Phytosterol butyrate as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemical synthesis, and relates to phytosterol butyrate as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing phytosterol (beta-sitosterol) and butyric acid in an organic solvent by taking an ionic liquid as a catalyst under a dry anhydrous condition, carrying out a stirring reaction at a certain temperature, and finally, carrying out centrifugal separation to obtain a phytosterol butyrate product. The phytosterol butyrate is synthesized for the first time, the reaction steps are few, the conditions are mild, the operation is simple, the yield is high, the product is easy to separate, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical synthesis and relates to a phytosterol butyrate and a preparation method and application thereof. Background Art

[0002] Phytosterol butyrate is a prodrug synthesized on the basis of natural medicinal organic compounds. After entering the human or animal body, it can be gradually hydrolyzed by esterase in the small intestine to release free phytosterols and butyric acid, which can not only show their own physiological activities, but also have synergistic effects. Phytosterols can effectively reduce plasma total cholesterol and low-density lipoprotein cholesterol, help prevent coronary artery sclerosis and reduce the risk of cardiovascular and cerebrovascular diseases. The role of phytosterols in animal nutrition and feed is mainly reflected in: as a natural active ingredient, it can promote the absorption and utilization of nutrients by animals, improve feed conversion rate, thereby accelerating the growth rate of animals, and at the same time it has the function of regulating the immune system, which can improve the immunity of animal bodies and reduce the incidence of diseases. Adding phytosterols to feed can help improve animal disease resistance, reduce the use of antibiotics, and ensure animal health; butyric acid is particularly important for colon health. It is the main energy source for epithelial cells in the colon wall, providing about 60-70% of the energy requirements. Butyric acid can promote epithelial cell growth and renewal, repair intestinal mucosal damage, improve cell wall transport of nutrients, regulate the absorption of sodium and water, enhance intestinal barrier function, reduce intestinal endotoxin absorption, reduce intestinal excessive spasm and movement, and inhibit the formation of colon adenomas and tumors. The application of butyric acid in animal feed has a significant effect. Butyric acid is a short-chain fatty acid that can reduce the pH value of the intestine and create an environment that is not conducive to the growth of harmful bacteria, thereby promoting the proliferation of beneficial bacteria. Adding butyric acid to feed can help animals maintain the balance of intestinal microecology and reduce the occurrence of intestinal diseases. At the same time, butyric acid can be directly absorbed by the animal intestine as an energy source, and it can also promote the digestion and absorption of other nutrients and improve the conversion rate of feed. This is of great significance for improving the growth rate and feed conversion rate of animals. Phytosterol butyrate combines the advantages of plant sterols and butyric acid and can be used as a new type of animal nutrition product.

[0003] There are several problems with the synthesis of sterol ester products: (1) Strong acids are usually used as catalysts in traditional chemical synthesis. Although this method can promote the esterification reaction to a certain extent, it also has some significant disadvantages. Strong acids are highly corrosive to reactors and other contact equipment. Long-term use will cause equipment damage, increase maintenance costs and replacement frequency. This not only affects the continuity of production, but also may cause safety hazards. (2) Enzymatic catalytic esterification is a more environmentally friendly and mild synthesis method. It uses specific enzymes as catalysts to promote the esterification reaction between fatty acids and alcohols. However, for short-chain fatty acids, due to their strong acidity, it is indeed easy to cause enzyme inactivation, thereby affecting the yield. For example, the enzymatic catalytic esterification reaction disclosed in patents CN201711337291.3 and WO2014183144A1 uses long-chain acids and glycerol under the catalysis of immobilized enzymes to synthesize long-chain triglycerides with a yield of 70% to 80%. These are all applications in the treatment of long-chain fatty acids. The acidity problem of short-chain fatty acids is still a huge challenge. Summary of the invention

[0004] One of the purposes of the present invention is to provide a phytosterol butyrate in order to address the problems existing in the prior art. The phytosterol butyrate is non-toxic, has the effects of promoting intestinal health, and can be used as a nutrient for humans and animals.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned phytosterol butyrate, which uses ionic liquid as a catalyst to catalyze the synthesis of phytosterol butyrate, has few reaction steps, mild conditions, simple operation, high yield, and easy product separation, and is suitable for industrial production.

[0006] The third object of the present invention is to provide the use of the above-mentioned phytosterol butyrate.

[0007] To this end, the first aspect of the present invention provides a phytosterol butyrate, the molecular structure of which is shown in formula (I):

[0008]

[0009] The second aspect of the present invention provides a method for preparing the phytosterol butyrate as described in the first aspect of the present invention, which comprises catalyzing the synthesis of phytosterol butyrate in an anhydrous system using phytosterol and butyric acid as reaction substrates and an ionic liquid as a catalyst.

[0010] According to the present invention, the preparation method comprises using toluene as a reaction solvent, mixing phytosterol and butyric acid, adding an ionic liquid as a catalyst, stirring the reaction, centrifuging and then washing and separating, and finally rotary evaporating to obtain a phytosterol butyrate product.

[0011] In some embodiments of the present invention, the molar ratio of phytosterols to butyric acid is 1:1 to 1:10.

[0012] Preferably, the mass of the ionic liquid accounts for 5% to 10% of the total mass of the reaction substrate.

[0013] Preferably, the mass volume ratio of the phytosterol to the reaction solvent is 0.105-0.42 g / mL.

[0014] According to the present invention, the reaction temperature is 60 to 120° C., the reaction time is 1 to 24 hours, and the stirring speed is 200 to 700 rpm.

[0015] Preferably, the rotation speed of the centrifugal rotary evaporation is 3000 to 7000 rpm.

[0016] In the present invention, the ionic liquid includes one or more of [BSO3HMimHSO4], [AMimCl], [VBimPF6], [VBimBF4], [VBimNF2], [BmimHSO4] and [EmimHSO4].

[0017] The third aspect of the present invention provides the use of the phytosterol butyrate as described in the first aspect of the present invention or the phytosterol butyrate prepared by the preparation method as described in the first aspect of the present invention as an animal feed additive.

[0018] The fourth aspect of the present invention provides the use of the phytosterol butyrate as described in the first aspect of the present invention or the phytosterol butyrate prepared by the preparation method as described in the first aspect of the present invention as a nutritional additive.

[0019] The present invention provides a method for synthesizing phytosterol butyrate using ionic liquid catalysis in an anhydrous system. In this method, under dry anhydrous conditions, ionic liquid is used as a catalyst, phytosterol (β-sitosterol) and butyric acid are mixed in an organic solvent, stirred at a certain temperature for reaction, and finally the phytosterol butyrate product is obtained by centrifugation. The present invention synthesizes phytosterol butyrate for the first time, with fewer reaction steps, mild conditions, simple operation, high yield, and easy product separation, which is suitable for industrial production. The phytosterol butyrate product synthesized by the present invention has the natural active function of sterols, and after the phytosterol butyrate is hydrolyzed, it also has the good characteristics of butyric acid, and butyric acid has the effects of promoting intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0021] Figure 1 The reaction formula for synthesizing phytosterol butyrate in the present invention is shown.

[0022] Figure 2 Infrared spectra of reactants and products.

[0023] Figure 3 This is the H NMR spectrum of the product after purification. DETAILED DESCRIPTION

[0024] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0025] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0026] As mentioned above, in the prior art, enzymatic synthesis is only suitable for the synthesis of long-chain fatty acids, such as oleic acid, palmitic acid, and stearic acid, but is not applicable to short-chain fatty acids; and traditional catalysts, such as highly corrosive catalysts such as concentrated sulfuric acid, will cause side reactions such as dehydration and etherification, and the excess acid needs to be neutralized, which will involve complex purification steps and affect the environment and equipment, and the reaction efficiency is also not ideal; in view of this, the inventors have conducted a lot of in-depth research on the synthesis technology of phytosterol butyrate.

[0027] The inventors have discovered and proposed for the first time the use of ionic liquids to catalyze the synthesis of plant sterol butyrates. Compared with enzymatic synthesis, enzymatic synthesis is only suitable for the synthesis of long-chain fatty acids, such as oleic acid, palmitic acid, and stearic acid, but not for short-chain fatty acids. The inventors have discovered that the use of ionic liquids as catalysts can successfully synthesize such short-chain plant sterol butyrates. Compared with traditional catalysts such as concentrated sulfuric acid, which are highly corrosive catalysts, the use of concentrated sulfuric acid will cause side reactions such as dehydration and etherification, and the excess acid needs to be neutralized, which will involve complex purification steps and affect the environment and equipment. The use of ionic liquid catalysis can enhance the activity of the acid catalyst, thereby accelerating the rate of the esterification reaction and increasing the yield. The inventors have discovered that since the acid sites in the ionic liquids may be more effective than traditional liquid acids, less catalysts can be used. At the same time, ionic liquids reduce the use of organic solvents and reduce pollution to the environment. Ionic liquids are easily separated from the reaction products and can be recycled multiple times. From the perspective of economic effects, ionic liquids can be used for esterification reactions under milder conditions, reducing energy consumption. Although the initial cost of ionic liquids is high, considering their recyclability and possible reduction in post-processing costs, they may be more economical in the long run. From the above, it can be seen that the use of ionic liquids to catalyze the synthesis of phytosterol butyrate is a new green and efficient method for synthesizing phytosterol butyrate, which solves the deficiencies and blank defects in the prior art.

[0028] Therefore, the preparation method of phytosterol butyrate involved in the present invention is a preparation method of phytosterol butyrate catalyzed by ionic liquid as a catalyst in an anhydrous system, and its reaction formula is as follows: Figure 1 As shown; the preparation method includes using toluene as a reaction solvent, mixing phytosterol [(β-sitosterol), compound (1)] and butyric acid [compound (2)], adding an ionic liquid as a catalyst, stirring the reaction at a certain temperature, centrifuging and then washing and separating, and finally rotary evaporating at 70°C to obtain a phytosterol butyrate [compound (3)] product.

[0029] As a preferred embodiment of the method for synthesizing phytosterol butyrate using ionic liquid as catalyst according to the present invention, the molar ratio of the phytosterol to butyric acid is 1:1 to 1:10.

[0030] As a preferred embodiment of the method for synthesizing phytosterol butyrate using ionic liquid as catalyst according to the present invention, the mass of the ionic liquid accounts for 5% to 10% of the total mass of the substrate (phytosterol and butyric acid).

[0031] Preferably, the mass volume ratio of the phytosterol to the reaction solvent is 0.105-0.42 g / mL.

[0032] As a preferred embodiment of the method for synthesizing phytosterol butyrate using ionic liquid as catalyst according to the present invention, the certain temperature is 60 to 120°C.

[0033] As a preferred embodiment of the method for catalyzing the synthesis of phytosterol butyrate using ionic liquid as a catalyst as described in the present invention, the ionic liquid includes one or more of [BSO3HMimHSO4], [AMimCl], [VBimPF6], [VBimBF4], [VBimNF2], [BmimHSO4], [EmimHSO4], etc.

[0034] As a preferred embodiment of the method for synthesizing phytosterol butyrate using ionic liquid as catalyst described in the present invention, the stirring reaction has a reaction time of 1 to 24 hours and a rotation speed of 200 to 700 rpm.

[0035] As a preferred embodiment of the method for synthesizing phytosterol butyrate using ionic liquid as catalyst described in the present invention, the rotation speed of the centrifugal separation is 3000 to 7000 rpm.

[0036] The present invention also relates to phytosterol butyrate prepared by the above preparation method, the molecular structure of which is shown in formula (I):

[0037]

[0038] The research results show that the phytosterol butyrate product synthesized for the first time in the present invention not only has the natural active functions of sterols, but also has the good properties of butyric acid after the phytosterol butyrate is hydrolyzed. Butyric acid has the effect of promoting intestinal health.

[0039] The beneficial effects of the present invention are:

[0040] In the method for catalyzing the synthesis of phytosterol butyrate using ionic liquid as a catalyst described in the present invention, compared with enzymatic synthesis, the ionic liquid catalytic synthesis remains stable at a higher temperature and pH range, while the enzyme may be inactivated at high temperature, extreme pH value or certain organic solvents.

[0041] In the method for synthesizing phytosterol butyrate using ionic liquid as catalyst of the present invention, the ionic liquid provides a specific catalytic environment, enhances the solubility and reactivity of the reactants, thereby greatly improving the catalytic efficiency, and the reaction time is reduced from 24 hours to 7 hours to complete the reaction. Although the enzyme has high specificity, its catalytic efficiency may be limited by the reaction conditions.

[0042] In the method for synthesizing phytosterol butyrate using ionic liquid as a catalyst described in the present invention, the reaction product catalyzed by ionic liquid is more easily separated by simple physical methods, while the product synthesized by enzyme method requires a more complicated separation process.

[0043] In the method for synthesizing phytosterol butyrate using ionic liquid as catalyst of the present invention, although the initial cost of ionic liquid is relatively high, it can be reused many times and may be more cost-effective in the long run. The production and purification process of enzymes is relatively costly.

[0044] In the method for synthesizing phytosterol butyrate using ionic liquid as catalyst described in the present invention, the substrate range catalyzed by ionic liquid is usually wider and is not limited to specific biological molecules, while enzymes are usually highly selective for substrates and only act on specific molecules.

[0045] The invention has the good property that butyric acid, phytosterol butyrate and ionic liquid are mutually immiscible, and butyric acid is soluble in water, so that the butyric acid and ionic liquid can be reused and the product can be purified and separated through distillation and centrifugation, which is conducive to industrial production.

[0046] In the method for synthesizing phytosterol butyrate using ionic liquid as catalyst described in the present invention, ionic liquid catalysis is more suitable for large-scale industrial production because they can be used in a continuous flow system, while enzymatic synthesis may be more suitable for small-scale or specific biopharmaceutical applications.

[0047] In the method for synthesizing phytosterol butyrate using ionic liquid as a catalyst described in the present invention, the first synthesized product phytosterol butyrate not only has the natural active functions of sterols, but also has the good properties of butyric acid after the phytosterol butyrate is hydrolyzed. Butyric acid has the effect of promoting intestinal health.

[0048] The results of the study indicate that the plant sterol butyrate can be used as an animal nutrition product as an animal feed additive and can also be used as a human nutritional supplement.

[0049] In some specific embodiments of the present invention, the phytosterol butyrate can be used as an animal feed additive.

[0050] In some specific embodiments of the present invention, the phytosterol butyrate can be used as a nutritional additive.

[0051] II. Examples

[0052] The present invention is described in detail below through specific examples. The experimental methods described below are all routine laboratory methods unless otherwise specified. The experimental materials described below are all available from commercial channels unless otherwise specified.

[0053] Embodiment 1:

[0054] Take 2.1g of phytosterols (the mass volume ratio of phytosterols to reaction solvent is 0.42g / mL) and 4.4g of butyric acid (the molar ratio of phytosterols to butyric acid is 1:10), add 5mL of toluene and 0.39g of ionic liquid (BSO3HMimHSO4) (the mass of the ionic liquid accounts for 6% of the total mass of the reaction substrate), dissolve phytosterols and butyric acid, the reaction temperature is 110°C, the stirring speed is 400rpm, the reaction time is 7h, and the reaction is centrifuged at 4500rpm for 10min. After washing and separation, the organic solvent is removed by rotary evaporation at 70°C to obtain the reaction product, phytosterol butyrate. The esterification rate is measured by trace1300 gas chromatograph (Thermo Fisher) and is 97.2%.

[0055] Figure 2 The infrared spectra of the reactants and products in the above example (Thermo Fisher Scientific Nicolet iS20 Fourier transform infrared spectrometer FTIR, USA); 3422 cm -1 It is a broad absorption peak of the intermolecular hydrogen bond OH stretching vibration. The infrared spectrum of the product shows 1740 cm -1 , which is the stretching vibration absorption peak of C=O, indicating that an ester bond is formed in the reaction, indicating that phytosterol butyrate is generated.

[0056] Figure 3 This is the nuclear magnetic hydrogen spectrum of the product in the above example after centrifugal rotary evaporation, washing, separation and purification (Bruker liquid nuclear magnetic resonance spectrometer (NMR), 400MHz, Germany). Through spectrum analysis, it can be seen that the hydrogen on the C-3 of phytosterol migrates to 4.6ppm, indicating that esterification reaction occurs between phytosterol and butyric acid; the hydrogen at 5.37ppm is the hydrogen on the double bond of phytosterol, and 2.31ppm and 2.25ppm represent the hydrogen on the two -CH2 of butyric acid, respectively. The integral area ratio shows that the generated substance is the corresponding phytosterol butyrate.

[0057] Embodiment 2:

[0058] Take 3.1g of phytosterols and 8.4g of butyric acid, add 10mL of toluene and 0.39g of ionic liquid (BSO3HMimHSO4) to dissolve phytosterols and butyric acid. The reaction temperature is 100°C, the stirring speed is 500rpm, the reaction time is 8h, and the mixture is centrifuged at 4500rpm for 10min. After washing and separation, the organic solvent is removed by rotary evaporation at 70°C to obtain the reaction product, phytosterol butyrate, and the esterification rate is measured to be 84.72%.

[0059] Embodiment 3:

[0060] Take 2.1g of phytosterols and 2.64g of butyric acid, add 5mL of toluene and 0.39g of ionic liquid (BSO3HMimHSO4) to dissolve phytosterols and butyric acid. The reaction temperature is 90°C, the stirring speed is 400rpm, the reaction time is 6h, and the mixture is centrifuged at 4500rpm for 15min. After washing and separation, the organic solvent is removed by rotary evaporation at 70°C to obtain the reaction product, phytosterol butyrate, and the esterification rate is measured to be 81.1%.

[0061] Embodiment 4:

[0062] Take 2.1g of phytosterols and 10.4g of butyric acid, add 10mL of toluene and 0.39g of ionic liquid (BmimHSO4) to dissolve phytosterols and butyric acid. The reaction temperature is 70°C, the stirring speed is 600rpm, the reaction time is 7h, and the mixture is centrifuged at 4500rpm for 20min. After washing and separation, the organic solvent is removed by rotary evaporation at 70°C to obtain the reaction product, phytosterol butyrate, and the esterification rate is measured to be 78.9%.

[0063] The phytosterol butyrate obtained in the above embodiment was subjected to relevant animal experiments, and the results of toxicity test showed that the product was non-toxic; the results of pig feeding experiments showed that the phytosterol butyrate was beneficial to the growth of livestock such as pigs, the weight of pigs increased significantly, and the intestinal physiological state was healthier and more active.

[0064] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A phytosterol butyrate, the molecular structure of which is shown in formula (I):

2. The method for preparing phytosterol butyrate as claimed in claim 1, comprising catalyzing the synthesis of phytosterol butyrate in an anhydrous system using phytosterol and butyric acid as reaction substrates and ionic liquid as a catalyst.

3. The preparation method according to claim 2, characterized in that: The preparation method comprises the following steps: using toluene as a reaction solvent, mixing phytosterol and butyric acid, adding an ionic liquid as a catalyst, stirring for reaction, centrifuging, washing and separating, and finally rotary evaporating to obtain a phytosterol butyrate product.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the phytosterols to butyric acid is 1:1 to 1:

10.

5. The preparation method according to claim 3, characterized in that: The mass of the ionic liquid accounts for 5% to 10% of the total mass of the reaction substrate; and / or the mass volume ratio of the phytosterol to the reaction solvent is 0.105-0.42 g / mL.

6. The preparation method according to claim 3, characterized in that: The reaction temperature is 60 to 120° C., the reaction time is 1 to 24 hours, and the stirring speed is 200 to 700 rpm.

7. The preparation method according to claim 3, characterized in that: The rotation speed of the centrifugal rotary evaporation is 3000 to 7000 rpm.

8. The preparation method according to any one of claims 2 to 7, characterized in that: The ionic liquid includes one or more of [BSO3HMimHSO4], [AMimCl], [VBimPF6], [VBimBF4], [VBimNF2], [BmimHSO4] and [EmimHSO4].

9. Use of the phytosterol butyrate according to claim 1 or the phytosterol butyrate prepared by the preparation method according to any one of claims 2 to 8 as an animal feed additive.

10. Use of the phytosterol butyrate according to claim 1 or the phytosterol butyrate prepared by the preparation method according to any one of claims 2 to 8 as a nutritional additive.

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