A method and use of semi-enzymatic catalysis of sialic acid to synthesize sialic acid ester

Abstract

The invention discloses a method and use of semi-enzymatic catalysis of sialic acid to synthesize sialic acid esters, belongs to the field of biosynthesis, takes sialic acid and C1-C4 alcohol as reaction substrates, takes lipase and acid accounting for 0.05-5‰ of substrate sialic acid mass as catalyst, reaction rate is greatly accelerated, far superior to single lipase or acid catalysis, the selection of the reaction substrate C1-C4 alcohol depends on the target sialic acid esters, adds molecular sieve water absorbent, carries out esterification reaction under the condition of temperature of 30-60 DEG C and oscillation speed of 200-300r / min, after reaction ends, reaction solution is separated and purified to obtain sialic acid esters. The method of the invention has the advantages of strong regioselectivity of enzyme, fast reaction, high reaction conversion rate, simple downstream separation, low energy consumption, reaction solvent recovery, environmentally friendly, suitable for industrial production, and high yield.

Description

Technical Field

[0001] The invention belongs to the field of biosynthesis, and particularly relates to a method and use of semi-enzymatically catalyzing sialic acid to synthesize sialic acid ester. Background Art

[0002] Sialic acid (SA) refers to a series of carboxylated monosaccharide acylated derivatives containing 9 carbon atoms. It is found in animals and some bacteria, usually in the form of oligosaccharides, glycolipids or glycoproteins. Sialic acid was first isolated from the mucin of the submandibular gland, hence the name. There are many types of sialic acid, and more than 50 types are currently known. In mammals, the most common sialic acid derivative is N-acetylneuraminic acid (Neu5Ac). In the human body, the brain has the highest sialic acid content, among which the sialic acid content in the gray matter of the brain is 15 times that of internal organs such as the liver and lungs. The main food source of sialic acid is breast milk, and it is also present in milk, eggs and cheese. Its chemical structure is shown below:

[0003]

[0004] Sialic acid compounds have important biological functions and are directly involved in the interactions between cells, cells and microorganisms, cells and toxins, and cells and antibodies in many important physiological, biochemical, and pathological processes of living organisms. They have important application value in the fields of anti-inflammatory, anti-tumor, anti-viral, and anti-Alzheimer's.

[0005] Sialic acid has been approved as a food additive by the European Union (2018) and my country (2019), mainly as an ingredient in infant food and a nutritional supplement. In 2021, it was approved as a cosmetic raw material by my country. Sialic acid can be obtained from chemical synthesis, extraction and purification from natural dairy products, microbial fermentation, and bio-enzyme catalytic synthesis, each with its own advantages and disadvantages. The preparation of sialic acid for use as a food additive or cosmetic raw material requires high standards and no harmful substances can remain. Therefore, sialic acid from chemical synthesis is not suitable as a food additive or cosmetic raw material.

[0006] Because sialic acid contains carboxyl groups, it generates negative charges in the body and is easily soluble in water, soluble in methanol, slightly soluble in ethanol, and insoluble in ether, acetone, and chloroform. The carboxyl groups of sialic acid are esterified to form sialic acid esters, which reduce polarity and increase solubility in methanol or ethanol. At the same time, due to the presence of multiple hydroxyl groups, it is easy to form hydrogen bonds with water, so the solubility in water is also good. The chemical structure of sialic acid esters is shown below.

[0007]

[0008] Sialic acid plays an important role in cell-to-cell interactions, such as pathogen-host recognition, tumor metastasis, toxin-receptor interaction, cell differentiation and proliferation. Sialic acid has a large polarity, which is not conducive to cell absorption as a drug or nutrient, and has a low bioavailability. By esterifying sialic acid and using it as a prodrug, the polarity can be reduced, which is conducive to the rapid absorption and utilization of sialic acid by cells. The synthesis of traditional sialic acid esters is chemical synthesis, such as the synthesis of methyl sialate, which uses methanol as a solvent and adds trifluoroacetic acid (TFA) as a catalyst, with a high yield. However, the sialic acid ester prepared by chemical synthesis takes 3-7 days to react, and the time cost of production is large. In addition, a large amount of trifluoroacetic acid needs to be used, and a trace amount of trifluoroacetate may remain in the final product, and there are many by-products, which are not suitable for use as food additives, medicines or cosmetic raw materials.

[0009]

[0010] Traditionally, sialic acid esters are synthesized chemically. Due to the solubility of sialic acid, a catalytic amount (1%-10%) of trifluoroacetic acid (TFA) is added, and the reaction is carried out at room temperature for at least 3 days in anhydrous solvent, with a yield of more than 90%. If lipase is used alone, the reaction is carried out at 40°C for at least 7 days. The industrial production of sialic acid esters using traditional known methods is time-consuming, has low production efficiency, and high production costs. A new method for catalytic synthesis of sialic acid esters is urgently needed to solve the above problems. Summary of the invention

[0011] The purpose of the present invention is to solve the shortcomings of the above-mentioned chemical synthesis method, using lipase and a very small amount of 0.05-5‰ trifluoroacetic acid or hydrochloric acid or sulfuric acid (TFA or HCl or H2SO4 etc) as a catalyst, using a semi-enzymatic method to synthesize sialic acid esters, and there is no existing literature or patent report on the use of lipase to catalyze the synthesis of sialic acid esters. The present invention has the advantages of mild production process conditions, simple steps, high product yield, environmental friendliness, energy saving and environmental protection. The present invention first applies the semi-enzymatic method, using lipase and a very small amount of acid to catalyze the synthesis of sialic acid esters with sialic acid, which is safe and efficient, and the reaction rate is greatly accelerated. The catalyst can be recycled for multiple times (this experiment has been repeated more than 50 times to verify that the catalytic rate remains above 98%), and the solvent is recycled and reused, which is environmentally friendly, green and pollution-free, and has no toxic substance residues. It is suitable for industrial production such as batch production, and is suitable for uses such as food additives, medicines or cosmetic raw materials.

[0012] In order to achieve the purpose of the invention, the technical solution adopted by the present invention is:

[0013] A method for semi-enzymatically catalyzing sialic acid to synthesize sialic acid esters: sialic acid and C1-C4 alcohol are used as reaction substrates, lipase and acid are used as catalysts, and the amount of acid used is 0.05-5‰ of the corresponding mass of the substrate sialic acid, the selection of the reaction substrate C1-C4 alcohol depends on the target sialic acid esters, and the C1-C4 alcohol is also used as an organic solvent medium; sialic acid is dissolved in C1-C4 alcohol, and then lipase, acid accounting for 0.05-5‰ of the mass of the substrate sialic acid, The molecular sieve water absorbent is subjected to esterification reaction at a temperature of 30-60°C and an oscillation speed of 200-300 r / min. After the reaction is completed, the reaction liquid is separated and purified to obtain sialic acid ester.

[0014] Preferably, the sialic acid is one of N-acetylneuraminic acid, N-glycolylneuraminic acid and polysialic acid.

[0015] Preferably, the lipase is an immobilized lipase, specifically selected from Novozymes 435 of Denmark, an immobilized lipase derived from Candida Antarctica B, which is made by deep fermentation of a genetically modified Aspergillus oryzae microorganism and adsorption on a macroporous acrylic resin. The commercial enzyme is easy to obtain, has high catalytic efficiency, good stability, a particle size of 0.3-0.9 mm, a bulk density of 430 kg / m3, a moisture content of 1-2%, a wide substrate range, an optimal catalytic temperature of 40-60°C, and an enzyme activity of 10000 U / g.

[0016] Preferably, the C1-C4 alcohol is one of methanol, ethanol, propanol, isopropanol, n-butanol, sec-butanol, tert-butanol and isobutanol.

[0017] Preferably, the acid is ionizable to produce H + An organic acid or inorganic acid, including one of trifluoroacetic acid, hydrochloric acid and sulfuric acid.

[0018] Preferably, the lipase, sialic acid, C1-C4 alcohol, The molecular sieve water absorbent is used in a ratio of (0.5-1) g: 1 g: (100-125) mL: 10 g, and the trifluoroacetic acid or hydrochloric acid or sulfuric acid (TFA or HCl or H2SO4etc) is used in a ratio of 0.05-5‰ of the mass of the substrate sialic acid.

[0019] Preferably, the esterification reaction time is 6-20 hours.

[0020] Preferably, the method for separating and purifying the reaction liquid is: after the reaction is completed, the reaction liquid is filtered to remove the lipase and the water absorbent, and the filtrate is distilled under reduced pressure until there is no distillate to obtain the white solid sialic acid ester.

[0021] The present invention further provides the use of the sialic acid ester synthesized by the above method in the preparation of nutritional supplements, foods, medicines, feeds, food additives, feed additives, cosmetics, and skin care products.

[0022] The present invention further provides the use of sialic acid esters synthesized by the above method in the preparation of drugs for preventing and treating viral diseases, preventing and treating keloids, treating autoimmune diseases, treating tumors, treating rhinitis, treating asthma, treating nervous system diseases, treating inflammatory diseases, treating herpes zoster, sequelae of new coronary diseases, and anti-aging drugs.

[0023] Beneficial effects of the present invention:

[0024] 1. The present invention is the first to use the commercial immobilized lipase Novozyme 435 and an acid accounting for 0.05-5‰ of the mass of the reaction substrate sialic acid as a catalyst to catalyze the synthesis of sialic acid esters by a semi-enzymatic method. The inventors have found through innovative research that in the catalytic synthesis process, in a reaction system where the dosage ratio of lipase, sialic acid, C1-C4 alcohol, and water absorbent is (0.5-1) g:1 g:(100-125) mL:10 g, only 0.05-5‰ of the mass of the reaction substrate sialic acid (or hydrochloric acid or sulfuric acid, etc., which can ionize H + The present invention upgrades and optimizes the production process of sialic acid esters, and greatly improves the productivity of sialic acid esters. In addition, compared with the reaction of sialic acid esters synthesized by traditional chemical synthesis (the traditional chemical synthesis method requires 1-10wt% of trifluoroacetic acid and other acids), the amount of trifluoroacetic acid and other acids is greatly reduced, and the residual traces of trifluoroacetic acid and other acids in the final product are or are absent, and the by-product content is extremely low, and the final product is suitable for use in food additives, medicines or cosmetic raw materials.

[0025] 2. The present invention is the first to utilize the commercialized immobilized lipase Novozyme 435 and an acid accounting for 0.05-5‰ of the mass of the reaction substrate sialic acid as a catalyst to catalyze the synthesis of sialic acid esters by a semi-enzymatic method. The enzyme has strong regional selectivity, high reaction conversion rate, safety and efficiency, the catalyst can be recycled and reused multiple times, the solvent medium can be recycled and reused, it is environmentally friendly, green and pollution-free, no toxic substances remain, and is suitable for mass production.

[0026] 3. The present invention uses the reaction substrate C1-C4 alcohol as the organic solvent reaction medium. The selection of the reaction substrate C1-C4 alcohol depends on the target sialic acid esters. The downstream separation is simple. The immobilized lipase and Molecular sieves can be removed by filtration and recycled. The molecular sieve is activated by high-temperature water removal, with low energy consumption, little environmental pollution, no harmful substance residue and three wastes, suitable for industrial production, and the sialic acid conversion rate exceeds 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a picture of a capsule product prepared from sialic acid methyl ester obtained in Example 1;

[0028] Figure 2 The high performance liquid chromatogram (HPLC) of the product ethyl sialate prepared in Example 2;

[0029] Figure 3 is a diagram of herpes zoster symptoms of patient 1 in Example 7;

[0030] Figure 4 is a diagram of herpes zoster symptoms of patient 2 in Example 7;

[0031] Figure 5 is a diagram of herpes zoster symptoms of patient 3 in Example 7;

[0032] Figure 6 This is a facial skin picture of the test subject in Example 9 before using the sialic acid methyl ester solution for skin care;

[0033] Figure 7 This is a picture of the facial skin of the test subject in Example 9 after using methyl sialic acid solution for skin care. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0035] The present invention provides a method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid: sialic acid and C1-C4 alcohol are reaction substrates, lipase is used as a catalyst, the selection of the reaction substrate C1-C4 alcohol depends on the target sialic acid esters, and the C1-C4 alcohol is also used as an organic solvent medium; sialic acid is dissolved in the C1-C4 alcohol, and then lipase, trifluoroacetic acid (TFA) or hydrochloric acid (HCl) or sulfuric acid (H2SO4) accounting for 0.05-5‰ of the mass of the substrate sialic acid are added. The molecular sieve water absorbent is subjected to esterification reaction at a temperature of 30-60°C and an oscillation speed of 200-300 r / min for 8-15 hours. After the reaction is completed, the reaction solution is separated and purified to obtain sialic acid ester;

[0036] The sialic acid is one of N-acetylneuraminic acid, N-glycolylneuraminic acid and polysialic acid; the lipase is an immobilized lipase, specifically selected from immobilized Antarctic Candida antarctica lipase B (CAL-B), with a trade name of Novozyme 435. The commercial enzyme is cheap and easy to obtain, has high catalytic efficiency and good stability, and has a particle size of 0.3-0.9 mm and a bulk density of 430 kg / m 3 , the moisture content is 1-2%, it has a wide substrate range, the optimal catalytic temperature is 40-60℃, and the enzyme activity is 10000U / g.

[0037] The C1-C4 alcohol is one of methanol, ethanol, propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, and isobutanol; the lipase, sialic acid, C1-C4 alcohol, The usage ratio of molecular sieve water absorbent, trifluoroacetic acid or hydrochloric acid or sulfuric acid is (0.5-1)g:1g:(100-125)mL:10g:(0.00005-0.005)g;

[0038] The method for separating and purifying the reaction liquid is as follows: after the reaction is completed, the reaction liquid is filtered to remove the immobilized lipase and The filtrate is distilled under reduced pressure until no distillate is produced, thereby obtaining the sialic acid ester.

[0039] The sialic acid ester synthesized by the method of this embodiment is used in the preparation of nutritional supplements, foods, medicines, feeds, food additives, feed additives, cosmetics, and skin care products.

[0040] The sialic acid ester synthesized by the method of this embodiment is used in the preparation of drugs for preventing and treating viral diseases, preventing and treating keloids, treating autoimmune diseases, treating tumors, treating rhinitis, treating asthma, treating nervous system diseases, treating inflammatory diseases, treating herpes zoster, sequelae of COVID-19, and anti-aging drugs.

[0041] The lipase used in the following Examples 1-5 is an immobilized lipase, specifically immobilized Candida antarctica lipase B (CAL-B), trade name Novozyme 435, produced by Novozymes of Denmark, with an enzyme activity of 10000 U / g.

[0042] In the following Examples 1 to 5, the amount of trifluoroacetic acid (TFA) or hydrochloric acid (HCl) or sulfuric acid (H2SO4) used is 0.05-5‰ of the mass of the substrate sialic acid.

[0043] The liquid chromatography (HPLC) determination conditions of sialic acid and sialic acid esters in the following Examples 1-5 and Comparative Examples 1-3 are: ZORBAX SB C18 250×4.6 mm chromatographic column; mobile phase: pH 3.0 sulfuric acid aqueous solution; mobile phase flow rate: 1.0 mL / min; detector: UV 210 nm; injection volume: 5.0 μL.

[0044] Example 1

[0045] A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid: 4.0 g N-acetylneuraminic acid and 400 mL anhydrous methanol are added to a 500 mL conical flask, stirred to dissolve, and then 2.0 g lipase Novozyme 435, 40.0 g Molecular sieves and 0.01 mL trifluoroacetic acid (determined by mass volume conversion according to the molecular weight of trifluoroacetic acid), constant temperature oscillation reaction at 30 ° C for 15 h, the oscillation speed is 200 r / min, after the reaction is completed, the reaction solution is filtered to remove lipase Novozyme 435 and The filtrate was distilled under reduced pressure until no distillate was produced, and then a sample was taken for liquid chromatography analysis, which showed that the yield of methyl sialate was 95.6%.

[0046] Example 2

[0047] A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid: 4.0 g N-acetylneuraminic acid and 400.0 mL anhydrous ethanol are added to a 1000 mL conical flask, stirred to dissolve, and then 4.0 g lipase Novozyme 435, 40.0 g Molecular sieves and 0.005 mL of 1 M hydrochloric acid (determined by mass volume conversion according to the molecular weight of hydrochloric acid), constant temperature oscillation reaction at 40 ° C for 10 hours, the oscillation speed is 200 r / min, after the reaction is completed, the reaction solution is filtered to remove lipase Novozyme 435 and The filtrate was distilled under reduced pressure until no distillate was produced, and then a sample was taken for liquid chromatography analysis, which showed that the yield of ethyl sialate was 94.3%.

[0048] Example 3

[0049] A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid: 4.0 g N-glycolylneuraminic acid and 400.0 mL anhydrous ethanol are added to a 1000 mL conical flask, stirred to dissolve, and then 4.0 g lipase Novozyme 435, 40.0 g Molecular sieves and 0.0025 mL of 1 M sulfuric acid (determined by mass volume conversion according to the molecular weight of sulfuric acid), 50 ° C constant temperature oscillation reaction for 12 hours, the oscillation speed is 300r / min, after the reaction is completed, the reaction solution is filtered to remove lipase Novozyme 435 and The filtrate was distilled under reduced pressure until no distillate was produced, and then a sample was taken for liquid chromatography analysis, which showed that the yield of ethyl sialate was 95.9%.

[0050] Example 4

[0051] A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid: 4.0 g N-glycolylneuraminic acid and 500.0 mL anhydrous propanol were added to a 1000 mL conical flask, stirred to dissolve, and then 2.0 g lipase Novozyme 435, 40.0 g Molecular sieves and 0.002 mL trifluoroacetic acid (determined by mass volume conversion according to the molecular weight of trifluoroacetic acid), constant temperature oscillation reaction at 60 ° C for 8 hours, the oscillation speed is 200 r / min, after the reaction is completed, the reaction solution is filtered to remove lipase Novozyme 435 and The filtrate was distilled under reduced pressure until no distillate was produced, and then a sample was taken for liquid chromatography analysis, which showed that the yield of sialyl propyl ester was 92.8%.

[0052] Example 5

[0053] A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid: 4.0 g polysialic acid and 500.0 mL anhydrous butanol were added into a 1000 mL conical flask, stirred and dissolved, and then 2.0 g lipase Novozyme 435, 40.0 g Molecular sieves and 0.001 mL trifluoroacetic acid (determined by mass volume conversion according to the molecular weight of trifluoroacetic acid), 50 ° C constant temperature oscillation reaction for 12 hours, the oscillation speed is 200r / min, after the reaction is completed, the reaction solution is filtered to remove lipase Novozyme 435 and The filtrate was distilled under reduced pressure until no distillate was produced, and then a sample was taken for liquid chromatography analysis, which showed that the yield of butyl sialate was 91.5%.

[0054] Comparative Examples 1-5

[0055] Comparative Examples 1-5 are the same as Example 1, except that different specific lipases are used, and the sialic acid conversion rate is calculated according to 100%×(initial sialic acid mass-reacted sialic acid mass) / initial sialic acid mass, as shown in Table 1:

[0056]

[0057]

[0058] As shown in Table 1, when different lipases are used as catalysts and other conditions remain unchanged, the catalytic effect of the lipase Novozyme 435 in Example 1 is better than that of other lipases in the semi-enzymatic esterification of sialic acid.

[0059] Comparative Example 6

[0060] Comparative Example 6 is the same as Example 1, except that trifluoroacetic acid is not added dropwise during the reaction. When the sialic acid methyl ester yield of the reaction system of Comparative Example 6 reaches 95.6%, the required reaction time is recorded.

[0061] Test results and analysis: When the pure enzyme method of Comparative Example 6 achieved the same 95.6% sialic acid methyl ester yield as Example 1, it took 8 days of reaction time, proving that the very small amount of trifluoroacetic acid in Example 1 produced a huge synergistic effect on the enzymatic catalysis of lipase.

[0062] Comparative Example 7

[0063] Comparative Example 7 is the same as Example 1, except that 2.0 g of lipase Novozyme 435 is not added during the reaction, and the reaction time is 6 days.

[0064] Test results and analysis: In comparative example 7, lipase Novozyme 435 was not added during the reaction. After 6 days of oscillation reaction, the yield of sialic acid methyl ester was only 0.4%, indicating that the catalytic effect of a very small amount of trifluoroacetic acid on the synthesis of sialic acid esters was negligible.

[0065] Comparative Example 8

[0066] A method for synthesizing sialic acid esters by catalyzing sialic acid with trifluoroacetic acid: 4.0 g of N-acetylneuraminic acid and 400.0 mL of anhydrous methanol are added to a 1000 mL conical flask, stirred and dissolved, and 40.0 g of Molecular sieve and 0.26mL trifluoroacetic acid (determined by mass volume conversion according to the molecular weight of trifluoroacetic acid), 30℃ constant temperature oscillation reaction for 5 days, oscillation speed 200r / min, after the reaction is completed, the reaction solution is filtered to remove The filtrate was distilled under reduced pressure until no distillate was produced, and then a sample was taken for liquid chromatography analysis, which showed that the yield of methyl sialate was 88.4%.

[0067] Test results and analysis: Compared with the reaction of synthesizing sialic acid esters catalyzed by conventional trifluoroacetic acid in comparative example 8, the semi-enzymatic catalytic synthesis method of embodiment 1 compresses the reaction time to within 15 h, and the yield of the target product of embodiment 1 is also significantly improved, which proves that the semi-enzymatic catalytic synthesis method of embodiment 1 combining lipase and trifluoroacetic acid effectively improves the reaction rate and the yield of the target product sialic acid ester; at the same time, the amount of trifluoroacetic acid used is greatly reduced (the amount of trifluoroacetic acid used in the chemical synthesis method of comparative example 8 accounts for about 10% of the mass of the reaction substrate N-acetylneuraminic acid, while only about 3.375‰ is required in embodiment 1), and there is a trace or no residual trifluoroacetic acid in the final product, and the content of by-products is extremely low.

[0068] Based on Examples 1-5 and Comparative Examples 1-8, it can be seen that Examples 1-5 creatively utilize the commercialized immobilized lipase Novozyme 435 for the first time and trifluoroacetic acid (or hydrochloric acid or sulfuric acid, etc., which can ionize H + The inventors have found through innovative research that in the catalytic synthesis process, in a reaction system where the dosage ratio of lipase, sialic acid, C1-C4 alcohol, and water absorbent is (0.5-1) g:1 g:(100-125) mL:10 g, only 0.05-5‰ of trifluoroacetic acid (or hydrochloric acid or sulfuric acid, etc., which can ionize H) is added dropwise to the reaction substrate N-acetylneuraminic acid. + The reaction time can be shortened to 6-20h by adding organic acid or inorganic acid). Compared with the traditional single trifluoroacetic acid catalysis method in Comparative Example 8 (requiring 5d reaction time) or the single lipase enzymatic method in Comparative Example 6 (requiring 8d reaction time), the semi-enzymatic method of the present invention significantly improves the reaction efficiency of the entire reaction system and greatly shortens the reaction time, proving that lipase and trifluoroacetic acid produce a synergistic catalytic effect. The reason may be that trifluoroacetic acid (or hydrochloric acid or sulfuric acid, etc., which can ionize H + The organic acid or inorganic acid) is used as a proton acid catalyst and acts as a proton donor during the reaction. The carboxyl group of the lipase accepts protons to form an ionized lipase. The ionized lipase may be a key factor in improving the catalytic efficiency. On the one hand, the ionized lipase further reduces the activation energy in the enzymatic synthesis of sialic acid esters. On the other hand, trifluoroacetic acid (or hydrochloric acid or sulfuric acid) can ionize H + The ionic intermediates formed by the organic or inorganic acid) and sialic acid and C1-C4 alcohol are easier to combine with the ionized lipase, which increases the effective contact area between the lipase and the reaction substrate, thereby achieving the reaction between the lipase and trifluoroacetic acid (or hydrochloric acid or sulfuric acid, etc., which can ionize H +The organic acid or inorganic acid) can synergistically and rapidly catalyze the synthesis of sialic acid esters, thereby upgrading and optimizing the production process of sialic acid esters and greatly improving the productivity of sialic acid esters.

[0069] Example 6

[0070] Using the sialic acid methyl ester obtained in Example 1 as a raw material, capsules with a sialic acid methyl ester content of 70 mg / capsule were prepared. Figure 1 As shown, it is used to treat herpes zoster, inflammatory diseases, etc.

[0071] Example 7

[0072] The sialic acid methyl ester capsules prepared in Example 6 were used to treat patients with herpes zoster:

[0073] Treatment plan 1: Patient 1, male, 60 years old, herpes zoster for more than 1 week (symptoms as shown in the figure Figure 3 The patient had severe neuralgia in the affected area, accompanied by restlessness. Oral and external application of various drugs were ineffective. The sialic acid methyl ester capsule prepared in Example 6 was taken orally on an empty stomach, 140 mg / time, once / day, for 3 consecutive days.

[0074] Treatment effect: The pain was significantly relieved 3.5 hours after the first oral administration of methyl sialate capsules. Telephone follow-up showed that the pain and skin lesions gradually decreased, and the symptoms did not worsen after stopping the medication.

[0075] Treatment plan 2: Patient 2, female, 48 years old, initial onset of herpes zoster (symptoms as shown in the figure Figure 4 The skin of the affected area was burning and painful. No medication was used for treatment. The sialic acid methyl ester capsule prepared in Example 6 was taken orally on an empty stomach, 140 mg / time, once / day, for 3 consecutive days.

[0076] Treatment effect: The pain was relieved 4.5 hours after the first oral administration of methyl sialate capsules. Telephone follow-up showed that the pain and skin lesions gradually decreased, and the symptoms did not worsen after stopping the medication.

[0077] Treatment plan 3: Patient 3, female, 75 years old, initial onset of herpes zoster (symptoms as shown in the figure Figure 5 The affected area and the surrounding skin were burning and hyperesthetized, but the pain was tolerable. Oral acetaminophen was ineffective. The sialic acid methyl ester capsules prepared in Example 6 were taken orally on an empty stomach, 140 mg / time, once / day, for 3 consecutive days.

[0078] Treatment effect: The pain was relieved 5 hours after the first oral administration of methyl sialate capsules. Telephone follow-up showed that the pain and skin lesions gradually decreased, and the patient was cured after 5 days.

[0079] Example 8

[0080] The sialic acid methyl ester capsules prepared in Example 6 are used to treat inflammatory diseases:

[0081] Patient 4, female, 51 years old, had swollen lymph node area at the root of the right thigh, no local skin temperature, accompanied by mild to moderate pain and discomfort. The local pain and discomfort intensified when walking, sitting for a long time or tired, and the discomfort radiated to the back corresponding to the right lower abdomen. Self-treatment with multiple antibiotics was ineffective.

[0082] Treatment plan: Take 70 mg of sialic acid methyl ester capsule orally on an empty stomach before bedtime, once a day, for 10 consecutive days.

[0083] Treatment effect: The swelling in the lymph node area at the root of the right thigh was significantly reduced, the pain basically disappeared, and the local discomfort was not obvious after walking, sitting for a long time or fatigue.

[0084] Example 9

[0085] The sialic acid methyl ester prepared in Example 1 is used for skin moisturizing and maintenance:

[0086] Usage: Use the sialic acid methyl ester obtained in Example 1 as a raw material to prepare a physiological saline solution with a sialic acid methyl ester content of 3%, spray it on the facial skin, and gently massage it for moisturizing and maintaining the facial skin.

[0087] Skin care effect: 30 minutes after the facial spray application of 3% sialic acid methyl ester saline solution, compared with before use (such as Figure 6 As shown), the skin is noticeably moisturized and smooth, and fine wrinkles are significantly reduced (skin moisturizing effect as shown Figure 7 shown).

[0088] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid, characterized in that: Sialic acid and C1-C4 alcohol are used as reaction substrates, lipase and acid are used as catalysts, and the amount of acid is 0.05-5‰ of the corresponding mass of substrate sialic acid, wherein the selection of C1-C4 alcohol depends on the target sialic acid esters; sialic acid is dissolved in C1-C4 alcohol, and then lipase, acid, The molecular sieve water absorbent is subjected to esterification reaction at a temperature of 30-60°C and an oscillation speed of 200-300 r / min. After the reaction is completed, the reaction solution is separated and purified to obtain pure sialic acid ester; The lipase is immobilized Antarctic Candida lipase B.

2. A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid as claimed in claim 1, characterized in that: The sialic acid is one of N-acetylneuraminic acid, N-glycolylneuraminic acid and polysialic acid.

3. A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid as claimed in claim 1, characterized in that: The C1-C4 alcohol is one of methanol, ethanol, propanol, isopropanol, n-butanol, sec-butanol, tert-butanol and isobutanol.

4. A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid as claimed in claim 1, characterized in that: The acid is ionizable to release H + An organic acid or inorganic acid, including one of trifluoroacetic acid, hydrochloric acid and sulfuric acid.

5. A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid according to any one of claims 1 to 4, characterized in that: The lipase, sialic acid, C1-C4 alcohol, The usage ratio of molecular sieve water absorbent and acid is (0.5-1)g:1g:(100-125)mL:10g:(0.00005-0.005)g.

6. A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid as claimed in claim 1, characterized in that: The esterification reaction time is 6-20h.

7. A method for synthesizing sialic acid esters by semi-enzymatic catalysis of sialic acid as claimed in claim 1, characterized in that: The method for separating and purifying the reaction liquid is as follows: after the reaction is completed, the reaction liquid is filtered to remove the lipase and the water absorbent, and the filtrate is distilled under reduced pressure until there is no distillate, to obtain a white solid as the sialic acid ester.

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