Application of tannic acid in preparation of anti-atherosclerosis medicine and preparation of tannic acid

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of tannic acid in preparation of an anti-atherosclerosis medicine and a preparation of the tannic acid. The invention finds that the tannic acid has an anti-atherosclerosis effect for the first time through research, and mouse experiments find that the tannic acid can effectively inhibit the formation of ApoE- / -mouse atherosclerosis plaques induced by high fat diet. The tannic acid microcapsule prepared by the preparation method disclosed by the invention has good thermal stability. The microcapsule is stable in structure, the core material is not easy to leak, the tannic acid after microcapsule effectively solves the problem of poor acceptability, and after microencapsulation, the astringent taste of the tannic acid is masked, and the tannic acid is isolated from the external environment, so that the biological activity of the tannic acid is favorably maintained, and the processing and utilization of the tannic acid are facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of tannic acid in the preparation of anti-atherosclerotic drugs and its formulation. Background Technology

[0002] Tannic acid, CAS: 1401-55-4, chemical formula C 76 H 52 O 46 Tannic acid, also known as tannin, is a colorless or yellowish-brown powder with a complex structure. It is well-soluble in water and ethanol, has a strong astringent taste, and is very low in toxicity. It is abundant, found in plants such as gallnut and pomegranate, and is currently most widely used in industrial leather making. The reducing properties, free radical scavenging activity, and numerous derivatization reactions of tannic acid have also led to significant advancements in its applications in the medical field. Recent studies have found that tannic acid not only possesses antibacterial, antiviral, anti-inflammatory, and antioxidant effects, and has a certain inhibitory effect on cancer and tumors, but also exhibits physiological activities such as lowering blood sugar and blood pressure.

[0003] Tannic acid itself has a strong astringent taste, is highly water-soluble, and is easily affected by external environmental factors such as light, temperature, humidity, and oxygen. It is also easily oxidized and hydrolyzed, which limits its practical application and its bioavailability needs to be improved. Furthermore, there is no existing record of using tannic acid in the treatment of atherosclerosis. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an application of tannic acid in the preparation of lipid-lowering and anti-atherosclerotic drugs.

[0005] Another object of the present invention is to provide tannic acid microcapsules for lowering blood lipids and preventing atherosclerosis.

[0006] The present invention further provides a method for preparing the above-mentioned tannic acid microcapsules.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides the application of tannic acid in the preparation of lipid-lowering and anti-atherosclerotic drugs.

[0008] This invention also provides a tannic acid microcapsule for lowering blood lipids and preventing atherosclerosis, the tannic acid microcapsule being composed of the following components: Core materials: tannic acid, EM90, kelp extract; Wall material: Shiitake mushroom protein, inulin, mixed alcohol.

[0009] Preferably, in the wall material, the mixed alcohol is composed of ethanol and glycerol in a volume ratio of 3:1.

[0010] This invention also provides a method for preparing the above-mentioned tannic acid microcapsules, comprising the following steps: (1) After dissolving tannic acid in water, an aqueous solution of tannic acid is obtained. Then EM90 is added and stirred evenly. Then kelp extract is added and stirred evenly again to obtain a core material solution for later use. (2) After mixing shiitake mushroom protein, inulin and mixed alcohol evenly, a wall material solution is obtained; (3) The core material solution is slowly added to the wall material solution, stirred evenly, and then subjected to high-speed homogenization and vacuum drying to obtain tannic acid microcapsules.

[0011] Preferably, in step (1), the mass ratio of tannic acid to water is 1-10:80; the amount of EM90 added accounts for 0.2% of the mass of the tannic acid aqueous solution; and the amount of kelp extract added accounts for 0.5-1% of the mass of the tannic acid aqueous solution.

[0012] Preferably, in step (2), the mass ratio of shiitake mushroom protein, inulin and mixed alcohol is 1-2:0.5:50-60.

[0013] Preferably, in step (3), the mass ratio of the core material solution to the wall material solution is 16:84.

[0014] The kelp extract used in this invention is prepared by the following method: fresh kelp is pulped, saturated lime water (solid-liquid ratio 1 g: 5 mL) is added to the pulp, and after stirring evenly, 0.5-1 mol / L NaOH solution (solid-liquid ratio 1 g: 25 mL) is added again, and the mixture is stirred at a constant temperature of 40-60℃ for 1-2 h. Then, acetic acid is added to neutralize to pH 6-7 to obtain the kelp extract.

[0015] The beneficial effects of this invention are as follows: (1) This invention is the first to discover that tannic acid has an anti-atherosclerotic effect. Through mouse experiments, it was found that tannic acid can effectively inhibit the formation of atherosclerotic plaques induced by a high-fat diet in ApoE- / - mice.

[0016] (2) The tannic acid microcapsules prepared by this invention have good thermal stability. Moreover, the microcapsule structure is stable and the core material is not easy to leak. The tannic acid after microencapsulation effectively solves the problem of poor acceptability. In addition, after microencapsulation, its astringent taste is masked and the contact between tannic acid and the external environment is isolated, which is conducive to maintaining the biological activity of tannic acid and helps the processing and utilization of tannic acid. Attached Figure Description

[0017] Figure 1 Effects of tannic acid on the activity of THP-1 macrophages; Figure 2The effect of tannic acid on ox-LDL-induced foaming of THP-1 macrophages; Figure 3 Effects of tannic acid co-incubation on the ability of THP-1 macrophages to take up ox-LDL; Figure 4 Tannins affect lipid metabolism factors in THP-1 macrophages ( ABCA1 , ABCG1 , CD36 , MSR The effect of mRNA expression; Figure 5 Tannic acid affects ox-LDL-induced lipid metabolism factors in THP-1 macrophages ( ABCA1 , ABCG1 , CD36 , MSR The effect of mRNA expression; Figure 6 Effects of tannic acid pretreatment on the ability of THP-1 macrophages to take up ox-LDL; Figure 7 Effects of tannic acid on blood lipids (total cholesterol TC, triglycerides TG, LDL, HDL) in mice with acute hyperlipidemia; Figure 8 Tannic acid's effect on ApoE - / - The effects of atherosclerotic lesions in the aorta of mice. Detailed Implementation

[0018] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0019] Example 1: Effect of tannic acid on the transformation of macrophages into foam cells Experimental methods: (1) Cell culture: Human THP-1 mononuclear cells were purchased from the China National Biomedical Experimental Cell Resource Bank. All cells were cultured in a cell culture incubator at 37°C with 5% CO2 and maintained in RPMI 1640 medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0020] (2) Cell differentiation induction: THP-1 monocytes were induced to differentiate into macrophages by 100 nM PMA for 48 h.

[0021] (3) Cell viability detection: The effect of tannic acid on the cell viability of THP-1 macrophages was detected by CCK-8 assay.

[0022] (4) RNA extraction and RT-PCR qPCR: Total RNA was extracted using the Biotech High Purity Total RNA Rapid Extraction Kit (both 260 / 230 and 260 / 280 RNA were between 1.8 and 2.0 μg). 0.8–1 μg of RNA was reverse transcribed into cDNA using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. RT-PCR was performed using an ABI 7500 real-time quantitative PCR instrument. q PCR.

[0023] (5) High-content analysis of macrophage foaming: THP-1 monocytes were seeded in 96-well plates (Costar 3603) with black walls and clear bottoms and induced to differentiate into macrophages. They were exposed to tannic acid (0.6-10 μM) alone for 48 h. After compound exposure, the cells were washed three times with PBS and incubated with oxLDL (30 μg / mL). After incubation, the cells were washed three times with PBS at room temperature, then fixed with 4% formaldehyde for 10 min, permeabilized with 0.1% Triton for 10 min, stained with BODIPY at room temperature in the dark for 10 min, and stained with Hoechst 33342 for 10 min. Fluorescence images were collected and analyzed using the Thermo Scientific™ CellInsight™ CX7 high-content analysis platform. Fluorescence images of 16 fields of view were collected from each well, including at least 7000 cells for fluorescence signal quantification analysis. The average BODIPY fluorescence intensity was obtained using HCS Studio 3.0 software to characterize lipid deposition in THP-1 macrophages.

[0024] (6) High-content analysis of macrophage uptake of oxLDL: The cells were exposed to tannic acid (0.6-10 μM) alone for 48 h. After exposure, the cells were washed three times with PBS and incubated with 30 μg / mL Dil-oxLDL working solution for 6 h. After incubation, the cells were washed three times with PBS, then fixed with 4% formaldehyde for 10 min, permeabilized with 0.1% Triton for 10 min, and stained with Hoechst 33342 for 10 min. Fluorescence images were acquired and analyzed using the ThermoScientific™ CellInsight™ CX7 high-content analysis platform. Fluorescence images of 16 fields of view were acquired from each well, including at least 7000 cells for fluorescence signal quantification analysis. The average Dil-oxLDL fluorescence intensity per well was determined using HCS Studio 3.0 software to accurately quantify the Dil-oxLDL uptake by THP-1 macrophages in that well.

[0025] Results and Analysis: Using the CCK-8 assay, the maximum safe concentration of tannic acid at the cellular level was determined to be 10 μM. Figure 1High-content analysis showed that treatment with 2.5-10 μM tannic acid significantly inhibited ox-LDL-induced foaming of THP-1 macrophages in a concentration-dependent manner compared to the model group. Figure 2 This indicates that tannic acid has a good potential to inhibit the transformation of macrophages into foam cells. Dil-ox-LDL cell uptake experiments showed that after co-incubation with 0.6-10 μM tannic acid and Dil-ox-LDL, the fluorescence signal of Dil-ox-LDL in THP-1 macrophages was significantly reduced compared to the untreated model group. Figure 3 This indicates that tannic acid can effectively inhibit the uptake of ox-LDL by macrophages. At the molecular level, the molecular mechanism by which tannic acid inhibits foam cell formation was preliminarily explored. RT-qPCR analysis showed that, compared to the control group, tannic acid treatment alone significantly reduced ox-LDL uptake in THP-1 macrophages. CD36 and MSR ) and cholesterol excretion ( ABCA1 and ABCG1 The mRNA expression levels of related genes did not change significantly. Figure 4 After Ox-LDL treatment, THP-1 macrophages CD36 and MSR The mRNA level is elevated, while ABCA1 and ABCG1 The mRNA level decreased, but tannic acid treatment had no significant effect on this change. Figure 5 Consistent with the above molecular-level results, tannic acid pretreatment for 24 h had no significant effect on the ability of THP-1 macrophages to take up ox-LDL. Figure 6 The above results indicate that tannic acid can effectively inhibit the transformation of macrophages into foam cells under ox-LDL treatment, which is speculated to be related to the direct binding of ox-LDL to macrophages, thereby inhibiting the uptake of ox-LDL by macrophages.

[0026] Example 2: Effects of tannic acid on blood lipids in mice with acute hyperlipidemia Experimental methods: Eight-week-old male C57BL / 6 mice were housed in an SPF-grade animal facility at a temperature of 20-26°C and a humidity of 40%-70%, with 12 hours of light and 12 hours of darkness. The mice had free access to food and water.

[0027] (1) Mouse grouping: C57BL / 6 mice were randomly divided into control group (n=6), model group (n=6), and tannic acid administration group (n=6) after 7 days of acclimatization.

[0028] (2) Mouse modeling: 15 mL of fresh egg yolk was mixed with 5 mL of physiological saline to prepare 75% egg yolk emulsion. The emulsion was prepared fresh each time it was used. 0.5 mL was injected into each mouse via intraperitoneal injection.

[0029] (3) Mouse administration: Tannic acid (50 mg / kg) was administered via tail vein injection 20 h after injection of 75% egg yolk emulsion.

[0030] (4) Blood collection from mice: Blood was collected by enucleation 3 h after drug administration. After blood collection, the mice were allowed to stand for 10 min, centrifuged at 3000 rpm for 10 min, and the supernatant was aliquoted and stored at -80℃.

[0031] (5) Detection indicators: HDL-C and LDL-C were detected using a kit.

[0032] Results and Analysis: Accumulation of lipid components such as cholesterol and triglycerides in the blood is an important pathological mechanism in the development of atherosclerosis. This invention established an acute hyperlipidemic mouse model using 75% egg yolk emulsion and administered tannic acid (50 mg / kg) via tail vein injection. Results showed that after administration, serum total cholesterol, triglycerides, LDL, and HDL cholesterol in the hyperlipidemic mice were significantly reduced. Figure 7 This indicates that tannic acid has the potential to lower blood lipid levels after entering the bloodstream.

[0033] Example 3: Tannins and ApoE - / - Effects of atherosclerotic lesions in mice Experimental methods: 8-week-old male ApoE - / - Mice were housed in an SPF-grade animal facility at a temperature of 20-26°C and a humidity of 40%-70%, with 12 hours of light and 12 hours of darkness. Mice had free access to food and water, and their diet consisted of a high-fat rat diet (21% milk fat, 0.15% cholesterol) that promotes atherosclerosis.

[0034] (1) Mouse grouping: After 1 week of acclimatization, ApoE mice were grouped into groups. - / - Mice were randomly divided into three groups: a model group (physiological saline), and low (5 mg / kg) and high (25 mg / kg) dose groups of tannic acid.

[0035] (2) Mouse administration: The mice were fed a high-fat diet (MD12017) and simultaneously administered the drug via intraperitoneal injection at a ratio of 0.1 mL / 10 g. The administration frequency was once a day for 8 consecutive weeks.

[0036] (3) Mouse tissue collection: Blood was collected by enucleating the eyeballs after treatment. After blood collection, the blood was allowed to stand for 10 min, centrifuged at 3000 rpm for 10 min, and the supernatant was aliquoted and stored at -80℃. The aorta was then separated after perfusion until the liver turned white.

[0037] (4) Lipid plaque analysis: The mouse aorta was longitudinally sectioned and stained with Oil Red O, photographed under a light microscope, and quantitatively analyzed using ImageJ software to assess the formation of atherosclerotic plaques in the aorta.

[0038] Results and Analysis: Based on the results of Examples 1 and 2, an ApoE was constructed using a pro-atherosclerotic high-fat diet. - / - A mouse model of atherosclerosis was established, and the effects of tannic acid on plaque formation in mice were investigated. Mice were fed a high-fat diet and then administered intraperitoneal injections of tannic acid at doses of 5 mg / kg / d and 25 mg / kg / d for 60 consecutive days. The control group received an equal volume of physiological saline. After the administration period, mice were sacrificed, and the aorta was dissected for pathological analysis. Gross Oil Red O staining results showed that, compared to the model group, the high-dose group (25 mg / kg / d) and the low-dose group (5 mg / kg / d) showed a decreasing trend in vascular lipid plaque formation. Figure 8 This indicates that tannic acid can effectively inhibit ApoE induced by a high-fat diet. - / - Atherosclerotic plaque formation in mice.

[0039] Example 4 Microcapsule Optimization Preparation method of tannic acid microcapsules: (1) Fresh kelp is pulped, saturated lime water (solid-liquid ratio 1 g: 5 mL) is added to the pulp, and after stirring evenly, 0.5 mol / L NaOH solution (solid-liquid ratio 1 g: 25 mL) is added again. The mixture is stirred at a constant temperature of 50℃ for 1.5 h, and then acetic acid is added to neutralize to pH 7 to obtain kelp extract.

[0040] (2) After dissolving tannic acid in 15 times the amount of water, an aqueous solution of tannic acid is obtained. Then, 0.2% of EM90 based on the mass of the aqueous solution of tannic acid is added and stirred evenly. Then, 1% of kelp extract based on the mass of the aqueous solution of tannic acid is added and stirred evenly again to obtain a core material solution for later use. (3) Mix shiitake mushroom protein, inulin and mixed alcohol in a mass ratio of 1.8:0.5:55 to obtain a wall material solution; (4) The core material solution is slowly added to the wall material solution (core material: wall material = 16:84), stirred evenly, and then subjected to high-speed homogenization and vacuum drying to obtain tannic acid microcapsules.

[0041] Comparative Example 1 Preparation method of tannic acid microcapsules: (1) After dissolving tannic acid in 15 times the amount of water, an aqueous solution of tannic acid is obtained. Then, 0.2% of EM90 based on the mass of the aqueous solution of tannic acid is added and stirred evenly. Then, 1% of guar gum based on the mass of the aqueous solution of tannic acid is added and stirred evenly again to obtain a core material solution for later use. (2) Mix shiitake mushroom protein, inulin and mixed alcohol in a mass ratio of 1.8:0.5:55 to obtain a wall material solution; (3) The core material solution is slowly added to the wall material solution (core material: wall material = 16:84), stirred evenly, and then subjected to high-speed homogenization and vacuum drying to obtain tannic acid microcapsules.

[0042] Effect verification (a) The encapsulation efficiency and drug loading of the microcapsules prepared in Example 4 and Comparative Example 1 were tested. The specific testing methods are as follows: 1. Plotting the standard curve of tannic acid (1) Accurately weigh 10 mg of tannic acid standard and dilute to 100 mL with deionized water to obtain a 0.1 mg / mL standard stock solution.

[0043] (2) Take 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL of stock solution into 10 mL colorimetric tubes respectively, add water to 5 mL, add 0.5 mL of Folin-phenol reagent, shake well and let stand for 3 min, then add 2 mL of 10% Na2CO3 solution, add water to make up to 10 mL, and react in the dark for 60 min.

[0044] (3) Using blank reagent (5 mL water + 0.5 mL Folin-phenol + 2 mL Na2CO3) as a reference, the absorbance (A) was measured at a wavelength of 765 nm. A standard curve was plotted with tannic acid concentration (C, μg / mL) as the abscissa and A as the ordinate. The regression equation was obtained as A = 0.0082C + 0.012, R² ≥ 0.995.

[0045] 2. Determination of free tannic acid content (W1) (1) Accurately weigh 1.0 g of tannic acid microcapsule sample, add 50 mL of deionized water, and stir magnetically for 30 min (25℃, 300 r / min) to fully dissolve the free tannic acid on the surface.

[0046] (2) Take the above suspension, centrifuge at 8000 r / min for 20 min, take 1 mL of supernatant, measure the absorbance according to the colorimetric method of "standard curve plotting", substitute it into the regression equation to calculate the tannic acid concentration in the supernatant, and then calculate the total free tannic acid content W1 (mg) in the sample.

[0047] 3. Total tannin content (W) 总 ) Measurement (1) Accurately weigh the same mass of microcapsule sample as in step (2), add 50 mL of 70% ethanol solution, and ultrasonically break it for 30 min (power 200 W, temperature 40℃) to destroy the wall material structure and release all tannic acid.

[0048] (2) Centrifuge at 10000 r / min for 15 min, take 1 mL of the supernatant, dilute to a suitable concentration, and determine the absorbance using the colorimetric method. Calculate the total tannic acid content W in the sample. 总 (mg).

[0049] 4. Calculation formula (1) Encapsulation efficiency (EE, %) = (W 总 -W1) / W 总 ×100% (2) Drug loading (LC, %) = (W 总 -W1) / Microcapsule sample mass (g) × 100% The specific results are shown in Table 1.

[0050] Table 1

Claims

1. Use of tannin in the preparation of a medicine for reducing blood lipid and anti-atherosclerosis.

2. A tannic acid microcapsule for reducing blood lipids and anti-atherosclerosis, characterized by, The tannin microcapsule is composed of the following components: Core material: tannin, EM90, kelp extract; Wall material: lentinan protein, inulin, mixed alcohol.

3. The tannin acid microcapsule according to claim 2, characterized by, In the wall material, the mixed alcohol is composed of ethanol and glycerol in a volume ratio of 3:

1.

4. A process for the preparation of tannin microcapsules as claimed in claim 2 or 3, characterised in that, The following steps are included: (1) Dissolve tannin in water to obtain a tannin aqueous solution, then add EM90, stir until uniform, then add kelp extract, stir again until uniform, and obtain a core material solution for standby; (2) Mix lentinan protein, inulin and mixed alcohol uniformly to obtain a wall material solution; (3) Slowly add the core material solution to the wall material solution, stir until uniform, then perform high-speed homogenization treatment, vacuum drying, and obtain the tannin microcapsule.

5. The production method according to claim 4, characterized by, In step (1), the mass ratio of tannin to water is 1-10:80; the addition amount of EM90 accounts for 0.2% of the mass of the tannin aqueous solution; the addition amount of kelp extract accounts for 0.5-1% of the mass of the tannin aqueous solution.

6. The preparation method according to claim 4, characterized in that, In step (2), the mass ratio of lentinan protein, inulin and mixed alcohol is 1-2:0.5:50-60.

7. The method of any one of claims 4-6, wherein, In step (3), the mass ratio of the core material solution to the wall material solution is 16:84.

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