phytosterols are steroid substances widely present in various natural foods. Commercially available phytosterols are mainly extracted from the deodorized distillate of soybean oil and mainly consist of β-sitosterol, a small amount of stigmasterol and campesterol. The structural formulas of their components are as follows:

[0003]

[0004] Multiple studies have shown that phytosterols have various physiological activities such as regulating blood lipids, anti-inflammatory, antioxidant, enhancing immunity and protecting cardiovascular health, and have been widely used in the fields of pharmaceuticals, health foods, feeds, etc. However, phytosterols have poor water solubility (almost insoluble in water) and low bioavailability (taking β-sitosterol as an example, only 0.41%), which limits their scope of application.

[0005] In view of the problems of poor water solubility and low bioavailability of phytosterols, the inventors of the present application have proposed a phytosterol-cholic acid composition, which significantly improves the solubility and bioavailability of phytosterols and has better physiological activity in reducing blood lipids. Summary of the Invention

[0006] In order to improve the bioavailability of phytosterols, the present invention combines phytosterols with cholic acid to improve the dissolution and dissolution performance of phytosterols, thereby improving their bioavailability.

[0007] One object of the present invention is to provide a phytosterol-cholic acid composition.

[0008] Another object of the present invention is to provide a preparation method of the phytosterol-cholic acid composition.

[0009] A third object of the present invention is to provide the beneficial effect of the phytosterol-cholic acid composition in regulating blood lipids.

[0010] A fourth object of the present invention is to provide a preparation comprising the phytosterol-cholic acid composition.

[0011] A fifth object of the present invention is to provide the use of a preparation comprising the phytosterol-cholic acid composition in the preparation of foods, cosmetics, pharmaceuticals, and health products for regulating blood lipids.

[0012] Among them, the structural formula of cholic acid is shown as follows:

[0013]

[0014] According to one aspect of the present invention, there is provided a composition of a phytosterol and a bile acid, wherein the molar ratio of the phytosterol to the bile acid in the composition is from 10:1 to 1:10, preferably 1:1.

[0015] According to one embodiment of the present invention, the phytosterol is selected from β-sitosterol, stigmasterol, campesterol, and mixtures thereof.

[0016] When the phytosterol is a mixture of β-sitosterol, stigmasterol, and campesterol, the molar ratio is calculated according to the sum of the molar ratios of each sterol.

[0017] The phytosterol mixture comprises a combination of two or three of β-sitosterol, stigmasterol, and campesterol.

[0018] When the phytosterol mixture comprises two of β-sitosterol, stigmasterol, and campesterol, the weight ratio of the two is from 1:10 to 10:1, preferably from 1:5 to 5:1, more preferably from 1:3 to 3:1.

[0019] When the phytosterol mixture comprises all three of β-sitosterol, stigmasterol, and campesterol, by weight, the content of β-sitosterol is 40% to 65%, the content of stigmasterol is 5% to 30%, and the content of campesterol is 20% to 35%.

[0020] According to one embodiment of the present invention, the composition contains a phytosterol-bile acid eutectic.

[0021] According to one embodiment of the present invention, the unit cell parameters of the phytosterol-bile acid eutectic are α = 90°, β = 97.042°, γ = 90°.

[0022] According to one embodiment of the present invention, the X-ray powder diffraction pattern of the phytosterol-bile acid eutectic expressed in 2θ angle has characteristic peaks at least at 6.4° ± 0.2°, 7.9° ± 0.2°, 10.8° ± 0.2°, 12.2° ± 0.2°, 12.6° ± 0.2°, 13.5° ± 0.2°, 14.1° ± 0.2°, 17.2° ± 0.2°, 18.2° ± 0.2°, 21.2° ± 0.2°. Preferably, it has an X-ray powder diffraction pattern substantially as Figure 1 shown.

[0023] According to one embodiment of the present invention, the phytosterol-bile acid eutectic has an endothermic peak at 168°C ± 2°C as determined by differential scanning calorimetry. Preferably, it has a differential scanning calorimetry pattern substantially as Figure 2 shown.

[0024] According to an embodiment of the present invention, for the phytosterol-cholic acid co-crystal, its infrared absorption spectrum has absorption peaks at least at 3351 cm -1 , 2923 cm -1 , 2865 cm -1 , 1705 cm -1 , 1442 cm -1 , 1373 cm -1 , 1330 cm -1 , 1214 cm -1 , 1041 cm -1 , 949 cm -1 . Preferably, it has an infrared spectrum substantially as Figure 3 shown.

[0025] Compared with phytosterol, the phytosterol-cholic acid co-crystal prepared by the present invention has significantly improved dissolution performance.

[0026] Compared with phytosterol, the phytosterol-cholic acid co-crystal prepared by the present invention has higher bioavailability.

[0027] According to an embodiment of the present invention, the unit cell parameters of the β-sitosterol-cholic acid co-crystal are α = 90°, β = 96.7510°, γ = 90°.

[0028] According to an embodiment of the present invention, for the β-sitosterol-cholic acid co-crystal, the X-ray powder diffraction pattern expressed in 2θ angle has characteristic peaks at least at 6.3° ± 0.2°, 7.8° ± 0.2°, 10.7° ± 0.2°, 12.1° ± 0.2°, 12.5° ± 0.2°, 13.5° ± 0.2°, 14.1° ± 0.2°, 17.1° ± 0.2°, 18.2° ± 0.2°, 21.1° ± 0.2°. Preferably, it has an X-ray powder diffraction pattern substantially as Figure 4 shown.

[0029] According to an embodiment of the present invention, for the β-sitosterol-cholic acid co-crystal, measured by differential scanning calorimetry, it has an endothermic peak at 164°C ± 2°C. Preferably, it has a differential scanning calorimetry pattern substantially as Figure 5 shown.

[0030] According to an embodiment of the present invention, for the β-sitosterol-cholic acid co-crystal, its infrared absorption spectrum has absorption peaks at least at 3335 cm -1 , 2937 cm -1 , 2865 cm -1 , 1705 cm-1 , 1443 cm -1 , 1373 cm -1 , 1330 cm -1 , 1228 cm -1 , 1077 cm -1 , 1041 cm -1 has absorption peaks at. Preferably, it has an infrared spectrum substantially as Figure 6 shown.

[0031] According to one embodiment of the present invention, the unit cell parameters of the stigmasterol-cholic acid co-crystal are α = 90°, β = 96.763°, γ = 90°.

[0032] According to one embodiment of the present invention, the X-ray powder diffraction pattern of the stigmasterol-cholic acid co-crystal expressed in 2θ angle has characteristic peaks at least at 6.2° ± 0.2°, 7.8° ± 0.2°, 10.8° ± 0.2°, 12.0° ± 0.2°, 12.3° ± 0.2°, 13.3° ± 0.2°, 14.0° ± 0.2°, 17.5° ± 0.2°, 18.0° ± 0.2°, 21.0° ± 0.2°. Preferably, it has an X-ray powder diffraction pattern substantially as Figure 7 shown.

[0033] According to one embodiment of the present invention, the stigmasterol-cholic acid co-crystal has an endothermic peak at 184°C ± 2°C as determined by differential scanning calorimetry. Preferably, it has a differential scanning calorimetry pattern substantially as Figure 8 shown.

[0034] According to one embodiment of the present invention, the stigmasterol-cholic acid co-crystal has infrared absorption spectra at least at 3343 cm -1 , 2933 cm -1 , 2863 cm -1 , 1704 cm -1 , 1443 cm -1 , 1373 cm -1 , 1330 cm -1 , 1228 cm -1 , 1041 cm -1 , 949 cm -1 has absorption peaks at. Preferably, it has an infrared spectrum substantially as Figure 9 shown.

[0035] According to the second aspect of the present invention, it provides a method for preparing the phytosterol-cholic acid composition, and the method is one of the following Method 1 or Method 2:

[0036] Method 1: Recrystallize phytosterol and cholic acid with a molar ratio of 10:1 to 1:10 in a solvent, and the precipitate is separated and dried to obtain the composition; or

[0037] Method 2: Ball-mill phytosterol and cholic acid with a molar ratio of 10:1 to 1:10 in a solvent for more than 10 minutes, and then dry the obtained solid to obtain the composition.

[0038] According to an embodiment of the present invention, the solvent is selected from one or more of water, C1-C6 alcohols, C3-C8 ketones, esters formed by C1-C6 fatty acids and C1-C6 fatty alcohols, C5-C16 alkanes, C6-C20 aromatic hydrocarbons, and halogenated C5-C16 alkanes; preferably, the solvent is acetone or ethyl acetate.

[0039] According to the third aspect of the present invention, it provides the use of the phytosterol-cholic acid composition in regulating blood lipids, for example, helping to reduce high blood lipid levels and / or maintain normal plasma or blood lipid levels.

[0040] Compared with phytosterol, the phytosterol-cholic acid eutectic prepared by the present invention has a better effect on regulating blood lipids.

[0041] According to the fourth aspect of the present invention, it provides a preparation, comprising the above-mentioned phytosterol-cholic acid composition, and excipients acceptable in foods, cosmetics, drugs, health products or feeds.

[0042] According to the fifth aspect of the present invention, it provides the application of the phytosterol-cholic acid composition or a formulation containing the phytosterol-cholic acid composition in the preparation of foods, cosmetics, drugs, health products or feeds for regulating blood lipids.

[0043] The beneficial effects of the present invention at least include:

[0044] The phytosterol-cholic acid composition of the present invention improves the water solubility of phytosterol at the molecular level by utilizing the emulsifying properties of cholic acid. Therefore, compared with phytosterol itself, the dissolution properties of the phytosterol-cholic acid composition of the present invention are significantly improved, and the phytosterol-cholic acid composition of the present invention has higher bioavailability and better blood lipid regulation effect. Description of the Drawings

[0045] Figure 1 is the X-ray powder diffraction (PXRD) pattern of the phytosterol I-cholic acid eutectic in Example 1 of the present invention;

[0046] Figure 2 is the differential scanning calorimetry (DSC) pattern of the phytosterol I-cholic acid eutectic in Example 1 of the present invention;

[0047] Figure 3 is an infrared spectrum (IR) of the phytosterol I-cholic acid cocrystal of Example 1 of the present invention;

[0048] Figure 4 is an X-ray powder diffraction (PXRD) pattern of the β-sitosterol-cholic acid cocrystal of Example 2 of the present invention;

[0049] Figure 5 is a differential scanning calorimetry (DSC) diagram of the β-sitosterol-cholic acid cocrystal of Example 2 of the present invention;

[0050] Figure 6 is an infrared spectrum (IR) of the β-sitosterol-cholic acid cocrystal of Example 2 of the present invention;

[0051] Figure 7 is an X-ray powder diffraction (PXRD) pattern of the stigmasterol-cholic acid cocrystal of Example 3 of the present invention;

[0052] Figure 8 is a differential scanning calorimetry (DSC) diagram of the stigmasterol-cholic acid cocrystal of Example 3 of the present invention;

[0053] Figure 9 is an infrared spectrum (IR) of the stigmasterol-cholic acid cocrystal of Example 3 of the present invention;

[0054] Figure 10 This is the dissolution curve of the phytosterol I-cholic acid cocrystal and the phytosterol I raw material in Example 1 of the present invention in pH 2.0 buffer (containing 0.5% sodium lauryl sulfate);

[0055] Figure 11 This is the dissolution curve of the phytosterol I-cholic acid cocrystal and the phytosterol I raw material in Example 1 of the present invention in a pH 4.5 buffer solution (containing 0.5% sodium lauryl sulfate);

[0056] Figure 12 This is the dissolution curve of the phytosterol I-cholic acid cocrystal and the phytosterol I raw material in Example 1 of the present invention in a pH 6.8 buffer solution (containing 0.5% sodium lauryl sulfate);

[0057] Figure 13 This is the plasma drug-time curve of the phytosterol I-cholic acid cocrystal and the phytosterol I raw material in Example 1 of the present invention in SD rats. DETAILED DESCRIPTION

[0058] 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.

[0059] Reagents and instruments

[0060] In the embodiment of the present invention, the X-ray powder diffraction pattern was obtained by an X-ray powder diffractometer of the Bruker D2 Phaser model, and the instrument used Cu-Kα irradiation. The scanning range was from 3° to 40° in the 2θ interval, and the scanning speed was 2° / minute.

[0061] Differential scanning calorimetry was performed using a TA DSC Q2000 device, and the heating rate was 10 K / min;

[0062] Thermogravimetric analysis was performed using a TGA-55 thermogravimetric analyzer from TA Instruments, USA;

[0063] Fourier transform infrared spectroscopy was performed using a Thermo Scientific Nicolet 6700;

[0064] A microdissolution tester was used, namely Mini-IDR microdissolution tester;

[0065] High performance liquid chromatography was performed using an Agilent 1260 series high performance liquid chromatograph from Agilent Technologies;

[0066] Mass spectrometry analysis was performed using a Triple Quad 4500 LC-MS from AB SCIEX TM 4500LC-MS;

[0067] Phytosterol I was purchased from Shaanxi Jiahe Biotechnology Co., Ltd., with a purity of ≥95%;

[0068] According to the product description, the phytosterol I consists of β-sitosterol, a small amount of stigmasterol and campesterol.

[0069] Phytosterol II was purchased from Shandong New Element Biotechnology Co., Ltd., with a purity of ≥95%;

[0070] According to the product description, the phytosterol II consists of β-sitosterol, a small amount of stigmasterol and campesterol.

[0071] Phytosterol III was purchased from Shaanxi Hisfort Bioengineering Co., Ltd., with a purity of ≥95%;

[0072] According to the product description, the phytosterol III consists of β-sitosterol, a small amount of stigmasterol and campesterol.

[0073] β-sitosterol was purchased from Shaanxi Xutai Biotechnology Co., Ltd., with a purity of ≥95%;

[0074] Stigmasterol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of ≥90%;

[0075] Cholic acid was purchased from Aladdin Biochemical Technology Co., Ltd., with a purity of ≥95%.

[0076] Example 1

[0077] Weigh 828 mg (about 2 mmol) of phytosterol I and 816 mg (about 2 mmol) of cholic acid, place them in a round-bottom flask, add 200 mL of acetone, and sonicate for 30 min to completely dissolve them. Then, under the condition of a water bath at 55 °C, perform rotary evaporation at a speed of 90 rpm until the solvent is completely evaporated. Dry the obtained solid in a vacuum drying oven at room temperature for 12 h to obtain 1.53 g of the phytosterol I-cholic acid cocrystal, with a yield of about 93.1%.

[0078] The mother liquor was volatilized and crystallized to obtain single crystals. The unit cell parameters were determined by an X-ray single crystal diffractometer as follows: α = 90°, β = 97.042°, γ = 90°.

[0079] The above experimental data verified that the molar ratio of phytosterol I to cholic acid in the cocrystal was 1:1.

[0080] This cocrystal was characterized by X-ray powder diffraction (PXRD), differential scanning calorimetry, and infrared spectroscopy. The experimental results are as Figures 1 - 3 shown.

[0081] Example 2

[0082] Weigh 828 mg (about 2 mmol) of phytosterol II and 816 mg (about 2 mmol) of cholic acid, place them in a round-bottom flask, add 200 mL of acetone, and sonicate for 30 min to completely dissolve them. Then, under the condition of a water bath at 55 °C, perform rotary evaporation at a speed of 90 rpm until the solvent is completely evaporated. Dry the obtained solid in a vacuum drying oven at room temperature for 12 h to obtain 1.47 g of the phytosterol II-cholic acid cocrystal, with a yield of about 89.4%.

[0083] Example 3

[0084] Weigh 828 mg (about 2 mmol) of phytosterol III and 816 mg (about 2 mmol) of cholic acid, place them in a round-bottom flask, add 200 mL of acetone, and sonicate for 30 min to completely dissolve them. Then, under the condition of a water bath at 55 °C, perform rotary evaporation at a speed of 90 rpm until the solvent is completely evaporated. Dry the obtained solid in a vacuum drying oven at room temperature for 12 h to obtain 1.51 g of the phytosterol III-cholic acid cocrystal, with a yield of about 91.8%.

[0085] Example 4

[0086] Weigh 828 mg (about 2 mmol) of β-sitosterol and 816 mg (about 2 mmol) of cholic acid, place them in a round-bottom flask, add 200 mL of acetone, and ultrasonicate for 30 min to completely dissolve them. Then, under the condition of a water bath at 55 °C, perform rotary evaporation at a speed of 90 rpm until the solvent is completely evaporated. Dry the obtained solid in a vacuum drying oven at room temperature for 12 h to obtain 1.51 g of β-sitosterol-cholic acid cocrystal, with a yield of about 91.9%.

[0087] The mother liquor was volatilized and crystallized to obtain single crystals. The unit cell parameters were determined by an X-ray single crystal diffractometer as follows: α = 90°, β = 96.7510°, γ = 90°.

[0088] The above experimental data verified that the molar ratio of β-sitosterol to cholic acid in the cocrystal was 1:1.

[0089] This cocrystal was characterized by X-ray powder diffraction (PXRD), differential scanning calorimetry, and infrared spectroscopy. The experimental results are as Figures 4 - 6 shown.

[0090] Example 5

[0091] Weigh 824 mg (about 2 mmol) of stigmasterol and 816 mg (about 2 mmol) of cholic acid, place them in a round-bottom flask, add 200 mL of acetone, and ultrasonicate at 40 °C for 30 min to completely dissolve them. Then, under the condition of a water bath at 55 °C, perform rotary evaporation at a speed of 90 rpm until the solvent is completely evaporated. Dry the obtained solid in a vacuum drying oven at room temperature for 12 h to obtain 1.48 g of stigmasterol-cholic acid cocrystal, with a yield of about 90.2%.

[0092] The mother liquor was volatilized and crystallized to obtain single crystals. The unit cell parameters were determined by an X-ray single crystal diffractometer as follows: α = 90°, β = 96.763°, γ = 90°.

[0093] The above experimental data verified that the molar ratio of stigmasterol to cholic acid in the cocrystal was 1:1.

[0094] This cocrystal was characterized by X-ray powder diffraction (PXRD), differential scanning calorimetry, and infrared spectroscopy. The experimental results are as Figures 7 - 9 shown.

[0095] Example 6

[0096] Weigh 8.28 g of phytosterol Ⅰ and 8.16 g of cholic acid (molar ratio 1:1), add them to a flask, add 50 mL of acetone, stir at 100 rpm, heat to 80 °C, stir for 1 h, then cool to room temperature and stir for 1 h. Filter the precipitate through a Buchner funnel and place it in a vacuum drying oven to dry at room temperature for 12 h to obtain 12.3 g of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 74.8%.

[0097] Example 7

[0098] Weigh 8.28 g of phytosterol Ⅰ and 8.16 g of cholic acid (molar ratio 1:1), add them to a flask, add 100 mL of ethyl acetate, stir at 100 rpm, heat to 80 °C, stir for 1 h, then cool to room temperature and stir for 1 h. Filter the precipitate through a Buchner funnel and place it in a vacuum drying oven to dry at room temperature for 12 h to obtain 11.7 g of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 71.2%.

[0099] Example 8

[0100] Weigh 82.8 g of phytosterol Ⅰ and 81.6 g of cholic acid (molar ratio 1:1) in a reaction kettle, add 0.6 L of acetone, heat to 60 °C, stir for 1 h, cool to 25 °C, and continue to stir for 12 h. Filter the solution and dry it in vacuum for 24 h to obtain 148.1 g of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 90.1%.

[0101] Example 9

[0102] Weigh 828 mg (about 2 mmol) of phytosterol Ⅰ and 816 mg (about 2 mmol) of cholic acid, add 5 stainless steel balls of 6 mm and 80 μL of acetone, and grind in a metal ball mill at a frequency of 40 Hz for 60 min (10 min each time, with an interval of 1 min). After grinding, dry the obtained solid in a vacuum drying oven at room temperature for 12 h to obtain 1.58 g of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 96.1%.

[0103] Example 10

[0104] Weigh 828 mg (about 2 mmol) of phytosterol Ⅰ and 816 mg (about 2 mmol) of cholic acid, add 5 stainless steel balls of 6 mm and 100 μL of ethyl acetate, and grind in a metal ball mill at a frequency of 40 Hz for 60 min (10 min each time, with an interval of 1 min). After grinding, dry the obtained solid in a vacuum drying oven at room temperature for 12 h to obtain 1.56 g of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 94.9%.

[0105] Example 11

[0106] Weigh 4.14 g of phytosterol Ⅰ and 4.08 g of cholic acid (molar ratio 1:1) and place them in a beaker. Add 800 mL of acetone, stir at 100 rpm, heat up to 80 °C at a rate of 5 °C / min and keep for 30 min until clear. Add 50 mL of pure water, cool down to 20 °C at a rate of -2 °C / min (add 0.1% seed crystals when cooling down to 30 °C), keep at a constant temperature for 2 h, then cool down to -10 °C at a rate of -2 °C / min and keep at a constant temperature for 6 h. White precipitates gradually precipitate out. Filter the precipitates through a Buchner funnel, and place the solid in a vacuum drying oven to dry at room temperature for 18 h to obtain 6.1 g of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 74.2%.

[0107] Example 12

[0108] Weigh 828 mg of phytosterol Ⅰ and 816 mg of cholic acid (molar ratio 1:1) and place them in a beaker. Add 160 mL of acetone, stir at 600 rpm, heat up to 100 °C until completely clear, cool down to -20 °C at a rate of -5 °C / min. White precipitates gradually precipitate out. Filter the precipitates through a Buchner funnel, and place the solid in a vacuum drying oven to dry at room temperature for 18 h to obtain 782 mg of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 47.6%.

[0109] Example 13

[0110] Weigh 414 mg of phytosterol Ⅰ and 408 mg of cholic acid (molar ratio 1:1) and place them in a beaker. Add 400 mL of ethyl acetate, stir at 600 rpm, heat up to 100 °C until completely clear, cool down to -20 °C at a rate of -5 °C / min. White precipitates gradually precipitate out. Filter the precipitates through a Buchner funnel, and place the solid in a vacuum drying oven to dry at room temperature for 18 h to obtain 337 mg of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 41.0%.

[0111] Example 14

[0112] Weigh 82.8 mg of phytosterol Ⅰ and 81.6 mg of cholic acid (molar ratio 1:1) and place them in a beaker. Add 20 mL of acetone, stir at 600 rpm, heat up to 100 °C until completely clear, then slowly cool and volatilize at room temperature for 48 h. White precipitates gradually precipitate out. Place the solid in a vacuum drying oven to dry at room temperature for 18 h to obtain 132 mg of phytosterol Ⅰ-cholic acid cocrystal, with a yield of approximately 80.3%.

[0113] Example 15

[0114] Weigh 41.4 g of phytosterol I and 8.2 g of cholic acid (molar ratio is approximately 5:1), add them to a flask, add 200 mL of acetone, stir at 100 rpm, heat to 80 °C, stir for 1 h, then cool to room temperature and stir for 1 h. Filter the precipitate through a Buchner funnel and place it in a vacuum drying oven to dry at room temperature for 12 h to obtain 40.9 g of the phytosterol I-cholic acid composition, with a yield of approximately 82.4%.

[0115] Example 16

[0116] Weigh 82.8 g of phytosterol I and 8.2 g of cholic acid (molar ratio is approximately 10:1), add them to a flask, add 500 mL of acetone, stir at 100 rpm, heat to 80 °C, stir for 1 h, then cool to room temperature and stir for 1 h. Filter the precipitate through a Buchner funnel and place it in a vacuum drying oven to dry at room temperature for 12 h to obtain 72.5 g of the phytosterol I-cholic acid composition, with a yield of approximately 79.7%.

[0117] Example 17

[0118] Weigh 8.3 g of phytosterol I and 40.8 g of cholic acid (molar ratio is approximately 1:5), add them to a flask, add 200 mL of acetone, stir at 100 rpm, heat to 80 °C, stir for 1 h, then cool to room temperature and stir for 1 h. Filter the precipitate through a Buchner funnel and place it in a vacuum drying oven to dry at room temperature for 12 h to obtain 39.7 g of the phytosterol I-cholic acid composition, with a yield of approximately 80.9%.

[0119] Example 18

[0120] Weigh 8.3 g of phytosterol I and 81.6 g of cholic acid (molar ratio is approximately 1:10), add them to a flask, add 500 mL of acetone, stir at 100 rpm, heat to 80 °C, stir for 1 h, then cool to room temperature and stir for 1 h. Filter the precipitate through a Buchner funnel and place it in a vacuum drying oven to dry at room temperature for 12 h to obtain 71.6 g of the phytosterol I-cholic acid composition, with a yield of approximately 79.6%.

[0121] Example 19

[0122] Weigh 414 mg (about 1 mmol) of phytosterol I, 408 mg (about 1 mmol) of cholic acid and 1248 mg of β-cyclodextrin, add 5 6-mm stainless steel balls and 80 μL of acetone, and grind in a metal ball mill at a frequency of 40 Hz for 30 min (10 min each time, with an interval of 1 min). After grinding, dry the obtained solid in a vacuum drying oven at room temperature for 12 h to obtain 1.86 g of the phytosterol I-cholic acid composition preparation, with a yield of approximately 89.9%.

[0123] Test Example 1

[0124] Compare the equilibrium solubilities of phytosterol I, phytosterol II, phytosterol III, β-sitosterol, stigmasterol, the phytosterol I-cholic acid eutectic obtained in Example 1, the phytosterol II-cholic acid eutectic obtained in Example 2, the phytosterol III-cholic acid eutectic obtained in Example 3, the β-sitosterol-cholic acid eutectic obtained in Example 4, the stigmasterol-cholic acid eutectic obtained in Example 5, the phytosterol I-cholic acid (molar ratio about 5:1) composition obtained in Example 15, the phytosterol I-cholic acid (molar ratio about 10:1) composition obtained in Example 16, the phytosterol I-cholic acid (molar ratio about 1:5) composition obtained in Example 17, and the phytosterol I-cholic acid (molar ratio about 1:10) composition obtained in Example 18 in a buffer solution at pH 6.8 (added with 0.5 wt% sodium dodecyl sulfate).

[0125] Weigh approximately 10 mg of each of the above samples in triplicate and add them separately to 1 mL of a buffer solution at pH 6.8 containing 0.5% sodium dodecyl sulfate. Stir at 200 rpm at room temperature for 24 h. Subsequently, centrifuge the suspension at 14000 rpm for 5 min, collect 0.4 mL of the supernatant, and dilute it to a final volume of 0.8 mL with an equal volume of pure water. Determine the concentration using high performance liquid chromatography.

[0126] The test conditions are as follows:

[0127] Wavelength: 210 nm; Chromatographic column: Agilent ZORBAX SB-C8 column (4.6×150 mm, 5 μm); Column temperature: 35 °C; Injection volume: 10 μL; Flow rate: 1 mL / min; Mobile phase: pure water (eluent A) and acetonitrile (eluent B), isocratic elution with 12% A and 88% B; Sampling time: 30 min.

[0128] It was determined that phytosterol I contains 55.7% β-sitosterol, 14.0% stigmasterol, and 28.7% campesterol; phytosterol II contains 49.7% β-sitosterol, 19.6% stigmasterol, and 27.8% campesterol; phytosterol III contains 49.2% β-sitosterol, 20.1% stigmasterol, and 28.7% campesterol.

[0129] The experimental results are shown in the following table. The solubility of phytosterol (a mixture of β-sitosterol, stigmasterol, and campesterol) was calculated by adding the solubilities of these three components respectively. The results show that the equilibrium solubilities of the phytosterol-cholic acid compositions in the buffer solution at pH 6.8 (containing 0.5% sodium dodecyl sulfate) are all significantly improved compared to the prototypes.

[0130] Table 1 Phytosterol Equilibrium Solubility Test

[0131]

[0132]

[0133] Test Example 2

[0134] Compare the dissolution properties of phytosterol Ⅰ and the phytosterol Ⅰ-cholic acid co-crystal obtained in Example 1 in different buffer media.

[0135] Weigh about 100 mg each (non-sedimentation conditions) of phytosterol Ⅰ and the phytosterol Ⅰ-cholic acid co-crystal obtained in Example 1, in triplicate, and then add them separately to 15 mL of buffer solutions with pH 2.0, 4.5, and 6.8 containing 0.5% sodium dodecyl sulfate. Conduct powder dissolution experiments at 37 °C and 50 rpm. Take 600 μL of the liquid at 5, 10, 15, 30, 45, 60, and 120 min respectively. After centrifugation, take 400 μL of the supernatant and transfer it to a liquid phase bottle, and dilute it with 400 μL of pure water. Detect the concentration by HPLC.

[0136] The test conditions are as follows:

[0137] Wavelength: 210 nm; Chromatographic column: Agilent ZORBAX SB-C8 column (4.6×150 mm, 5 μm); Column temperature: 35 °C; Injection volume: 10 μL; Flow rate: 1 mL / min; Mobile phase: pure water (eluent A) and acetonitrile (eluent B), isocratic elution with 12% A and 88% B; Collection time: 30 min.

[0138] The experimental results are as Figures 10 - 12 shown, where the solubility of phytosterol (a mixture of β-sitosterol, stigmasterol, and campesterol) is calculated by adding the solubilities of these three components respectively.

[0139] It can be Figures 10 - 12 seen that the dissolution of the phytosterol-cholic acid co-crystal in buffer solutions (containing 0.5% sodium dodecyl sulfate) with pH 2.0, pH 4.5, and pH 6.8 has been significantly improved. For example, in the medium with pH 6.8, the co-crystal group can quickly reach a relatively high dissolution concentration (about 3.23 mg / mL at 5 min), which is approximately 163 times that of the raw material group (about 19.8 μg / mL at 5 min). Moreover, the co-crystal group can continuously maintain a high dissolution concentration within 2 h, and still has a 142-fold dissolution advantage at 120 min. And characterized by PXRD, after the dissolution is completed, the co-crystal still does not dissociate, which is consistent with the experimental phenomenon that the co-crystal can maintain a supersaturated concentration in the solution. Thus, it can be seen that the phytosterol-cholic acid co-crystal can significantly improve the in vitro dissolution performance of phytosterol.

[0140] Test Example 3

[0141] Compare the pharmacokinetic properties of phytosterol Ⅰ and the phytosterol Ⅰ-cholic acid co-crystal obtained in Example 1 in SD rats.

[0142] Seven Sprague-Dawley (SD) rats weighing 200-250 g were divided into one group, and were respectively given phytosterol I at 210 mg / kg (calculated as phytosterol) and an equal amount of the phytosterol I-cholic acid eutectic obtained in Example 1. The samples were suspended in a 0.5% Tween 80 aqueous solution and administered by gavage. The rats were purchased from Beijing Huafukang Biotechnology Co., Ltd., and the license number was SCXK (Jing) 2024-0003. They were fasted for 12 hours before administration and allowed to drink water ad libitum. According to the pharmacokinetic properties of phytosterol, about 400 μL of blood was taken from the suborbital venous plexus of the rats at 0 h (before administration) and at 1, 3, 5, 7, 9, 11, 13, and 24 h after administration, and collected into a centrifuge tube containing 20 μL of 1% sodium heparin. Then, after centrifuging the samples at 14,000 rpm for 10 min, 150 μL of the supernatant was taken and stored frozen in a refrigerator at -80 °C.

[0143] Plasma sample treatment: Accurately pipette 100 μL of the thawed plasma sample into a 1.5 mL centrifuge tube, and add 1000 μL of a mixed solution of methanol and methyl tert-butyl ether (volume ratio 1:1). The above mixture was vortexed on a vortex mixer for 30 min to extract and precipitate proteins, and then centrifuged at 14,000 rpm for 5 min. 800 μL of the supernatant was collected and purged with nitrogen until no liquid remained. Finally, it was re-dissolved with 400 μL of methanol and shaken for 20 min to ensure thorough mixing, and the content of the solution was analyzed by HPLC-MS.

[0144] HPLC-MS analysis method: Chromatographic column: Agilent Poroshell 120 EC-C8 (2.1×50 mm, 2.7 μm); Mobile phase: 0.1% formic acid-aqueous solution (eluent A) and 0.1% formic acid-methanol solution (eluent B), gradient elution, and the specific conditions are shown in the following table; Sampling time 9 min; Flow rate: 0.5 mL / min; Column temperature: 40 °C; Injection volume: 10 μL; Ion source: Atmospheric pressure chemical ionization source (APCI), positive ion mode; Scanning mode: MRM, β-sitosterol m / z 397.5→147.1, stigmasterol m / z 383.5→146.9, campesterol m / z 395.5→83.1.

[0145] Since there is a certain amount of phytosterol in the rats' bodies before administration, in order to ensure the accuracy of comparative analysis during data processing, all data need to subtract the baseline concentration at 0 h. The experimental results are as Figure 13As shown, the plasma concentration of phytosterols (a mixture of β-sitosterol, stigmasterol, and campesterol) was calculated by adding the concentrations of these three components individually. The results showed that the phytosterol-cholic acid co-crystal obtained in Example 1 had higher bioavailability than the phytosterol prototype at the same dosing dose, and C max was 4 times that of it, and the AUC 1-24 was 6 times that of it.

[0146] Table 2 Mobile phase gradient

[0147] Time (min) Proportion of Phase A (%) Proportion of Phase B (%) 0 30 70 1 30 70 2 10 90 7 10 90 7.1 30 70 9 30 70

[0148] Test Example 4

[0149] Compare the lipid-lowering effects of phytosterol I and the phytosterol I-cholic acid co-crystal obtained in Example 1 on hyperlipidemic model rats.

[0150] Animal model preparation: A model was established using the high-fat diet induction method. Eighty male SD rats with an initial body weight of 180 to 220 g were randomly divided into two groups. One group was used as the normal control group (n = 8) and fed a normal standard diet; the other group was used as the model group (n = 72) and received a high-fat diet. The rats were purchased from Beijing Huafukang Biotechnology Co., Ltd., with the license number SCXK (Jing) 2024-0003. The rats had free access to food and water and underwent a one-week diet adaptation period. After four weeks of feeding, the rats were fasted but not water-deprived the night before the test, and then 400 μL of blood samples were collected from the fundus venous plexus of each rat. The collected blood samples were centrifuged at 14,000 revolutions per minute for 10 minutes, and the separated plasma was used to analyze four lipid indices, including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). The four lipid indices between the control group and the model group were compared statistically. If the levels of TG, TC, and LDL-C in the model group were significantly higher than those in the control group, it indicated that the model was successfully constructed.

[0151] Animal grouping and administration process: The 8 rats in the normal control group were continuously fed with ordinary control feed and gavaged with blank solvent (0.5% Tween 80) every day, serving as the blank control group. The 56 successfully modeled rats were randomly divided into 7 groups according to the initial indicators, with 8 rats in each group, ensuring that there were no significant differences in the initial conditions among the groups. The rats in the first group were given high-fat feed and gavaged with blank solvent every day, serving as the model control group; the rats in the second group were given high-fat feed and gavaged with 210 mg / kg fish oil every day, serving as the fish oil intervention group; the rats in the third group were given high-fat feed and gavaged with 200 mg / kg curcumin every day, serving as the curcumin intervention group; the rats in the fourth group were given high-fat feed and gavaged with 210 mg / kg phytosterol every day, serving as the low-dose phytosterol intervention group; the rats in the fifth group were given high-fat feed and gavaged with 315 mg / kg phytosterol every day, serving as the high-dose phytosterol intervention group; the rats in the sixth group were given high-fat feed and gavaged with 210 mg / kg phytosterol-cholic acid eutectic equivalent to phytosterol every day, serving as the low-dose eutectic intervention group; the rats in the seventh group were given high-fat feed and gavaged with 315 mg / kg phytosterol-cholic acid eutectic equivalent to phytosterol every day, serving as the high-dose eutectic intervention group. All rats were administered by oral gavage, and the samples were suspended in 0.5% Tween 80 for 4 weeks. The body weights of the rats were recorded regularly every week, and the administration doses were precisely adjusted according to the body weight changes.

[0152] Testing of related indicators: After four weeks of administration, 400 μL of blood samples were collected from the fundus venous plexus of each rat. The collected blood samples were centrifuged at a speed of 14,000 revolutions per minute for 10 minutes, and the separated plasma was used to analyze four blood lipid indicators, including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C).

[0153] Animal sampling: After blood collection on the 28th day of the administration period in each group, the rats continued to be administered until sampling. Before sampling, the rats were weighed, and then the rats were sacrificed without fasting. The perirenal and epididymal adipose tissues were separated, washed with normal saline, dried and weighed, and observed and photographed. Fat index (mg / g) = fat weight (mg) / final body weight (g).

[0154] The experimental results are shown in the following table. The results show that both phytosterol and phytosterol-cholic acid eutectic have certain effects on inhibiting body weight gain, reducing visceral fat and lowering blood lipids. However, the effect of phytosterol-cholic acid eutectic is more obvious, and the higher the dose, the more significant the effect.

[0155] Table 3 Body weights and fat indices of rats in each group

[0156]

[0157] TC, TG, LDL-C, and HDL-C levels in the plasma of rats in each group in Table 4

[0158]

[0159] Note: n = 8; t-tests were performed on the data using SPSS software. Compared with the rats in the blank control group # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the rats in the model control group * p < 0.05, ** p < 0.01,

[0160] *** p < 0.001.

[0161] This application is only a preferred embodiment of the present invention, but it is not used to limit the claims. Any modifications, equivalent replacements, and improvements made by any person skilled in the art without departing from the concept of this application shall fall within the protection scope of the present invention.

Claims

1. A composition of phytosterol and cholic acid, characterized in that, The molar ratio of phytosterol to cholic acid in the phytosterol-cholic acid composition ranges from 10:1 to 1:10, preferably 1:

1.

2. The phytosterol-cholic acid composition according to claim 1, wherein The phytosterol is selected from β-sitosterol, stigmasterol, campesterol and mixtures thereof.

3. The phytosterol-cholic acid composition according to claim 1, characterized in that, The composition contains a phytosterol-cholic acid co-crystal.

4. The phytosterol-cholic acid composition according to claim 3, wherein The phytosterol-cholic acid co-crystal is a co-crystal of a phytosterol mixture (including β-sitosterol, stigmasterol, campesterol)-cholic acid, and its X-ray powder diffraction expressed in 2θ angle has characteristic peaks at least at diffraction angles of 6.4°±0.2°, 7.9°±0.2°, 10.8°±0.2°, 12.2°±0.2°, 12.6°±0.2°, 13.5°±0.2°, 14.1°±0.2°, 17.2°±0.2°, 18.2°±0.2°, 21.2°±0.2°.

5. The phytosterol-cholic acid composition according to claim 3, wherein The phytosterol-cholic acid co-crystal is a β-sitosterol-cholic acid co-crystal, and its X-ray powder diffraction expressed in 2θ angle has characteristic peaks at least at diffraction angles of 6.3°±0.2°, 7.8°±0.2°, 10.7°±0.2°, 12.1°±0.2°, 12.5°±0.2°, 13.5°±0.2°, 14.1°±0.2°, 17.1°±0.2°, 18.2°±0.2°, 21.1°±0.2°.

6. The phytosterol-cholic acid composition according to claim 3, characterized in that, The phytosterol-cholic acid co-crystal is a stigmasterol-cholic acid co-crystal, and its X-ray powder diffraction expressed in 2θ angle has characteristic peaks at least at diffraction angles of 6.2°±0.2°, 7.8°±0.2°, 10.8°±0.2°, 12.0°±0.2°, 12.3°±0.2°, 13.3°±0.2°, 14.0°±0.2°, 17.5°±0.2°, 18.0°±0.2°, 21.0°±0.2°.

7. The preparation method of the phytosterol-cholic acid composition according to any one of claims 1 to 6, and the method is one of the following method one or method two: Method one: Recrystallize phytosterol and cholic acid with a molar ratio of 10:1 to 1:10 in a solvent, and the precipitate is separated and dried to obtain the composition; or Method two: Ball-mill phytosterol and cholic acid with a molar ratio of 10:1 to 1:10 in a solvent for more than 10 minutes, and then dry the obtained solid to obtain the composition.

8. The preparation method according to claim 7, characterized in that, The solvent is selected from one or more of water, C1-C6 alcohols, C3-C8 ketones, esters formed by C1-C6 fatty acids and C1-C6 fatty alcohols, C5-C16 alkanes, C6-C20 aromatic hydrocarbons and halogenated C5-C16 alkanes; preferably, the solvent is acetone or ethyl acetate.

9. A preparation, which contains the phytosterol-cholic acid composition according to any one of claims 1 to 6, and excipients acceptable in food, cosmetics, drugs, health products, or feeds.

10. The application of the phytosterol-cholic acid composition according to any one of claims 1 to 6 or the preparation according to claim 9 in the preparation of foods, cosmetics, drugs, health products or feeds for regulating blood lipids.

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