Plant-derived sterol short-chain fatty acid ester as well as preparation method and application thereof

Abstract

The invention provides plant-derived sterol short-chain fatty acid ester as well as a preparation method and application thereof, and the method comprises the following steps: respectively weighing plant-derived sterol, short-chain fatty acid or short-chain fatty acid ester, a catalyst and a molecular sieve, mixing, and adding a reaction solvent for reaction; after the reaction is finished, taking out reaction liquid, filtering to remove the catalyst and the molecular sieve, and removing the reaction solvent to obtain a plant-derived sterol short-chain fatty acid ester crude product; and extracting and separating to obtain a pure product of the plant-derived sterol short-chain fatty acid ester. The plant-derived sterol short-chain fatty acid ester is phytosterol short-chain fatty acid ester or phytostanol short-chain fatty acid ester. The method has the advantages of high conversion rate, simplicity in process operation, easiness in separation, environment friendliness and the like, and is good in safety, high in yield and low in cost. The plant-derived sterol short-chain fatty acid ester is applied to preparation of food, medicine or feed for relieving colitis, reducing cholesterol or losing weight, the relieving effect on colitis is good, and meanwhile, the plant-derived sterol short-chain fatty acid ester has good cholesterol-reducing and weight-losing effects.

Description

Technical Field

[0001] The present invention belongs to the technical fields of food, cosmetics, medicine and feed, and particularly relates to a plant-derived sterol short-chain fatty acid ester and a preparation method and application thereof. Background Art

[0002] While drugs like statins, ezetimibe, and cholestyramine can lower blood lipids, long-term use often results in significant side effects. In contrast, proper dietary intervention can effectively prevent cardiovascular disease and avoid the shortcomings of drug therapy.

[0003] In recent years, due to irregular work and rest schedules and eating habits, the resulting colitis is gradually developing into a new global intestinal inflammatory disease with a long course, difficult to cure, and drug treatment is often accompanied by many side effects. Although its pathogenesis has not yet been fully clarified, genetics, environment, immune system and intestinal flora have all been proven to be related to it. Intestinal homeostasis imbalance, especially changes in intestinal flora composition and metabolic disorders, are important characteristics of colitis. Short-chain fatty acids (such as acetate, propionate and butyrate) are important components for alleviating colitis as energy sources for colon epithelial cells and regulators of intestinal homeostasis. However, short-chain fatty acids are volatile, highly corrosive, have a pungent odor and are easily absorbed by the upper digestive tract, making it difficult to reach the colon directly, and have almost no effect on alleviating colitis. Although the corresponding derivatives can solve these problems, how to achieve colon-specific release remains a major challenge.

[0004] Phytosterols and phytostanols are plant-derived sterols that are active ingredients in vegetable oils, seeds, nuts, and grains. They have significant cholesterol-lowering and anti-inflammatory activities, and have low bioavailability and are not easily absorbed by the upper gastrointestinal tract. Combining short-chain fatty acids with phytosterols or phytostanols to form corresponding phytosterol esters or phytostanol esters can not only overcome the deficiency of short-chain fatty acids, but also have the potential to relieve colitis and lower blood lipids. In the existing published papers and open patent documents, no research on the synthesis of phytosterol short-chain fatty acid esters or phytostanol short-chain fatty acid esters has been found, nor has the application of phytosterol short-chain fatty acid esters or phytostanol short-chain fatty acid esters in relieving colitis, lowering cholesterol, and losing weight been found. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides a plant-derived sterol short-chain fatty acid ester, a preparation method thereof and an application thereof. For the first time, the plant-derived sterol short-chain fatty acid ester is disclosed. The plant-derived sterol short-chain fatty acid ester is a phytosterol short-chain fatty acid ester or a phytostanol short-chain fatty acid ester, and a preparation method of the product and an application thereof in the preparation of foods, medicines or feeds for relieving colitis, reducing cholesterol or losing weight are provided. The present invention uses a biological enzyme as a catalyst to catalyze the reaction of a short-chain fatty acid with phytosterol or phytostanol under certain conditions to synthesize a phytosterol short-chain fatty acid ester or a phytostanol short-chain fatty acid ester, which has the advantages of high conversion rate (≥90%), simple process operation, easy separation, environmental friendliness, etc., good safety, high yield (≥90%) and low cost, which is beneficial to the industrial production of phytosterol short-chain fatty acid ester and phytostanol short-chain fatty acid ester. The phytosterol short-chain fatty acid ester and the phytostanol short-chain fatty acid ester have good effects on relieving colitis, and at the same time have good cholesterol-lowering and weight-loss effects.

[0006] Note that the description of these objectives does not preclude the existence of other objectives. One embodiment of the present invention does not need to achieve all of the above objectives. Objectives other than the above can be extracted from the description of the specification, drawings and claims.

[0007] The present invention achieves the above technical objectives through the following technical means.

[0008] A plant-derived sterol short-chain fatty acid ester, the plant-derived sterol short-chain fatty acid ester has the following structure:

[0009]

[0010] Wherein, R1 is CH3, C2H5 or C3H7;

[0011] R2 is

[0012] A preparation method of the plant-derived sterol short-chain fatty acid ester according to the above, comprising the following steps:

[0013] Step S1, respectively weigh a plant-derived sterol, a short-chain fatty acid or a short-chain fatty acid ester, a catalyst and a molecular sieve, mix them, and add a reaction solvent to carry out a reaction;

[0014] Step S2, after the reaction is completed, take out the reaction solution, filter to remove the catalyst and the molecular sieve, and remove the reaction solvent to obtain a crude product of the plant-derived sterol short-chain fatty acid ester;

[0015] Step S3, obtain a pure product of the plant-derived sterol short-chain fatty acid ester through extraction and separation.

[0016] In the above scheme, the plant-derived sterol is phytosterol or phytostanol; the plant-derived sterol short-chain fatty acid ester is phytosterol short-chain fatty acid ester or phytostanol short-chain fatty acid ester.

[0017] Furthermore, the phytosterol is one or more of sitosterol, stigmasterol, ergosterol, campesterol, fucosterol in any proportion mixture; the phytostanol is one or more of sitostanol, stigmasterol, ergostanol, campestanol, fucostanol in any proportion mixture.

[0018] In the above scheme, the short-chain fatty acid is one or more of acetic acid, propionic acid, butyric acid in any proportion mixture; the short-chain fatty acid ester is one or more of methyl acetate, ethyl acetate, methyl propionate, ethyl propionate, methyl butyrate, ethyl butyrate, glyceryl triacetate, glyceryl tripropionate, glyceryl tributyrate in any proportion mixture.

[0019] In the above scheme, the catalyst is one or more of Candida antarctica lipase B, Rhizomucor miehei lipase, Candida lipase 99-125, porcine pancreatic lipase, Bacillus subtilis protease in any proportion mixture.

[0020] In the above scheme, the reaction solvent is one of n-hexane, isooctane, cyclohexane, tert-butanol, tert-amyl alcohol, acetone, tetrahydrofuran.

[0021] In the above scheme, the concentration of the plant-derived sterol is 20 mmol / L to 160 mmol / L, the molar ratio of the plant-derived sterol to the short-chain fatty acid is 1:1 to 1:5, the catalyst addition amount is 5 g / L to 120 g / L, the molecular sieve addition amount is 40 g / L to 200 g / L, the reaction solvent is 30 to 500 mL, the reaction temperature is 30 °C to 80 °C, and the reaction time is 6 h to 60 h.

[0022] Preferably, the concentration of the plant-derived sterol is 30 mmol / L to 150 mmol / L, the molar ratio of the plant-derived sterol to the short-chain fatty acid is 1:1 to 1:3, the catalyst addition amount is 10 g / L to 80 g / L, the molecular sieve addition amount is 40 g / L to 150 g / L, the reaction solvent is 50 to 400 mL, the reaction temperature is 40 °C to 70 °C, and the reaction time is 12 h to 48 h.

[0023] Application of a plant-derived sterol short-chain fatty acid ester, application of the plant-derived sterol short-chain fatty acid ester obtained according to the plant-derived sterol short-chain fatty acid ester or the preparation method of the plant-derived sterol short-chain fatty acid ester in the preparation of foods, medicines or feeds for relieving colitis, reducing cholesterol or losing weight. Specifically, application of phytosterol short-chain fatty acid esters and stanol short-chain fatty acid esters in the preparation of foods, medicines or feeds for relieving colitis, reducing cholesterol or losing weight has a good effect on relieving colitis, and at the same time has good cholesterol-lowering and weight-loss effects.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] The present invention first invented a new plant-derived sterol short-chain fatty acid ester: short-chain fatty acid derivative - phytosterol / stanol short-chain fatty acid ester. Compared with short-chain fatty acids, this new type of phytosterol short-chain fatty acid ester or stanol short-chain fatty acid ester not only makes up for the deficiencies of short-chain fatty acid products in practical applications, such as easy volatilization, strong corrosiveness, pungent odor, and difficulty in reaching the colon, but also has advantages such as colon-specific release, and also shows excellent effects in relieving colitis, reducing cholesterol and losing weight. It is a functional ingredient with good development prospects. In addition, the present invention also provides a preparation method for this new type of plant-derived sterol short-chain fatty acid ester. This method has the advantages of high conversion rate (≥90%), short time consumption (6h - 48h), simple process operation, low energy consumption, easy separation, mild reaction conditions, and environmental friendliness. The phytosterol / stanol short-chain fatty acid ester product obtained by separation has good safety, high yield (≥90%), and low cost, which is conducive to industrial production and is of great significance for improving the industrial production level of plant-derived sterol short-chain fatty acid esters or stanol short-chain fatty acid esters in China.

[0026] Note that the recording of these effects does not prevent the existence of other effects. A mode of the present invention does not necessarily have to have all of the above effects. Effects other than the above can be obviously seen and extracted from the descriptions in the specification, drawings, claims, etc. Description of the Drawings

[0027] Figure 1 It is a diagram showing the effects of different phytosterol fatty acid esters on DSS-induced colitis in mice. Among them, Figure 1 A shows the effect on body weight, Figure 1 B shows the effect on relative spleen weight, Figure 1 C shows the effect on colon morphology, Figure 1 D shows the effect on colon length. Detailed Embodiments

[0028] The present invention will be further described below in conjunction with specific embodiments. These embodiments are for illustrative purposes only and do not limit the scope and essence of the present invention. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified; the reagents, materials, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0029] A plant-derived sterol short-chain fatty acid ester, and the plant-derived sterol short-chain fatty acid ester has the following structure:

[0030]

[0031] Among them, R1 is CH3, C2H5 or C3H7;

[0032] R2 is

[0033] The plant-derived sterol short-chain fatty acid esters formed by different general formulas, combinations of R1 and R2 are shown in Table 1:

[0034] Table 1 Plant-derived sterol short-chain fatty acid esters formed by different general formulas, combinations of R1 and R2

[0035]

[0036]

[0037] A preparation method of the plant-derived sterol short-chain fatty acid ester according to claim 1, comprising the following steps:

[0038] Step S1, respectively weigh plant-derived sterol, short-chain fatty acid or short-chain fatty acid ester, catalyst and molecular sieve into a reaction flask, add a reaction solvent and carry out a reaction;

[0039] Step S2, after the reaction is completed, take out the reaction solution, filter to remove the catalyst and molecular sieve, and remove the reaction solvent to obtain a crude product of the plant-derived sterol short-chain fatty acid ester;

[0040] Step S3, obtain a pure product of the plant-derived sterol short-chain fatty acid ester through extraction and separation.

[0041] Preferably, the plant-derived sterol is phytosterol or phytostanol; the plant-derived sterol short-chain fatty acid ester is phytosterol short-chain fatty acid ester or phytostanol short-chain fatty acid ester.

[0042] Preferably, the phytosterol is one or more of sitosterol, stigmasterol, ergosterol, campesterol, fucosterol in any proportion; the phytostanol is one or more of sitostanol, stigmasterol, ergostanol, campestanol, fucostanol in any proportion.

[0043] Preferably, the short-chain fatty acid is one or more of acetic acid, propionic acid, and butyric acid in any proportion; the short-chain fatty acid ester is one or more of methyl acetate, ethyl acetate, methyl propionate, ethyl propionate, methyl butyrate, ethyl butyrate, triacetin, tripropionin, and tributyrin in any proportion.

[0044] Preferably, the catalyst is one or a mixture of more than one of Candida antarctica lipase B, Rhizomucor miehei lipase, Candida lipase 99-125, porcine pancreatic lipase, and Bacillus subtilis protease in any proportion.

[0045] Preferably, the reaction solvent is one of n-hexane, isooctane, cyclohexane, tert-butanol, tert-amyl alcohol, acetone, and tetrahydrofuran.

[0046] The concentration of the plant-derived sterols is 20 mmol / L to 160 mmol / L, the molar ratio of the plant-derived sterols to the short-chain fatty acids is 1:1 to 1:5, the amount of catalyst added is 5 g / L to 120 g / L, the amount of molecular sieve added is 40 g / L to 200 g / L, the reaction solvent is 30 to 500 mL, the reaction temperature is 30° C. to 80° C., and the reaction time is 6 h to 60 h.

[0047] Preferably, the concentration of the plant-derived sterols is 30 mmol / L to 150 mmol / L, the molar ratio of the plant-derived sterols to the short-chain fatty acids is 1:1 to 1:3, the amount of catalyst added is 10 g / L to 80 g / L, the amount of molecular sieve added is 40 g / L to 150 g / L, the reaction solvent is 50 to 400 mL, the reaction temperature is 40°C to 70°C, and the reaction time is 12 h to 48 h.

[0048] A use of a plant-derived sterol short-chain fatty acid ester, and the use of the plant-derived sterol short-chain fatty acid ester obtained according to the plant-derived sterol short-chain fatty acid ester or the preparation method of the plant-derived sterol short-chain fatty acid ester in the preparation of food, medicine or feed for relieving colitis, lowering cholesterol or losing weight.

[0049] Thin layer analysis: The developing solvent was petroleum ether (60-90°C):ethyl acetate (8:2, v / v), and the color was developed with iodine vapor.

[0050] Extraction and separation: After the reaction, centrifuge at 3000 rpm for 5 minutes to remove lipase and molecular sieves, add an equal volume of water, extract thoroughly, collect the organic phase, and remove the solvent by rotary evaporation to obtain the pure product.

[0051] Liquid chromatography analysis: Take 200 μL of the reaction solution, dilute it with 1 mL of n-hexane: absolute ethanol (1:1, v / v), and filter it through a 0.45 μm organic microporous membrane. Use an LC-20AD type high performance liquid chromatograph, Symmetry C 18 chromatographic column (5 μm, 4.6 mm × 150 mm, Waters), control the column temperature at 35 °C, the injection volume is 10 μL, the mobile phase is methanol: isopropanol (8:2, v / v), and the flow rate is 1.0 mL / min. Select a ZAM4000 type evaporative light scattering detector as the detector, use nitrogen as the carrier gas, the pressure is 0.6 bar, and control the detector temperature at 70 °C.

[0052] Mass spectrometry analysis: Use a Waters Xevo G2-XS QTof high resolution mass spectrometer, the injection volume is 1 μL, and the ionization method uses the electrospray positive ion mode (ESI + ), the capillary voltage is 3.5 KV, the ion source temperature is 100 °C, the desolvation temperature is 250 °C, the desolvation gas flow rate is 500 L / h, the cone gas flow rate is 50 L / h, the cone voltage is 20 V, the collision energy is 6 V, the detector voltage is 1700 V, and the mass range is 100 - 1200 m / z.

[0053] Infrared spectroscopy analysis: The instrument model is Nicolet iS50, use the attenuated total reflection method (ATR), the scanning range is 400 - 4000 cm -1 , the number of scans is 32 times, the number of background scans is 32 times, the resolution is 4 cm -1 , the sampling gain is 1, the moving mirror speed is 0.4747, and the aperture setting is 100.

[0054] Example 1

[0055] Put 20 mmol / L sitosterol, 20 mmol / L acetic acid, 5 g / L Candida antarctica lipase B and 50 g / L molecular sieve into the reaction flask in turn, add 50 mL of n-hexane, and place it in a constant temperature gas bath oscillator at 40 °C for reaction for 24 h. After the reaction is completed, filter off the lipase and molecular sieve, collect the supernatant, and rotary evaporate to remove n-hexane to obtain the crude product of sitosterol acetate. By high performance liquid chromatography analysis, the conversion rate of sitosterol acetate is 95.2%. After extraction and separation, the yield of the product is 93.1%.

[0056] The structure characterization data of the product is as follows. Infrared spectrum: The signal corresponding to C-H in =CH in the product is at 3019 cm -1 , 2850 - 2940 cm -1 is the asymmetric stretching vibration of methyl -CH3 and methylene -CH2, and 1733.1 cm -1is the characteristic peak of the stretching vibration of the carbonyl - C=O, 1183.7 cm -1 is the characteristic absorption peak of the carbon - oxygen single bond C - O. Compared with acetic acid, the characteristic signal of the carboxyl group does not appear in the product; compared with sitosterol, the characteristic absorption signal of the hydroxyl group does not appear, indicating that the product does not contain a hydroxyl group. In addition, the product shows the signals of the carbonyl (C=O) and carbon - oxygen single bond (C - O) of the ester bond at 1733.1 cm -1 and 1183.7 cm -1 , indicating that the product is sitosterol acetate.

[0057] Mass spectrometry: The molecular weight of acetic acid is 60, the molecular weight of sitosterol is 414, and the theoretical molecular weight of the product is 456. In the ES + mass spectrometry, m / z 479 is [M + Na] + , m / z 397 is [M - acetic acid + H] + , further confirming that the product is sitosterol acetate.

[0058] Example 2

[0059] 40 mmol / L stigmasterol, 80 mmol / L butyric acid, 50 g / L Rhizomucor miehei lipase, and 40 g / L molecular sieve were successively placed in a reaction flask, 30 mL of isooctane was added, and the mixture was reacted in a 30 °C constant - temperature gas - bath oscillator for 60 h. After the reaction was completed, the lipase and molecular sieve in the reaction flask were removed, the supernatant was collected, and the crude stigmasterol butyrate was obtained by rotary evaporation. By high - performance liquid chromatography analysis, the conversion rate of stigmasterol butyrate was 92.4%. The yield of the product after extraction and separation was 90.9%.

[0060] The structure characterization data of the product are as follows. Infrared spectrum: The signal at 3027 cm -1 in the product corresponds to the C - H signal in =CH, and 2851 - 2938 cm -1 is the antisymmetric stretching vibration of the C - H in the characteristic absorption methyl - CH3 and methylene - CH2. The stretching vibration characteristic absorption peak of the carbonyl - C=O - is at 1732.2 cm -1 (n -C＝O- ), and 1184.1 cm -1 is the characteristic absorption peak of the carbon - oxygen single bond C - O (n C-O ). Compared with butyric acid, the characteristic absorption signal of the carboxyl group does not appear in the product, indicating that there is no carboxyl group in the product. Compared with stigmasterol, the characteristic absorption signal of the hydroxyl group does not appear, indicating that the product does not contain a hydroxyl group. In addition, the product shows the signals of the carbonyl (C=O) and carbon - oxygen single bond (C - O) of the ester bond at 1732.2 cm -1 and 1184.1 cm -1 , indicating that the product is stigmasterol butyrate.

[0061] Mass spectrometry: The molecular weight of butyric acid is 88, the molecular weight of stigmasterol is 412, and the theoretical molecular weight of the product stigmasterol butyrate is 482. In the ES+ mass spectrometry, m / z 505 is the [M+Na] of stigmasterol butyrate + molecular ion peak, and m / z 395 is the signal of [M-butyric acid + H] + , further indicating that the product is stigmasterol butyrate.

[0062] Example 3

[0063] 80 mmol / L ergosterol, 240 mmol / L ethyl acetate, 60 g / L porcine pancreatic lipase and 120 g / L molecular sieve were placed in a reaction flask, 200 mL of cyclohexane was added, and the mixture was reacted in a 45 °C constant temperature air bath oscillator for 36 h. After the reaction was completed, the catalyst and molecular sieve were removed by filtration, and tert-butanol was removed by rotary evaporation to obtain a crude product of ergosterol acetate. The conversion rate analyzed by high performance liquid chromatography was 94.5%. After extraction and separation, the yield of the product was 92.0%.

[0064] The structural characterization data of the product are as follows. Infrared spectrum: The C-H signal of =CH in the product is at 3027.5 cm -1 , the C-H vibrations of methyl and methylene are at 2851-2937 cm -1 , the characteristic peak of carbonyl -C=O is at 1731.0 cm -1 , and the C-O characteristic peak is at 1181.0 cm -1 . The strong carbonyl (C=O) at 1743 cm and the strong characteristic signal peak of carbon-oxygen single bond (C-O) at 1098 cm -1 of ethyl acetate disappeared, while medium-intensity signal peaks appeared at 1731.0 cm -1 and 1085.0 cm -1 . Compared with ergosterol, the characteristic absorption signal of the hydroxyl group did not appear, indicating that there is no hydroxyl group. It shows that the product is ergosterol acetate. -1 Mass spectrometry: The molecular weight of ethyl acetate is 88, the molecular weight of ergosterol is 396, and the theoretical molecular weight of the product is 438. In the ES + mass spectrometry, m / z461 is [M+Na] + , m / z 379 is [M-ethyl acetate + H] + , further indicating that the product is ergosterol acetate. -1 -1 Example 4

[0066] 120 mmol / L campesterol, 480 mmol / L methyl butyrate, 30 g / L Candida rugosa lipase 99-125 and 100 g / L

[0067] were placed in a reaction flask, and 200 mL of cyclohexane was added. The mixture was reacted in a 45 °C constant temperature air bath oscillator for 36 h. After the reaction was completed, the catalyst and molecular sieve were removed by filtration, and tert-butanol was removed by rotary evaporation to obtain a crude product of campesterol butyrate. The conversion rate analyzed by high performance liquid chromatography was 93.0%. After extraction and separation, the yield of the product was 90.5%. The molecular sieve was placed in a reaction flask, 150 mL of tert-butanol was added, and the mixture was reacted in a constant temperature air bath shaker at 60 °C for 12 h. After the reaction was completed, the catalyst and the molecular sieve were removed by filtration, and isooctane was removed by rotary evaporation to obtain a crude product of campesteryl butyrate. The conversion rate analyzed by high performance liquid chromatography was 90.6%. After extraction and separation, the yield of the product was 88.7%.

[0068] The structure characterization data of the product are as follows. Infrared spectrum: The C-H signal of =CH in the product is at 3026 cm -1 The C-H vibrations of methyl and methylene are at 2851 - 2939 cm -1 The characteristic peak of the carbonyl group -C=O is at 1732.4 cm -1 The characteristic peak of the C-O group is at 1184.5 cm -1 The strong carbonyl (C=O) and 1097 cm -1 The characteristic signal peaks of the strong carbon-oxygen single bond (C-O) of methyl butyrate disappear, while medium-intensity signal peaks appear at 1732.4 cm -1 and 1184.5 cm -1 Compared with campesterol, the characteristic absorption signal of the hydroxyl group does not appear, indicating that there is no hydroxyl group. It shows that the product is campesteryl butyrate. -1

[0069] Mass spectrometry: The molecular weight of methyl butyrate is 102, the molecular weight of campesterol is 400, and the theoretical molecular weight of the product is 470. In the ES + mass spectrometry, m / z 493 is [M + Na] + and m / z 383 is [M - butyric acid + H] + which further confirms that the product is campesteryl butyrate.

[0070] Example 5

[0071] 160 mmol / L of fucosterol, 240 mmol / L of glyceryl tripropionate, 50 g / L of Bacillus subtilis protease and 140 g / L The molecular sieve was placed in a reaction flask, 250 mL of tert-amyl alcohol was added, and the mixture was reacted in a constant temperature air bath shaker at 55 °C for 30 h. After the reaction was completed, the catalyst and the molecular sieve were removed by filtration, and tert-amyl alcohol was removed by rotary evaporation to obtain a crude product of fucosterol propionate. The conversion rate analyzed by high performance liquid chromatography was 91.2%. After extraction and separation, the yield of the product was 89.0%.

[0072] The structure characterization data of the product are as follows. Infrared spectrum: The C-H signal of =CH in the product is at 3011 cm -1 The C-H vibrations of methyl and methylene are at 2849 - 2938 cm -1 The characteristic peak of the carbonyl group is at 1733.6 cm -1 ​is the characteristic peak of carbonyl - C=O, 1084.5 cm -1 is the characteristic peak of C - O. Glycerol tripropionate shows strong carbonyl (C=O) at 1748 cm -1 and strong characteristic signal peaks of carbon - oxygen single bond (C - O) at 1127 cm -1 disappear, while medium - intensity signal peaks appear at 1733.6 cm -1 and 1084.5 cm -1 Compared with fucosterol, the characteristic absorption signal of the hydroxyl group does not appear, indicating the absence of a hydroxyl group. It shows that the product is fucosterol propionate.

[0073] Mass spectrometry: The molecular weight of glycerol tripropionate is 260, the molecular weight of fucosterol is 412, and the theoretical molecular weight of the product is 468. In the ES + mass spectrometry, m / z 491 is [M + Na] + , m / z 395 is [M - propionic acid + H] + , further indicating that the product is fucosterol propionate.

[0074] Example 6

[0075] Put 20 mmol / L stigmastanol, 100 mmol / L propionic acid, 120 g / L porcine pancreatic lipase and 80 g / L molecular sieve into a reaction flask, add 100 mL of acetone, and place it in a constant - temperature gas - bath oscillator at 50 °C for 24 h. After the reaction is completed, filter to remove the catalyst and molecular sieve, and rotary evaporate to remove acetone to obtain the crude product of stigmastanol propionate. High - performance liquid chromatography analysis shows that the conversion rate is 93.8%. After extraction and separation, the yield of the product is 91.5%.

[0076] The structural characterization data of the product are as follows. Infrared spectrum: In the product, 2852 - 2935 cm -1 is the stretching vibration of C - H of methyl and methylene groups, 1733.5 cm -1 is the characteristic peak of carbonyl - C=O, 1185.0 cm -1 is the characteristic peak of C - O. Compared with propionic acid, the characteristic signal of the carboxyl group does not appear in the product; compared with stigmastanol, the characteristic absorption signal of the hydroxyl group does not appear, indicating the absence of a hydroxyl group. In addition, the product shows the carbonyl (C=O) and carbon - oxygen single bond (C - O) signals of the ester bond at 1733.5 cm -1 and 1185.0 cm -1 , indicating that the product is stigmastanol propionate.

[0077] Mass spectrometry: The molecular weight of propionic acid is 74, the molecular weight of stigmastanol is 416, and the theoretical molecular weight of the product is 472. In the ES + mass spectrometry, m / z495 is [M + Na] + , m / z 399 is [M - propionic acid + H]+ , further confirmed that the product was stigmastanyl propionate.

[0078] Example 7

[0079] 40 mmol / L of phytosterols (phytosterols include 46% sitosterol, 29% stigmasterol, and 25% campesterol by mass percentage), 80 mmol / L of propionic acid, 80 g / L of Candida rugosa lipase 99 - 125, and 200 g / L molecular sieve were placed in a reaction flask, 80 mL of tetrahydrofuran was added, and the mixture was reacted in a 50 °C constant temperature air bath shaker for 20 h. After the reaction was completed, the catalyst and molecular sieve were removed by filtration, and tetrahydrofuran was removed by rotary evaporation to obtain a crude mixture of esters. The conversion rate analyzed by high performance liquid chromatography was 93.0%. After extraction and separation, the yield of the product was 90.8%.

[0080] The structure characterization data of the product are as follows. Infrared spectrum: The C-H signal of =CH in the product was at 3026 cm -1 , the C-H vibrations of methyl and methylene were at 2850 - 2940 cm -1 , the characteristic peak of carbonyl - C=O was at 1734.5 cm -1 , and the C-O characteristic peak was at 1182.0 cm -1 . Compared with propionic acid, the characteristic signal of carboxyl group did not appear in the product; compared with phytosterols, the characteristic absorption signal of hydroxyl group did not appear, indicating the absence of hydroxyl group. In addition, the carbonyl (C=O) and carbon-oxygen single bond (C-O) signals of the ester bond appeared at 1734.5 cm -1 and 1182.0 cm -1 , indicating that the product was phytosterol propionate.

[0081] Mass spectrometry: The molecular weight of propionic acid was 74, and the molecular weights of sitosterol, stigmasterol, and campesterol in phytosterols were 414, 412, and 400 respectively. The theoretical molecular weights of the products were 470, 468, and 456 respectively. In the ES + mass spectrometry, m / z 493, m / z 491, and m / z 479 corresponded to [M+Na] of different components in phytosterol propionate + , and m / z 397, m / z 395, and m / z 383 corresponded to [M - propionic acid + H] of different components in phytosterol propionate + , further confirming that the product was phytosterol propionate.

[0082] Example 8

[0083] 140 mmol / L of phytosterol (phytosterol includes 75% sitosterol and 25% campesterol by mass percentage), 280 mmol / L of tributyrin, 80 g / L of subtilisin, and 180 g / L The molecular sieve was placed in a reaction flask, 400 mL of n-hexane was added, and the mixture was reacted in a constant temperature air bath oscillator at 80 °C for 42 h. After the reaction ended, the catalyst and molecular sieve were removed by filtration, and n-hexane was removed by rotary evaporation to obtain a crude product of phytosterol butyrate. The conversion rate analyzed by high performance liquid chromatography was 90.8%. After extraction and separation, the yield of the product was 88.5%.

[0084] The structural characterization data of the product are as follows. Infrared spectrum: In the product, the vibrations of C-H of methyl and methylene are at 2851 - 2939 cm -1 and the characteristic peak of carbonyl - C=O is at 1732.0 cm -1 and the characteristic peak of C - O is at 1084.0 cm -1 For tributyrin, the strong characteristic signal peaks of carbonyl (C=O) at 1745 cm -1 and strong carbon - oxygen single bond (C - O) at 1107 cm -1 disappeared, while medium - intensity signal peaks appeared at 1732.0 cm -1 and 1084.0 cm -1 Compared with phytosterol, the characteristic absorption signal of hydroxyl did not appear, indicating the absence of hydroxyl. It shows that the product is phytosterol butyrate.

[0085] Mass spectrum: The molecular weight of tributyrin is 302, the molecular weights of sitosterol and campesterol in phytosterol are 416 and 402 respectively, and the molecular weights of the theoretical products are 486 and 474 respectively. In the ES + mass spectrum, m / z 509 and m / z 495 correspond to [M + Na] of different components in phytosterol butyrate + respectively, and m / z 399 and m / z 385 correspond to [M - butyric acid + H] of different components in phytosterol butyrate + respectively, further indicating that the product is phytosterol butyrate.

[0086] Example 9

[0087] Thirty-six SPF male C57BL / 6J mice (6 - 8 weeks old, weighing 20 - 22 g) were provided by the Experimental Animal Center of Jiangsu University. The feeding environment was as follows: temperature 20 ± 2°C, relative humidity 50 ± 10%, and a 12 h light / dark cycle. The mice had free access to food and water, and fresh feed was changed daily, while bedding was changed regularly every week. After 1 week of adaptive feeding, they were randomly divided into a low-fat group (LF, 6 mice per group), a high-fat group (HF, 6 mice per group), and four sample groups (stigmasteryl butyrate group SB, sitosteryl propionate group SP, ergosteryl acetate group EA, and phytosterol butyrate group PB, 6 mice per group). Among them, the low-fat group was fed a low-fat diet (10% of energy from fat), the high-fat group was fed a high-fat diet (60% of energy from fat, supplemented with 1% cholesterol), and the sample groups were fed high-fat diets supplemented with 0.5% stigmasteryl butyrate, 0.5% sitosteryl propionate, 0.5% ergosteryl acetate, or 0.5% phytosterol butyrate, respectively. The formula of the low-fat diet was: casein 200 parts, L-cystine 3 parts, corn starch 452.2 parts, maltodextrin 75 parts, sucrose 176.8 parts, cellulose 50 parts, soybean oil 25 parts, lard 20 parts, mixed minerals 50 parts, mixed vitamins 1 part, choline bitartrate 2 parts. The formula of the high-fat diet was: casein 200 parts, L-cystine 3 parts, corn starch 72.8 parts, maltodextrin 100 parts, sucrose 176.8 parts, cellulose 50 parts, soybean oil 25 parts, lard 177.5 parts, mixed minerals 50 parts, mixed vitamins 1 part, choline bitartrate 2 parts, potassium bitartrate 8.69 parts, sodium cholate 1.74 parts. The formulas of the four sample groups were all high-fat diets, with an additional 0.5% (w / w) stigmasteryl butyrate, 0.5% (w / w) sitosteryl propionate, 0.5% (w / w) ergosteryl acetate, or 0.5% (w / w) phytosterol butyrate added to the high-fat diet. The feeds for each group were prepared by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. and fed for 12 weeks, and the body weights of the mice were measured regularly.

[0088] At the end of feeding, the mice were fasted but allowed free access to water for 12 h. The mice were anesthetized with isoflurane by respiration, blood was collected by eye enucleation, and the mice were sacrificed by cervical dislocation. Tissues such as perirenal fat and epididymal fat of the mouse liver were separated, and all samples were stored in a -80 °C refrigerator. The fat coefficient was expressed as the percentage of fat weight to the final body weight of the mice. After the blood was allowed to stand for 4 h, the serum was collected by centrifugation at 3000 rpm for 10 min. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in the serum were measured using the corresponding kits (Nanjing Jiancheng Bioengineering Institute), and the specific measurement steps were in accordance with the kit instructions. The experimental results were expressed as mean ± standard deviation, and the experimental data were analyzed by one-way ANOVA using SPSS 20 software to compare the significant differences between groups. The data were labeled with different letters (a, b, c) (p < 0.05), indicating significant differences between the corresponding groups. The indexes of each group of mice are shown in Table 2.

[0089] Table 2 Body weight, fat coefficient and blood lipid levels of each group of mice

[0090]

[0091] At the beginning of the experiment, there were no significant differences in the body weights of each group of mice. After 12 weeks of dietary intervention, the body weights of each group of mice increased significantly. Specifically, the final body weight of the high-fat group was significantly higher than that of the low-fat group, increasing by 21.9%. Compared with the high-fat group, the final body weights of the four sample groups were significantly reduced, by 11.6%, 13.2%, 10.3% and 11.3% respectively. Similarly, in terms of body weight gain, the high-fat group was significantly higher than the low-fat group, and the four sample groups were all significantly lower than the high-fat group. Compared with the low-fat group, the perirenal fat coefficient and epididymal fat coefficient of the high-fat group were both significantly increased. Supplementing 0.5% stigmasterol butyrate, 0.5% sitosterol propionate, 0.5% ergosterol acetate and 0.5% phytosterol butyrate in the diet all caused significant decreases in the perirenal fat coefficient and epididymal fat coefficient. According to the results of the final body weight, body weight gain, perirenal fat coefficient and epididymal fat coefficient, a mouse obesity model was successfully established by feeding a high-fat diet for 12 weeks. The supplementation of stigmasterol butyrate, sitosterol propionate, ergosterol acetate and phytosterol butyrate all significantly inhibited the body weight increase induced by a high-fat diet, indicating that they have significant weight loss effects.

[0092] Compared with the low-fat group, the serum TC, TG, and LDL-C levels in the high-fat group were significantly increased (p<0.05), while the HDL-C level was significantly decreased (p<0.05), indicating that a high-fat diet successfully established hyperlipidemia and hypercholesterolemia models. Compared with the high-fat group, the serum TC in the four sample groups was decreased by 16.9% (p<0.05), 13.5% (p<0.05), 20.3% (p<0.05), and 23.7% (p<0.05), respectively. Compared with the high-fat group, the supplementation of stigmasteryl butyrate, sitosteryl propionate, ergosterol acetate, and phytosteryl butyrate significantly decreased the serum LDL-C and TG levels. This indicates that stigmasteryl butyrate, sitosteryl propionate, ergosterol acetate, and phytosteryl butyrate have significant effects on reducing serum TG and cholesterol.

[0093] Example 10

[0094] Experimental animals and grouping. Thirty-six SPF-grade male C57BL / 6J mice (6 - 8 weeks old, weighing 20 - 22 g) were provided by the Experimental Animal Center of Jiangsu University. Before the experiment, the mice were adaptively fed for one week and then randomly divided into 6 groups (6 mice in each group): control group (CTL), model group (DSS), and four sample groups (stigmasteryl butyrate group SB, sitosteryl propionate group SP, ergosterol acetate group EA, phytosteryl butyrate group PB).

[0095] Feed formulation and treatment. The CTL group and the DSS group were fed a basal diet with the following formulation: casein 200 parts, L-cystine 3 parts, corn starch 452.2 parts, maltodextrin 75 parts, sucrose 176.8 parts, cellulose 50 parts, soybean oil 25 parts, lard 20 parts, mixed minerals 50 parts, mixed vitamins 1 part, choline bitartrate 2 parts. The four sample groups in the intervention groups were fed the basal diet supplemented with 0.5% (w / w) stigmasteryl butyrate (SB), sitosteryl propionate (SP), ergosterol acetate (EA), or phytosteryl butyrate (PB), respectively. All feeds were prepared by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. and sterilized by 60 Co-γ ray irradiation.

[0096] Experimental design. 1) Pre-intervention phase (2 weeks): The CTL group and the DSS group were fed a basal diet and given ordinary water to drink. The SB, SP, EA, and PB groups were fed a basal diet containing 0.5% (w / w) of the corresponding sample and given ordinary water to drink. 2) DSS-induced colitis model phase (1 week): A dextran sulfate sodium (DSS, relative molecular weight 40,000 Da) was used to induce a colitis model. The specific protocol was as follows: CTL group: basal diet, ordinary drinking water. DSS group: basal diet, 2.5% (w / w) DSS drinking water. SB, SP, EA, PB groups: basal diet containing 0.5% (w / w) of the corresponding sample, 2.5% (w / w) DSS drinking water. During the model establishment period, the body weight of the mice was recorded at a fixed time every day, and the fecal status was observed. 3) Sample collection: After the model establishment was completed, the mice were euthanized under anesthesia, and the colon and spleen were dissected. The spleen was weighed and photographed, and the colon length was measured and photographed. About 1 cm from the anus of the colon tissue (about 0.5 cm) was taken and fixed in 4% paraformaldehyde solution for histological section analysis.

[0097] Colitis is usually accompanied by clinical symptoms such as weight loss, diarrhea, and bloody stools. Therefore, when using DSS to establish a colitis model, the severity of the symptoms is often judged by comprehensively evaluating the weight change and the disease activity index (DAI). As Figure 1 shown in Fig. A, as the number of days of model establishment increased, the body weight of the mice in the CTL group increased slowly, while the body weight of the mice in the DSS group decreased significantly. In particular, from the 3rd day to the 7th day, the body weight of the DSS group showed a linear downward trend; in contrast, the downward trend of the body weight of the four intervention groups of SB, SP, EA, and PB was relatively mild, and there was a significant difference compared with the DSS group (p < 0.05). This indicates that SB, SP, EA, and PB have good preventive and alleviating effects on the pathogenesis of colitis.

[0098] The main mechanism of DSS-induced colitis is to damage the colonic epithelial cells and mucosal layer through toxic effects. The morphological changes of the colon after dissection can directly reflect the degree of colon injury. As Figure 1 shown in Fig. C, the colon wall of the mice in the DSS group was swollen and bleeding, and the feces were blood-red; while the colon morphology of the mice in the four intervention groups was close to that of healthy mice, and the feces were black granular. Figure 1 Fig. D further shows that the colon length of the mice in the DSS group was significantly shorter than that in the CTL group (p < 0.01); the SB, SP, EA, and PB intervention groups significantly inhibited the shortening of the colon, and there was a statistical difference compared with the DSS group (p < 0.05). In addition, DSS-induced colitis can also cause systemic inflammation, mainly manifested as splenomegaly caused by bacterial translocation in the colonic lamina propria. As Figure 1 shown in Fig. B, the spleen of the mice in the DSS group was significantly enlarged, and there were significant differences compared with the CTL group (p < 0.01) and the four intervention groups (p < 0.05).

[0099] In summary, the interventions of the four samples, namely SB, SP, EA, and PB, can effectively delay the onset process of colitis and significantly relieve the related clinical symptoms, providing potential value for the prevention and treatment of colitis.

[0100] It should be understood that although this specification is described according to each embodiment, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0101] The series of detailed descriptions listed above are only specific descriptions of the feasible embodiments of the present invention, and they are not intended to limit the protection scope of the present invention. Any equivalent embodiments or modifications made without departing from the spirit of the art of the present invention should be included in the protection scope of the present invention.

Claims

1. A plant-derived sterol short-chain fatty acid ester, characterized in that, The plant-derived sterol short-chain fatty acid ester has the following structure: wherein, R1 is CH3, C2H5 or C3H7; R2 is 2. A method for preparing a phytosterol short-chain fatty acid ester according to claim 1, characterized in that, It includes the following steps: Step S1: Weigh plant-derived sterol, short-chain fatty acid or short-chain fatty acid ester, catalyst and molecular sieve respectively, mix them, and add a reaction solvent to carry out the reaction; Step S2: After the reaction is completed, take out the reaction solution, filter to remove the catalyst and molecular sieve, and remove the reaction solvent to obtain the crude product of plant-derived sterol short-chain fatty acid ester; Step S3: Obtain the pure product of plant-derived sterol short-chain fatty acid ester through extraction and separation.

3. The preparation method of the phytosterol short-chain fatty acid ester according to claim 2, wherein, The plant-derived sterol is phytosterol or phytostanol; the plant-derived sterol short-chain fatty acid ester is phytosterol short-chain fatty acid ester or phytostanol short-chain fatty acid ester.

4. The preparation method of the phytosterol short-chain fatty acid ester according to claim 3, characterized in that, The phytosterol is one or more of sitosterol, stigmasterol, ergosterol, campesterol, fucosterol in any proportion mixture; the phytostanol is one or more of sitostanol, stigmasterol, ergostanol, campestanol, fucostanol in any proportion mixture.

5. The preparation method of the phytosterol short-chain fatty acid ester according to claim 2, characterized in that, The short-chain fatty acid is one or more of acetic acid, propionic acid, butyric acid in any proportion mixture; the short-chain fatty acid ester is one or more of methyl acetate, ethyl acetate, methyl propionate, ethyl propionate, methyl butyrate, ethyl butyrate, glyceryl triacetate, glyceryl tripropionate, glyceryl tributyrate in any proportion mixture.

6. The preparation method of the phytosterol short-chain fatty acid ester according to claim 2, characterized in that, The catalyst is one or more of Candida antarctica lipase B, Rhizomucor miehei lipase, Candida lipase 99-125, porcine pancreatic lipase, Bacillus subtilis protease in any proportion mixture.

7. The preparation method of the phytosterol short-chain fatty acid ester according to claim 2, characterized in that, The reaction solvent is one of n-hexane, isooctane, cyclohexane, tert-butanol, tert-pentanol, acetone, tetrahydrofuran.

8. The preparation method of the phytosterol short-chain fatty acid ester according to claim 2, characterized in that, The concentration of the plant-derived sterol is 20 mmol / L to 160 mmol / L, the molar ratio of the plant-derived sterol to the short-chain fatty acid is 1:1 to 1:5, the addition amount of the catalyst is 5 g / L to 120 g / L, the addition amount of the molecular sieve is 40 g / L to 200 g / L, the reaction solvent is 30 to 500 mL, the reaction temperature is 30 °C to 80 °C, and the reaction time is 6 h to 60 h.

9. The preparation method of the phytosterol short-chain fatty acid ester according to claim 8, wherein The concentration of the plant-derived sterol is 30 mmol / L to 150 mmol / L, the molar ratio of the plant-derived sterol to the short-chain fatty acid is 1:1 to 1:3, the addition amount of the catalyst is 10 g / L to 80 g / L, the addition amount of the molecular sieve is 40 g / L to 150 g / L, the reaction solvent is 50 to 400 mL, the reaction temperature is 40 °C to 70 °C, and the reaction time is 12 h to 48 h.

10. Use of a phytosterol short-chain fatty acid ester, characterized in that, The application of the plant-derived sterol short-chain fatty acid ester obtained by the preparation method of the plant-derived sterol short-chain fatty acid ester according to claim 1 or any one of claims 2-9 in the preparation of foods, medicines or feeds for relieving colitis, reducing cholesterol or losing weight.

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