Preparation method and application of eicosapentaenoic acid-trimethoxy benzyl alcohol ester

Abstract

The invention belongs to the technical field of medicinal chemistry and organic synthesis, and relates to a preparation method and application of eicosapentaenoic acid-trimethoxy benzyl alcohol ester. According to the preparation method of the eicosapentaenoic acid-trimethoxy benzyl alcohol ester, provided by the invention, 3, 4, 5-trimethoxy benzyl alcohol and eicosapentaenoic acid are taken as raw materials, and lipase is added for reaction to synthesize the eicosapentaenoic acid-trimethoxy benzyl alcohol ester. The eicosapentaenoic acid-trimethoxy benzyl alcohol ester prepared by the invention not only has a remarkable blood fat reducing effect, but also can effectively block the pathological progress of inflammatory diseases through intramolecular synergistic anti-inflammatory and anti-oxidation action mechanisms, has a remarkable treatment effect on ulcerative colitis, and can be used for preparing a medicine for treating the ulcerative colitis. A more effective and safer treatment medicine is provided for treating the hyperlipidemia and the ulcerative colitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry and organic synthesis, and relates to a preparation method and application of trimethoxybenzyl alcohol eicosapentaenoate derivative, in particular to a preparation method and application of eicosapentaenoic acid-trimethoxybenzyl alcohol ester. BACKGROUND

[0002] EPA, as a core member of Omega-3 polyunsaturated fatty acids, plays an important role in cardiovascular protection, anti-inflammation and metabolic regulation, and has become an important raw material in the global pharmaceutical and functional food fields. At present, commercial preparations of EPA are mainly divided into two categories: ethyl ester type and glycerol type. Ethyl ester type (EE type) preparations, such as the prescription drug Vascepa®, significantly reduce triglyceride levels through high-purity (>96%) EPA ethyl ester, occupying the high-end pharmaceutical market.

[0003] Patent document CN105218367A discloses a preparation method of high-purity eicosapentaenoic acid ethyl ester concentrate. This method uses multi-stage purification processes such as urea inclusion, high-performance liquid chromatography separation and molecular distillation to control the content of trans-EPA and oxidation products and other impurities at a very low level. This method ensures the high purity and safety of the drug, but its application still has significant limitations. For example, after oral administration of ethyl ester type EPA, it needs to be hydrolyzed to free EPA by esterase to exert biological activity, resulting in a large influence of individual intestinal environment on its bioavailability, and some patients may have gastrointestinal side effects.

[0004] Glycerol triester type (TG type) preparations, especially reconstituted glycerol triesters (rTG), are considered to have better absorption characteristics because their structure is closer to natural fish oil, making them widely used in the field of health products and dietary supplements. Its preparation technology usually relies on enzyme catalysis. Patent document CN101348807B discloses a method for enriching n-3 polyunsaturated fatty acid glycerides from fish oil. This method first hydrolyzes fish oil to obtain free fatty acids, then purifies them, and then uses lipase to catalyze the esterification of EPA and glycerol in order to obtain high-content EPA triglycerides. However, the defect of this technology is that the final product is usually a mixture of EPA and DHA, making it difficult to achieve high-purity preparation of single EPA component, resulting in the need for a large dose to achieve the desired effect, which limits its application in precision medicine.

[0005] In order to break through the bottleneck of the above-mentioned traditional forms, the field is exploring the structural modification of polyunsaturated fatty acids at the molecular level. Therefore, how to precisely modify the molecular structure of EPA to solve the problems of purity, stability and bioavailability of traditional preparations is still a difficult problem that needs to be solved at present. SUMMARY

[0006] In order to solve the defects of the prior art, the present application provides a novel trimethoxybenzyl alcohol eicosapentaenoic acid ester derivative (EPA-TMB), in particular, a preparation method of eicosapentaenoic acid-trimethoxybenzyl alcohol ester and its application. The present application successfully constructs a novel trimethoxybenzyl alcohol eicosapentaenoic acid ester derivative (EPA-TMB) by coupling the 3,4,5-trimethoxyphenyl structural unit with eicosapentaenoic acid (EPA). The EPA-TMB prepared by the present application not only has significant hypolipidemic effect, but also can effectively block the pathological progression of inflammatory diseases through the synergistic anti-inflammatory and antioxidant mechanisms of the molecule, and has significant therapeutic effect on ulcerative colitis, thereby providing a more effective and safer therapeutic drug for the treatment of hyperlipidemia and ulcerative colitis.

[0007] In order to achieve the above-mentioned target, the technical scheme of the present application is as follows: The present application provides a preparation method of eicosapentaenoic acid-trimethoxybenzyl alcohol ester, comprising the following steps: Step S1, 3,4,5-trimethoxybenzyl alcohol and eicosapentaenoic acid are added to a container containing tetrahydrofuran solution, stirred at room temperature for 2-3 min, then lipase is added to the system, stirred at an oil bath temperature of 45-55 ℃ for 35-38 h, filtered, and the filter cake is taken; Step S2, the filter cake prepared in step S1 is washed with tetrahydrofuran for 3-4 times, the filtrate and washing liquid are collected and combined, and the combined solution is concentrated under reduced pressure to obtain an oily crude product; Step S3, the oily crude product prepared in step S2 is purified by silica gel column chromatography, the corresponding components are collected and combined, and concentrated under reduced pressure.

[0008] Further, the molar ratio of 3,4,5-trimethoxybenzyl alcohol to eicosapentaenoic acid in step S1 is 31.0 mmol:34.1 mmol.

[0009] Further, the feed liquid ratio of 3,4,5-trimethoxybenzyl alcohol to tetrahydrofuran solution in step S1 is (1-2) g:10 mL.

[0010] Further, the lipase in step S1 is immobilized lipase, and the immobilized lipase is immobilized lipase Novozym 435.

[0011] Further, the addition amount of the lipase in step S1 is 15-25% w / w of the mass of 3,4,5-trimethoxybenzyl alcohol.

[0012] Further, thin layer chromatography is used to monitor the reaction in the step S1, and the developing agent is petroleum ether / ethyl acetate=45:4 v / v, Rf=0.4, and the color reagent is phosphomolybdic acid.

[0013] Further, the gradient of 100% petroleum ether to ethyl acetate / petroleum ether=6:94 v / v is used to elute in the silica gel column chromatography in the step S3, and the silica gel is 200-300 mesh silica gel.

[0014] In addition, the present application also claims the eicosapentaenoic acid-trimethoxy benzyl alcohol ester prepared by the preparation method of the eicosapentaenoic acid-trimethoxy benzyl alcohol ester.

[0015] Further, the present application also provides the application of the eicosapentaenoic acid-trimethoxy benzyl alcohol ester in the preparation of drugs for preventing or treating hyperlipidemia.

[0016] Further, the present application also provides the application of the eicosapentaenoic acid-trimethoxy benzyl alcohol ester in the preparation of drugs for preventing or treating colitis, especially in the preparation of drugs for preventing or treating ulcerative colitis.

[0017] 3,4,5-trimethoxyphenyl structural unit is a recognized privileged scaffold, and its strong biological activity has been proved in many compounds. The present application successfully constructs eicosapentaenoic acid-trimethoxy benzyl alcohol ester (EPA-TMB) by the method of molecular coupling of 3,4,5-trimethoxyphenyl structural unit and EPA through structural design breakthrough. In the experimental process, the inventors accidentally find that eicosapentaenoic acid-trimethoxy benzyl alcohol ester not only has significant hypolipidemic effect, but also has anti-inflammatory and antioxidant effects, and can effectively block the pathological progression of inflammatory diseases, thereby providing a new drug for the treatment of hyperlipidemia and ulcerative colitis.

[0018] Specifically, the eicosapentaenoic acid-trimethoxy benzyl alcohol ester (EPA-TMB) prepared by the present application has the efficacy of treating hyperlipidemia. In the hyperlipidemia model mouse model, the EPA-TMB significantly improves the hyperlipidemia of the mice in a dose-dependent manner, and compared with natural EPA, the EPA-TMB has more advantages in improving hyperlipidemia, especially the blood lipid level of the high-dose group is close to the normal value, which is a high-efficiency lipid-lowering drug candidate and can be used as a therapeutic drug for hyperlipidemia.

[0019] Further, the eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared by the present application has the effect of treating colitis. In a colitis model mouse, the EPA-TMB has a significant inhibitory effect on intestinal barrier damage and inflammatory response. At the same time, the EPA-TMB can also reduce oxidative damage by inhibiting lipid peroxidation and enhancing antioxidant enzyme activity, and rebuild the mucosal barrier by up-regulating the expression of barrier proteins, thereby achieving efficient repair of intestinal structure and function damage, and having a significant therapeutic effect on ulcerative colitis, thereby breaking through the technical bottleneck of the functional limitation and insufficient bioavailability of existing EPA drugs. At the same time, the structural modification of the EPA-TMB can improve its targeting and metabolic stability in the intestinal tract, so that it can play the roles of anti-inflammatory, antioxidant and barrier repair at a low dose, thereby providing a potential optimization strategy for the treatment of ulcerative colitis.

[0020] In summary, compared with the prior art, the preparation method of eicosapentaenoic acid-trimethoxybenzyl alcohol ester provided by the present application has the following advantages: (1) The preparation method of eicosapentaenoic acid-trimethoxybenzyl alcohol ester provided by the present application has the following advantages:

[0021] (2) The preparation method of eicosapentaenoic acid-trimethoxybenzyl alcohol ester provided by the present application has the following advantages:

[0022] (3) The preparation method of eicosapentaenoic acid-trimethoxybenzyl alcohol ester provided by the present application has the following advantages: BRIEF DESCRIPTION OF DRAWINGS Figure 1 The synthesis route of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) is shown in the figure.

[0023] Figure 2 The synthesis route of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) is shown in the figure. 1 H spectrum.

[0024] Figure 3 The synthesis route of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) is shown in the figure. 13 C spectrum. DETAILED DESCRIPTION

[0025] The present application is further described by the following description of specific embodiments, which are not intended to limit the scope of the application. Various modifications or improvements can be made by those skilled in the art based on the basic idea of the present application, as long as they do not deviate from the basic idea of the present application, and are within the scope of the present application. The materials involved in the present application are all food-grade and can be obtained by commercial or conventional technical means in the art. For example: immobilized lipase Novozym 435 is purchased from Sigma-Aldrich, item number: 73940.

[0026] Example 1, a preparation method of eicosapentaenoic acid-trimethoxybenzyl alcohol ester Step S1, 6.15 g of 3,4,5-trimethoxybenzyl alcohol (31.0 mmol) and 10.3 g of eicosapentaenoic acid (EPA, 34.1 mmol, 1.1 equivalent) were added to a three-necked flask containing 50 mL of anhydrous tetrahydrofuran (THF), stirred at room temperature for 2 min, then 1.23 g of immobilized lipase Novozym 435 was added to the system, and the reaction was stirred at a temperature of 50 ℃ in an oil bath for 36 h. During the reaction, thin layer chromatography was used to monitor the reaction, and the developing agent of the thin layer chromatography was petroleum ether / ethyl acetate = 45:4 v / v, Rf=0.4, 10% phosphomolybdic acid ethanol solution coloration. After the reaction was completed, the mixture was transferred to a sand core funnel with diatomite on the surface while hot and filtered, the residual enzyme catalyst was removed by vacuum filtration, and the filter cake was taken; Step S2, the filter cake prepared in step S1 was washed with tetrahydrofuran, 10 mL each time for 3 times, the filtrate and washing liquid were collected and combined, and the combined solution was concentrated by rotary evaporation under reduced pressure to obtain a brown yellow oily crude product; Step S3, the brown yellow oily crude product prepared in step S2 was purified by silica gel column chromatography, the silica gel was 200-300 mesh silica gel, and elution was performed using a gradient of 100% petroleum ether to ethyl acetate / petroleum ether = 6:94 v / v, the corresponding fractions were collected, combined, and the solvent was evaporated using a rotary evaporator to obtain a light yellow oily eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB).

[0027] The synthetic route of the eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1 is shown in Figure 1 .

[0028] Example 2, a preparation method of eicosapentaenoic acid-trimethoxybenzyl alcohol ester Into a three-necked flask equipped with 50 mL of anhydrous tetrahydrofuran (THF), 6.15 g of 3,4,5-trimethoxybenzyl alcohol (31.0 mmol) and 10.3 g of eicosapentaenoic acid (EPA, 34.1 mmol, 1.1 equivalent) were added, stirred for 3 min at room temperature, then 1.23 g of immobilized lipase Novozym 435 was added to the system, and the reaction was stirred in an oil bath at a temperature of 45 °C for 38 h. During the reaction, thin layer chromatography was used to monitor the reaction, and the developing agent of the thin layer chromatography was petroleum ether / ethyl acetate = 45:4 v / v, Rf= 0.4, 10% phosphomolybdic acid ethanol solution coloration. After the reaction was completed, the mixture was transferred to a sand core funnel covered with diatomite on hot and filtered, and the residual enzyme catalyst was removed by vacuum filtration. The filter cake was collected; Step S2, the filter cake prepared in step S1 was washed with tetrahydrofuran, 10 mL each time for 4 times, the filtrate and washing liquid were collected and combined, and the combined solution was concentrated by rotary evaporator under reduced pressure to obtain brown yellow oil crude product; Step S3, the brown yellow oil crude product prepared in step S2 was purified by silica gel column chromatography, the silica gel was 200-300 mesh silica gel, and elution was performed using a gradient of 100% petroleum ether to ethyl acetate / petroleum ether = 6:94 v / v. The corresponding fractions were collected, combined, and the solvent was evaporated using a rotary evaporator to obtain eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) in the form of light yellow oil.

[0029] Experimental Example One, yield and structural identification experiment of eicosapentaenoic acid-trimethoxybenzyl alcohol ester 1. Experimental method: The eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1 was subjected to yield analysis and 1H NMR, 13C NMR and MS analysis.

[0030] 2. Experimental results: 2.1, the yield of eicosapentaenoic acid-trimethoxybenzyl alcohol ester prepared in Example 1: It was calculated that the yield of eicosapentaenoic acid-trimethoxybenzyl alcohol ester prepared in Example 1 was 71%.

[0031] 2.2, 1H NMR, 13C NMR and MS analysis results of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB): The 1H NMR spectrum of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1 is shown in Figure 1. 1 The 1H NMR spectrum of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1 is shown in Figure 1. Figure 2 The 1H NMR spectrum of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1 is shown in Figure 1.13 The1H NMR spectrum (in CDCl3) is as shown in Figure 1. Figure 3

[0032] In particular: (1) The1H NMR spectrum data of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1 is as follows: 1 1 d ) δ 6.58 (s, 2H), 5.46 - 5.26 (m, 10H), 5.04 (s, 2H), 3.85 (d, J = 7.2 Hz, 9H), 2.90 - 2.72 (m, 8H), 2.37 (t, J = 7.6 Hz, 2H), 2.17 - 2.01 (m, 4H), 1.73 (p, J = 7.6 Hz, 2H), 0.96 (t, J = 7.5 Hz, 3H).

[0033] (2) The13C NMR spectrum data of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1 is as follows: 13 13 d ) δ 173.43, 153.32, 132.06, 131.61, 128.91, 128.89, 128.59, 128.29, 128.18, 128.07, 127.86, 127.01, 105.45, 66.43, 60.85, 56.12, 33.70, 26.55, 25.63, 25.55, 24.80, 20.57, 14.30.

[0034] (3) The mass spectrum data of eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1 is as follows: MS (ESI, +ve): m / z 505.3 [M+Na + ].

[0035] Experimental Example Two, Analysis Experiment of Hypolipidemic Effect of Eicosapentaenoic Acid-trimethoxybenzyl Alcohol Ester Experimental Materials: Eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1.

[0036] ​​​​​Experimental method: 2.1, Model establishment: 8~10 weeks old male C57BL / 6 mice (body weight 18~22 g) were used, and after 5 days of adaptive feeding, they were randomly divided into five groups (10 in each group). The mice were standardized in the SPF level animal room (free intake of basic feed and sterilized water). Before the experiment, except for the normal control group (group A), the rest of the mice were induced by drinking 3% (w / v) dextran sulfate sodium (DSS) in sterilized water (pH 6.0) for 7 days to induce acute colitis model.

[0037] 2.2, Animal grouping: Each group of mice was given corresponding drugs or solvents (0.5% carboxymethylcellulose sodium, CMC-Na) by oral gavage from the first day. Among them: Group A: normal control group (NC): 0.5% CMC-Na by gavage and drinking sterilized water.

[0038] Group B: model control group (MC): 0.5% CMC-Na by gavage and drinking 3% DSS water.

[0039] Group C: EPA positive control group (EPA): EPA 60 mg / kg / day by gavage.

[0040] Group D: EPA-TMB low-dose group (LD): EPA-TMB 30 mg / kg / day by gavage.

[0041] Group E: EPA-TMB medium-dose group (MD): EPA-TMB 60 mg / kg / day by gavage.

[0042] Group F: EPA-TMB high-dose group (HD): EPA-TMB 90 mg / kg / day by gavage.

[0043] All dosing lasted for 7 days.

[0044] 2.3, Detection index: After the end of the administration, the experimental endpoint (day 8) was fasted for 12 hours, and blood samples were collected through the orbital venous plexus. After centrifugal separation of the serum, it was frozen at -80 ℃, and then the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were detected by colorimetric kit or automatic biochemical analyzer. The intervention effect of EPA-TMB on DSS-induced inflammatory stress state of blood lipid metabolism disorder was analyzed by comparison between groups, and the dose dependence and difference with positive control were evaluated.

[0045] 1, Experimental results: The experimental results are shown in Table 1.

[0046] Table 1: Serum lipid index detection results of each group of mice From Table 1, we can see that: (1) The total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels of the model control group (group B) were significantly increased (3.8 mmol / L, 2.1 mmol / L, 2.5 mmol / L, respectively), while the high-density lipoprotein cholesterol (HDL-C) was significantly reduced (0.3 mmol / L), indicating that the hyperlipidemia model was successfully induced.

[0047] (2) Compared with the EPA positive control group (group C), EPA-TMB significantly improved the hyperlipidemia of mice in a dose-dependent manner. As the dose of EPA-TMB increased from 30 mg / kg (group D) to 90 mg / kg (group F), the TC level decreased by 42%, 53% and 61% compared with the model group, respectively. The TC value of the high-dose group (90 mg / kg, group F) was close to the normal control group (group A), while the TC value of the EPA group (group C) was only 2.8 mmol / L (decrease of 26%).

[0048] (3) Compared with the EPA positive control group (group C), the high-dose EPA-TMB group (group F) significantly reduced the TG level to 0.6±0.2 mmol / L (decrease of 71%), close to the normal value level, while the EPA group (group C) only decreased to 1.5±0.1 mmol / L (decrease of 29%). Similarly, EPA-TMB was also superior to free EPA in reducing LDL-C and increasing HDL-C levels. For example, the same dose of EPA-TMB reduced LDL-C by 60%, while the EPA group only reduced by 28%. These research results show that EPA-TMB has more advantages than EPA in improving hyperlipidemia, especially the blood lipid level of the high-dose group is close to the normal value, which can become a more efficient lipid-lowering drug candidate.

[0049] Experimental Example Two, Analysis of the Treatment Effect of Eicosapentaenoic Acid-Trimethoxybenzyl Alcohol Ester on Colitis 1. Experimental materials: Eicosapentaenoic acid-trimethoxybenzyl alcohol ester (EPA-TMB) prepared in Example 1.

[0050] 2. Experimental method: 8~10 weeks old male C57BL / 6 mice (body weight 18~22 g) were used, and after 5 days of adaptive feeding, they were randomly divided into five groups (10 mice per group). The mice were standardized in the SPF level animal room (free intake of basic feed and sterilized water). Before the experiment, except for the normal control group (group A), the rest of the mice were induced by drinking 3% (w / v) dextran sulfate sodium (DSS) in sterilized water (pH 6.0) for 7 days to induce acute colitis model.

[0051] 2.2, animal grouping: Each group of mice was given the corresponding drug or solvent (0.5% carboxymethylcellulose sodium, CMC-Na) by oral gavage from the first day. Among them: Group A: normal control group (NC): 0.5% CMC-Na was given by gavage and sterilized water was drunk.

[0052] Group B: model control group (MC): 0.5% CMC-Na was given by gavage and 3% DSS water was drunk.

[0053] Group C: EPA positive control group (EPA): EPA 60 mg / kg / day was given by gavage.

[0054] Group D: EPA-TMB low-dose group (LD): EPA-TMB 30 mg / kg / day was given by gavage.

[0055] Group E: EPA-TMB medium-dose group (MD): EPA-TMB 60 mg / kg / day was given by gavage.

[0056] Group F: EPA-TMB high-dose group (HD): EPA-TMB 90 mg / kg / day was given by gavage.

[0057] All dosing lasted for 7 days.

[0058] 2.3, detection index: 2.2, disease activity index (DAI) monitoring and colon length: During the molding and administration period, all mice were monitored for clinical signs daily. Specifically including: (1) The body weight of each mouse was accurately weighed using an electronic balance, and the body weight change rate was calculated; (2) According to the stool shape and blood in the stool, the score was given, where the stool score rule was 0 points (normal), 2 points (diarrhea), 4 points (watery stool); The blood in the stool score rule is 0 points (no blood), 2 points (occult blood), 4 points (visible blood stool); (3) The above three indicators are combined to calculate the disease activity index (DAI), the formula is: DAI = [body weight loss (%) + stool shape score + blood in stool score] / 3. After the mice were sacrificed at the end of the experiment (8th day), the whole colon was quickly removed, and the colon length from the ileocecal junction to the anus was measured under tension-free conditions.

[0059] 2.2, Biochemical level of EPA-TMB on the level of inflammation in mouse colon tissue: The distal colon tissue (about 50~100 mg) was added to the RIPA lysis buffer containing protease inhibitors, and the homogenate was prepared by a high-speed tissue homogenizer. The homogenate was centrifuged at 12,000×g for 15 minutes at 4 ℃, and the supernatant was collected for subsequent detection. Commercial colorimetric method kit was used to determine the activity of myeloperoxidase (MPO) in colon tissue homogenate to evaluate the degree of neutrophil infiltration; at the same time, the protein concentration of key pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) was quantitatively detected by enzyme-linked immunosorbent assay (ELISA). By comparing the MPO activity and pro-inflammatory factor levels of each experimental group with the model control group, the inhibitory effect of EPA-TMB on local inflammatory response in colon and its mechanism were analyzed.

[0060] 2.3, Evaluation of oxidative stress status and barrier protein expression in colon tissue: Further analysis of colon tissue homogenate. First, the content of lipid peroxidation products was quantitatively determined using a malondialdehyde (MDA) detection kit to reflect the degree of oxidative damage, and the antioxidant capacity of the tissue was evaluated by superoxide dismutase (SOD) activity detection kit; then after extracting total protein, quantifying by BCA method, SDS-PAGE electrophoresis and PVDF membrane transfer, using primary antibodies against tight junction proteins such as Occludin, ZO-1 for immunoblotting, with β-actin as an internal reference, the protein expression intensity was semi-quantitatively analyzed by ECL development and ImageJ software. By integrating the oxidative stress indicators and barrier protein expression data, the protective mechanism of EPA-TMB on colon mucosal barrier function and antioxidant effect were explored.

[0061] 3, Experimental results: 3.1, The results of disease activity index (DAI) monitoring and colon length analysis are shown in Table 2: Table 2 Disease activity index of mice after different group treatment From Table 2, it can be seen that: (1) The disease activity index (DAI) of the model control group (group B) reached 8.2±1.1 points, compared with the normal control group (group A), the colon length was shortened by 44% compared with the normal group, and the body weight loss reached 18.7%, and was accompanied by severe stool shape abnormality and macroscopic blood stool (3.8±0.4 points), which indicated that the DSS-induced model mice showed a significant inflammatory phenotype.

[0062] (2) After EPA-TMB intervention, the inflammation of mice was improved to different degrees. Moreover, EPA-TMB intervention showed a dose-dependent effect, and the inflammation of mice was improved to a greater extent after administration of a higher concentration. Among them, the DAI of the high-dose EPA-TMB group (90 mg / kg, group F) was significantly reduced by 75%, the colon length returned to the normal level, the body weight loss was only 4.5±1.2%, and the blood stool score decreased to 0.9±0.3 points. The DAI of the medium-dose EPA-TMB group (60 mg / kg, group E) and the low-dose EPA-TMB group (30 mg / kg, group D) was reduced by 52% and 32%, respectively. This indicated that EPA-TMB had a significant inhibitory effect on intestinal barrier damage and inflammatory response.

[0063] (3) Although the EPA positive control group (60 mg / kg, group C) could partially alleviate the symptoms (DAI decreased by 23%, and the colon length recovered by 30%), the therapeutic effect was significantly weaker than that of the high-dose EPA-TMB group (90 mg / kg, group F). These results were consistent with the improvement trend of blood lipid indicators, indicating that EPA-TMB could achieve more efficient repair of intestinal structure and function damage. The structural modification of EPA-TMB enhanced its targeting and metabolic stability at the inflammation site, providing a potential optimization strategy for the treatment of ulcerative colitis.

[0064] 3.2, The biochemical level analysis results of the inflammation level of mouse colon tissue are shown in Table 3: Table 3 Biochemical level of mouse colon tissue inflammation level after treatment of different groups From Table 3, it can be seen that: (1) The MPO activity of the colon tissue of the model control group (group B) mice was increased by 18.9 times compared with the normal group, and the concentrations of TNF-a, IL-6 and IL-1b were increased by 10.4, 5.9 and 6.5 times, respectively, indicating that neutrophil infiltration and excessive release of pro-inflammatory factors were the core pathological features of colitis.

[0065] (2) After the intervention of EPA-TMB, the levels of each inflammatory marker showed different degrees of decline, and showed a significant dose-dependent effect. The levels of each marker in the high-dose group of EPA-TMB (90 mg / kg, group F) showed the largest decline, and the MPO activity decreased by 91.5% compared with the model group. The level was only 0.6 times higher than that of the normal control group (group A), and the levels of TNF-α, IL-6 and IL-1β were close to the normal level (only 10-30% higher), indicating that EPA-TMB had good anti-inflammatory effect.

[0066] (3) At the same concentration, the intervention effect of EPA-TMB was 2.5-3.6 times that of EPA, which indicated that the anti-inflammatory effect of EPA-TMB on colitis was significantly stronger than that of EPA.

[0067] 3.3, The results of the analysis of the oxidative stress state and the expression of barrier proteins in the colon tissue are shown in Table 4: Table 4 Effect of different treatments on oxidative stress and expression of tight junction proteins in colon tissue of mice From Table 4, we can know that: (1) The MDA in the model control group (group B) was significantly increased, and the SOD activity was decreased, indicating that oxidative stress was an important pathological mechanism of colitis, and the expression of tight junction proteins Occludin and ZO-1 was greatly reduced, indicating that the mucosal barrier function of mice was damaged.

[0068] (2) After the intervention of EPA-TMB, with the increase of the treatment dose, the level of MDA gradually decreased from 6.7 nmol / mg to 2.3 nmol / mg. At the same time, the activity of SOD increased significantly, and returned to the normal level in the high-dose group of EPA-TMB (90 mg / kg, group F). Interestingly, the EPA positive control group (60 mg / kg, group C) had limited improvement on MDA and SOD, which was only equivalent to the effect of the low-dose group of EPA-TMB (30 mg / kg, group D).

[0069] (3) Regarding barrier repair, the repair effect of the EPA positive control group (Group C) was less than 50% of that of the model control group (Group B). In the EPA-TMB medium-dose group (60 mg / kg, Group E), the expression of Occludin (0.89) and ZO-1 (0.76) had recovered to 90% and 76% of their near-normal levels, respectively. This difference indicates that EPA-TMB reduces oxidative damage by inhibiting lipid peroxidation and enhancing the activity of antioxidant enzymes, while rebuilding the mucosal barrier by upregulating barrier protein expression, thereby blocking the vicious cycle of "oxidative stress-barrier destruction-inflammation amplification". At the same time, the structural modification of EPA-TMB improves its targeting or stability in the intestine, enabling it to activate a series of antioxidant pathways at low doses and achieve more efficient barrier repair by regulating tight junction-related signals.

[0070] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing eicosapentaenoic acid-trimethoxybenzyl alcohol ester, characterized by, The method comprises the following steps: Step S1, adding 3,4,5-trimethoxybenzyl alcohol and eicosapentaenoic acid into a container containing tetrahydrofuran solution, stirring at room temperature for 2-3 min, then adding lipase into the system, stirring at an oil bath temperature of 45-55 ℃ for 35-38 h, filtering, and taking the filter cake; Step S2, washing the filter cake prepared in step S1 with tetrahydrofuran for 3-4 times, collecting the filtrate and washing liquid, combining, and concentrating the combined solution under reduced pressure to obtain an oily crude product; Step S3, purifying the oily crude product prepared in step S2 by silica gel column chromatography, collecting the corresponding fractions, combining, and concentrating under reduced pressure to obtain the product.

2. The method for preparing eicosapentaenoic acid-trimethoxybenzyl alcohol ester as described in claim 1, characterized in that, The molar ratio of 3,4,5-trimethoxybenzyl alcohol to eicosapentaenoic acid in step S1 is 31.0 mmol:34.1 mmol.

3. The method for preparing eicosapentaenoic acid-trimethoxybenzyl alcohol ester as described in claim 1, characterized in that, The feed ratio of 3,4,5-trimethoxybenzyl alcohol to tetrahydrofuran solution in step S1 is (1-2) g:10 mL.

4. The method for preparing eicosapentaenoic acid-trimethoxybenzyl alcohol ester as described in claim 1, characterized in that, The lipase in step S1 is Novozym 435 enzyme.

5. The method for preparing eicosapentaenoic acid-trimethoxybenzyl alcohol ester as described in claim 1, characterized in that, The addition amount of lipase in step S1 is 15-25% w / w of the mass of 3,4,5-trimethoxybenzyl alcohol.

6. The method for preparing eicosapentaenoic acid-trimethoxybenzyl alcohol ester as described in claim 1, characterized in that, The reaction is monitored by thin layer chromatography during the reaction in step S1, and the developing agent of the thin layer chromatography is petroleum ether / ethyl acetate=45:4 v / v, Rf=0.4, and the color developing agent is phosphomolybdic acid.

7. The method for preparing eicosapentaenoic acid-trimethoxybenzyl alcohol ester as described in claim 1, characterized in that, The silica gel column chromatography in step S3 is eluted using a gradient of 100% petroleum ether to ethyl acetate / petroleum ether=6:94 v / v.

8. The eicosapentaenoic acid-trimethoxybenzyl alcohol ester prepared by the method of any one of claims 1-7.

9. The use of the eicosapentaenoic acid-trimethoxybenzyl alcohol ester of claim 8 in the preparation of a drug for preventing or treating hyperlipidemia.

10. The use of the eicosapentaenoic acid-trimethoxybenzyl alcohol ester of claim 8 in the preparation of a drug for preventing or treating colitis.

Images