Polyphenol microcapsule, preparation method and application thereof

Abstract

The present invention discloses a polyphenol microcapsule, a preparation method and application thereof, and belongs to the technical field of nanomaterials. The polyphenol microcapsule of the present invention is prepared by emulsifying plant polyphenols as core materials, sodium alginate and chitosan as wall materials, and emulsifying with an emulsifier. The plant polyphenol-sodium alginate / chitosan nanocomposite microcapsules prepared by the present invention have a high encapsulation rate and a low particle size, which not only improves the stability of plant polyphenols and plays a sustained release effect, but also effectively improves the antioxidant and anti-allergic effects of plant polyphenols through synergistic enhancement.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and in particular relates to a polyphenol microcapsule, a preparation method and application thereof. Background Art

[0002] Plant polyphenols are antioxidant substances in nature, such as epicatechin gallate (EGCG), which have anti-inflammatory, anti-allergic, anti-tumor, and immune regulation effects, and are widely used in food, medicine, and cosmetics. However, natural plant polyphenols have problems such as poor stability, poor solubility, and low bioavailability. They are easily degraded during processing, which seriously restricts the application of plant polyphenols in the food industry. Therefore, improving the stability of plant polyphenols and achieving efficient utilization is a current focus. Summary of the invention

[0003] The present invention aims to construct plant polyphenol-sodium alginate / chitosan nanocomposite microcapsules with high encapsulation rate and low particle size using plant polyphenols as core materials and sodium alginate and chitosan as wall materials, so as to improve the stability of plant polyphenols, play a sustained release role, and achieve effective improvement of antioxidant and anti-allergic effects through synergistic enhancement.

[0004] The technical solution of the present invention is as follows:

[0005] The present invention provides a method for preparing polyphenol microcapsules, comprising the following steps:

[0006] (1) Preparation of sodium alginate / chitosan microcapsule wall materials

[0007] The calcium chloride solution and the sodium alginate solution are mixed and stirred; then the chitosan solution is added, stirred, the pH is adjusted, centrifuged, the nanoparticles are washed, and freeze-dried to obtain the sodium alginate / chitosan microcapsule wall material;

[0008] (2) Preparation of plant polyphenol-sodium alginate / chitosan microcapsules

[0009] The microcapsule wall material is dissolved in water to form a microcapsule wall material solution; then plant polyphenols and an emulsifier are added, stirred, centrifuged, the nanoparticles are washed, and freeze-dried to obtain plant polyphenol-sodium alginate / chitosan microcapsules.

[0010] In the above-mentioned method for preparing polyphenol microcapsules, the mass ratio of calcium chloride to sodium alginate is selected from 1:1 to 1:10, preferably 1:5.

[0011] In the above-mentioned method for preparing polyphenol microcapsules, the mass ratio of chitosan to sodium alginate is selected from 1:2 to 1:10, preferably 1:6.

[0012] In the above-mentioned method for preparing polyphenol microcapsules, the mass ratio of plant polyphenols to sodium alginate is selected from 1:1 to 1:5, preferably 1:3.

[0013] In the above-mentioned method for preparing polyphenol microcapsules, the emulsifier is selected from one or more of Tween 80, lecithin, lanolin, and gum arabic. The concentration of the emulsifier is selected from 0.1% to 5%, preferably 1%.

[0014] In the above method for preparing polyphenol microcapsules, the pH is selected from 2 to 7, preferably 5.5.

[0015] In the above-mentioned method for preparing polyphenol microcapsules, the centrifugation condition is selected from: 1000-5000 r / min for 5-20 min; preferably: 3000 r / min for 10 min.

[0016] In the above-mentioned method for preparing polyphenol microcapsules, the plant polyphenols can be selected from one or more of epicatechin gallate, chlorogenic acid, quercetin, anthocyanin, ellagic acid, arbutin, and quercetin; preferably, epicatechin gallate.

[0017] The invention provides polyphenol microcapsules prepared by the method.

[0018] The present invention provides the use of the polyphenol microcapsules in preparing preparations with anti-oxidation and / or anti-allergic effects.

[0019] The beneficial effects of the present invention are:

[0020] The present invention uses plant polyphenols as core materials, sodium alginate and chitosan as wall materials, and constructs plant polyphenol-sodium alginate / chitosan nanocomposite microcapsules with high encapsulation rate and low particle size, which not only improves the stability of plant polyphenols and plays a sustained release effect, but also effectively improves the antioxidant and anti-allergic effects of plant polyphenols through synergistic enhancement. Sodium alginate is a natural water-soluble acid-resistant anionic polysaccharide extracted from brown algae, which has strong biocompatibility and biodegradability. Chitosan is a natural cationic polysaccharide obtained by deacetylation of chitin, which is a non-toxic and tasteless polysaccharide, and can be decomposed into amino acids to be absorbed by the human body, and has high safety. Selecting sodium alginate and chitosan as load materials to encapsulate plant polyphenols is not only simple to operate, green and harmless, and has high economic benefits, but also has significant effects in sustained release, antioxidant and anti-allergic aspects, and can lay a certain foundation for the subsequent development of plant polyphenol products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effects of different mass ratios of chitosan and sodium alginate on the particle size (A), Zeta potential (B) and PDI (C) of microcapsules;

[0022] Figure 2The effect of different mass ratios of epicatechin gallate and sodium alginate on the encapsulation efficiency (A) and particle size (B) of microcapsules;

[0023] Figure 3 The scanning electron micrographs of epicatechin gallate-sodium alginate / chitosan microcapsules at different magnifications are shown;

[0024] Figure 4 is the release amount of epicatechin gallate in the simulated gastrointestinal fluid system;

[0025] Figure 5 DPPH scavenging capacity and ABTS of empty microcapsules, free EGCG and microcapsules with different EGCG-alginate / chitosan mass ratios + Clearing ability;

[0026] Figure 6 The effects of empty microcapsules, free EGCG and the optimal ratio of epicatechin gallate-sodium alginate / chitosan microcapsules (EGCG microcapsule group) on mouse serum specific IgE, IgG, IgG1, histamine and mMCP-1;

[0027] Figure 7 This is the effect of epicatechin gallate microcapsules on the duodenal tissue of mice; A: PBS group; B: TM group; C: EGCG group; D: EGCG microcapsule group; E: empty microcapsule group. DETAILED DESCRIPTION

[0028] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0029] Example 1

[0030] Polyphenol microcapsules were prepared using epicatechin gallate as the core material as follows:

[0031] (1) Preparation of sodium alginate / chitosan microcapsule wall materials

[0032] Take 6 groups of sodium alginate aqueous solutions (concentration of 1 mg / mL), 10 mL in each group, add 2.5 mL of calcium chloride solution (concentration of 0.8 mg / mL) respectively, so that the mass ratio of calcium chloride to sodium alginate is 1:5; stir with a magnetic stirrer at 3000 r / min for 20 min, then add 2.5 mL of chitosan solution of different concentrations to each solution system, so that the mass ratio of chitosan to sodium alginate (CS / SA) is 1:2, 1:4, 1:6, 1:8 and 1:10 respectively, stir for 20 min, and adjust the pH value to 5.5; then centrifuge the solution at 3000 r / min for 10 min, wash the nanoparticles with ultrapure water, freeze the washed nanoparticles in a -80°C ultra-low temperature refrigerator for 4 h, and then freeze-dry for 48 h to obtain microcapsule wall materials.

[0033] The particle size, PDI and Zeta of the empty microcapsules were analyzed to select the optimal microcapsule wall material, such as Figure 1 As shown:

[0034] Figure 1 The effect of CS / SA mass ratio on the particle size (Figure A), Zeta potential (Figure B) and PDI (Figure C) of microcapsule wall materials is shown. Figure 1 (A) It can be seen that the particle size of the microcapsule wall material ranges from 588 to 806 nm. When the CS / SA mass ratio increases from 1:2 to 1:10, the particle size first decreases and then increases. When the CS / SA mass ratio is 1:6, the minimum particle size is 588 nm. Figure 1 (B) It can be seen that the Zeta potential increases with the increase of the ratio of chitosan to sodium alginate. Figure 1 (C) It can be seen that the polydispersity index (PDI) decreases with the increase of the ratio of chitosan to sodium alginate. From the above results, it can be seen that when the mass ratio of chitosan to sodium alginate is 1:6, the particle size of the microcapsule wall material is the smallest, and the polydispersity index (PDI) is also small, about 0.156, so that the prepared nanoparticles are uniform and stable. Based on the above results, when the CS / SA mass ratio is 1:6, uniform, stable, and small-size nanoparticles can be prepared.

[0035] Epigallocatechin gallate (EGCG) is encapsulated with the microcapsule wall material having the best performance, as shown below.

[0036] (2) Preparation of epicatechin gallate-sodium alginate / chitosan microcapsules

[0037] The microcapsule wall material is dissolved in water to form a microcapsule wall material aqueous solution with a concentration of 0.5 mg / mL; 20 mL of the microcapsule wall material aqueous solution is added with epicatechin gallate so that the mass ratios of epicatechin gallate to sodium alginate are 1:1, 1:2, 1:3, 1:4 and 1:5, respectively, and then 1% Tween 80 is added, and the mixture is stirred at 1000 r / min; the solution is then centrifuged at 3000 r / min for 10 min, and the nanoparticles are washed with ultrapure water, the washed nanoparticles are placed in a -80°C ultra-low temperature refrigerator for 4 h, and then freeze-dried for 48 h to obtain nano-microcapsules encapsulating epicatechin gallate.

[0038] 1. Determination of physical indicators of microcapsules

[0039] 1. Particle size

[0040] The average particle size of the microcapsules was measured using a Malvern particle size analyzer.

[0041] 2. Encapsulation rate

[0042] First, the microcapsule dispersion was centrifuged at low speed to remove insoluble matter. 0.5 mL of the microcapsule dispersion was mixed with 1 mL of ethyl acetate, vortexed for 1 min, and then centrifuged to obtain the supernatant, and epicatechin gallate was extracted from the microcapsules into ethyl acetate. The supernatant was collected, dried with nitrogen, and then dissolved with acetonitrile. The EGCG content was detected using an HPLC system. The detection conditions were: the chromatographic column was a Sepax GP-C18 column (5 μm, 4.6 mm × 250 mm), the column temperature was 30 ° C, the flow rate was 1 mL / min, the mobile phase was acetonitrile-water solution (acetonitrile: water = 55:45, v / v), the detection wavelength was 305 nm, and the injection volume was 10 μL. The encapsulation efficiency of epicatechin gallate was calculated according to the following formula: Encapsulation efficiency % = C1 / C0×100%; wherein C0 is the content of epicatechin gallate added when preparing the sample, and C1 is the content of epicatechin gallate extracted from the nanoparticle dispersion and detected.

[0043] The test results of average particle size and encapsulation efficiency of microcapsules, such as Figure 2 As shown:

[0044] As the mass ratio of EGCG to sodium alginate decreases from 1:5 to 1:1, the encapsulation efficiency and particle size of EGCG show a downward trend. In the process of mass ratio from 1:5 to 1:3, the encapsulation efficiency does not decrease significantly, but the particle size becomes significantly smaller. When the mass ratio of EGCG to sodium alginate is 1:3, the encapsulation efficiency is as high as 86.6%. Therefore, the mass ratio of EGCG to sodium alginate of 1:3 can be used as the optimal condition for preparing microcapsule nanoparticles with high encapsulation efficiency and small particle size.

[0045] 3. Scanning electron microscope

[0046] Epicatechin gallate-sodium alginate / chitosan microcapsules (EGCG and sodium alginate mass ratio 1:3) were analyzed by scanning electron microscopy to determine the construction of the microcapsules.

[0047] The scan results are as follows Figure 3 As shown:

[0048] The microcapsules aggregate as a whole, and as the magnification increases, it can be clearly seen that the microcapsules are in a smooth round or oval state, which further demonstrates that the present invention successfully prepares epicatechin gallate-sodium alginate / chitosan microcapsules.

[0049] 2. In vitro simulated gastrointestinal fluid digestion experiment

[0050] 500 mg of EGCG microcapsules were placed in 50 mL of simulated gastric juice (2 mg / mL NaCl solution, 1000 U / mL pepsin), the pH was adjusted to 1.2, and the digestion was carried out in a 37°C constant temperature water bath with continuous shaking for 60 min. 0.5 mL of sample was taken at 15, 30, 45, and 60 min, and the amount of EGCG in the sample was extracted with petroleum ether, and the release amount of EGCG in each segment was calculated using a spectrophotometer.

[0051] The EGCG mixture after digestion in gastric juice for 60 minutes was transferred to 50 mL of simulated intestinal fluid (5 mg / mL K2HPO3, 1 mg / mL bile salt and 500 U / mL trypsin) for mixed incubation, adjusted to pH 7.4, and continuously shaken in a constant temperature water bath at 37°C for digestion for 240 minutes. 0.5 mL of samples were taken every 30 minutes to analyze the release of EGCG at each stage.

[0052] The test results are as follows Figure 4 As shown:

[0053] According to the digestion time of the human gastrointestinal tract, the digestion in gastric juice was set to 60 minutes and the digestion in intestinal juice was set to 240 minutes. Within 60 minutes, the release of EGCG in gastric juice was low, about 8.05%, and most of it remained in the intestine for absorption and utilization. The main reason is that sodium alginate can inhibit the activity of pepsin, thereby reducing the decomposition of EGCG microcapsules in the stomach, and the pH value in the gastric juice environment is close to the pKa of sodium alginate, so a dense and compact network particle structure will be formed, thereby delaying the release of EGCG in gastric juice. In addition, sodium alginate and chitosan can form a dense composite structure to prevent the destruction of microcapsules. 60 to 300 minutes is the intestinal digestion stage, and the release of EGCG begins to increase rapidly, and the final release reaches 90.7%. The possible reason is that the pH value in the intestine tends to be neutral, the EGCG microcapsules will degrade, and the surface wall material will be destroyed. Therefore, a higher EGCG release occurs during the digestion process of simulated intestinal juice, thereby improving the digestion, absorption and utilization rate of EGCG.

[0054] 3. Antioxidant Activity Determination

[0055] 1. Determination of DPPH free radical scavenging ability

[0056] Dissolve DPPH in 95% ethanol solution to prepare 0.1mmol / L DPPH·solution. Mix 4mL sample or deionized water (blank control group) with 4mL DPPH·working solution thoroughly, incubate at room temperature in the dark for 60min, and use a visible spectrophotometer to detect the absorbance of the reaction solution at 517nm.

[0057] Calculate DPPH clearance rate (%), the formula is as follows:

[0058] DPPH·scavenging percentage(%)=A c -(A s -A0) / A0×100%; where: A s is the absorbance value of the sample reaction solution; A c is the absorbance value of the blank control group; A0 is the absorbance value of the sample itself (consisting of 4 mL of sample and 4 mL of ethanol solution).

[0059] 2. ABTS + Free radical scavenging ability assay

[0060] Mix 5mmol / L ABTS·water solution and 5mmol / L K2S2O4 in equal volumes and react at room temperature in the dark for 24 hours to obtain ABTS+·working solution. Mix 150μL sample or ethanol (blank control group) with 4mL ABTS·working solution thoroughly, incubate at room temperature in the dark for 10min, and use a visible light spectrophotometer to detect the absorbance of the reaction solution at 734nm.

[0061] Calculate ABTS+ clearance rate (%), the formula is as follows:

[0062] DPPH·scavenging percentage(%)=A c -(A s -A0) / A0×100%; where: A s is the absorbance value of the sample reaction solution; A c is the absorbance value of the blank control group; A0 is the absorbance value of the sample itself (composed of 4 μL sample and 4 mL 95% ethanol solution).

[0063] The test results are as follows Figure 5 As shown:

[0064] The scavenging ability of free EGCG for DPPH· was 63.67%. After the construction of EGCG microcapsules, the scavenging ability of DPPH· gradually increased with the increase of the mass ratio of EGCG / sodium alginate. When the mass ratio of EGCG / sodium alginate was 1:3, the scavenging ability of EGCG microcapsules for DPPH· was 89.73%. With the further increase of the mass ratio of EGCG / sodium alginate, the change was not obvious. Similarly, the scavenging ability of free EGCG for ABTS+· was 60.33%. When the mass ratio of EGCG / sodium alginate was 1:3, the scavenging rate of EGCG microcapsules for ABTS+· was 80.62%. Overall, the antioxidant activity of empty microcapsules was low. The above results fully demonstrate that EGCG has strong antioxidant activity, and after EGCG is constructed into microcapsules, the antioxidant activity of EGCG can be further enhanced and exerted.

[0065] 4. Anti-allergic activity study

[0066] Mouse immunization protocol:

[0067] 6-8 week old SPF Balb / c mice were used as the research subjects. Antarctic krill tropomyosin (TM) was used as the allergen to sensitize mice and establish a mouse allergy model. The mice were divided into PBS negative control group (healthy group), TM positive control group, EGCG group, EGCG microcapsule group and empty microcapsule group, with 10 mice in each group. After one week of natural feeding, the TM positive control group, EGCG group, EGCG microcapsule group (EGCG / sodium alginate mass ratio of 1:3 group) and empty microcapsule group were injected with TM and alum adjuvant mixture (TM 200μg / mouse) on days 0, 7, 14 and 21, respectively. The PBS negative control group mice were injected with PBS and alum adjuvant mixture as negative control. The EGCG group, EGCG microcapsule group and empty microcapsule group were injected with EGCG, EGCG microcapsule and empty microcapsule (100μg / mouse, 100μL) three times a week starting from one week before sensitization. Starting from the 28th day, mice in the PBS group, TM group, EGCG group, EGCG microcapsule group and empty microcapsule group were gavaged with 10 mg TM (dissolved in 200 μL PBS solution) every 2 days, for a total of 6 times. Mice in the PBS group were gavaged with 200 μL PBS as a negative control, and the mice were killed on the 49th day to collect blood and tissues.

[0068] Detection of TM-specific IgE, IgG and IgG1 levels in mouse serum:

[0069] The purified TM was diluted to 100 mg / mL with coating solution, and 100 mL was added to 96-well plates and placed at 4°C for coating for 12 hours. Wash three times with PBST, add 200 mL / well of 5% skim milk, and block at 37°C for 2 hours. Then wash three times with PBST, add 200 mL / well of serum sample (diluted 1:5 with 1% skim milk), and incubate at 37°C for 2 hours. Wash three times with PBST, add 100 mL / well of secondary antibody (IgE diluted 1:1000; IgG diluted 1:5000; IgG1 diluted 1:5000, diluted with 1% skim milk), and incubate at 37°C for 2 hours. Wash three times with PBST, add TMB colorimetric solution, 100 mL / well, and place at 37°C for color development for 20 minutes. Take out the 96-well plate and add 2 mol / L H2SO4, 50 mL / well to terminate the color development reaction, and use an enzyme reader to detect the absorbance at a wavelength of 450 nm.

[0070] After obtaining mouse serum, the levels of TM-specific IgE, IgG and IgG1 in the serum were detected, and the content of histamine and mast cell protease (mMCP-1) in the serum was determined according to the instructions of the purchased kit. At the same time, the duodenum of the mouse was obtained and the duodenal tissue section was analyzed.

[0071] The test results are as follows Figure 6 and Figure 7 As shown:

[0072] Depend on Figure 6 It can be seen that the serum IgE level of mice in the blank group was normal, and the serum IgE level of mice in the EGCG group, EGCG microcapsule group (EGCG-SA NPs) and empty microcapsule group (SA NPs) was significantly decreased compared with the serum IgE level of mice in the TM allergy group. Moreover, the IgE level in the EGCG microcapsule group was more significantly reduced than that in the EGCG group. In addition, the serum IgG and IgG1 levels of mice in each group showed the same trend, and the serum IgG and IgG1 levels of mice in the EGCG microcapsule group decreased most significantly. Further analysis of the levels of histamine and mast cell protease (mMCP-1) in mouse serum showed that the serum histamine and mast cell protease (mMCP-1) levels of mice in the EGCG group, EGCG microcapsule group and empty microcapsule group were significantly decreased compared with those in the TM allergy group. In addition, the downward trend of the EGCG microcapsule group was more obvious. The above results preliminarily indicate that EGCG, empty microcapsules and EGCG microcapsules all have anti-allergic effects, but the EGCG microcapsules constructed by EGCG and empty microcapsules have better synergistic effects and achieve stronger anti-allergic effects.

[0073] Depend on Figure 7 It can be seen that the tissues of mice in the PBS group were normal and there were no pathological phenomena. The TM group showed obvious pathological phenomena of edema and lymphocyte infiltration, while the EGCG group and the empty microcapsule group showed that the allergic symptoms of mice were significantly alleviated, and the EGCG microcapsule group did not even show pathological symptoms. The above results fully demonstrate that the EGCG group, the empty microcapsule and the EGCG microcapsule group have obvious anti-allergic effects, and the encapsulated EGCG has achieved a stronger anti-allergic effect.

[0074] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing polyphenol microcapsules, characterized in that: Here are the steps: (1) Preparation of sodium alginate / chitosan microcapsule wall materials The calcium chloride solution and the sodium alginate solution are mixed and stirred; then the chitosan solution is added, stirred, the pH is adjusted, centrifuged, the nanoparticles are washed, and freeze-dried to obtain the sodium alginate / chitosan microcapsule wall material; (2) Preparation of plant polyphenol-sodium alginate / chitosan microcapsules The microcapsule wall material is dissolved in water to form a microcapsule wall material solution; then plant polyphenols and an emulsifier are added, stirred, centrifuged, the nanoparticles are washed, and freeze-dried to obtain plant polyphenol-sodium alginate / chitosan microcapsules; The mass ratio of calcium chloride to sodium alginate is selected from 1:5; the mass ratio of chitosan to sodium alginate is selected from 1:6; the mass ratio of plant polyphenols to sodium alginate is selected from 1:3; the emulsifier is Tween 80; the pH is 5.5; the centrifugation condition is: 3000r / min centrifugation for 10min; the plant polyphenols are epicatechin gallate.

2. The polyphenol microcapsules prepared by the method of claim 1.

3. Use of the polyphenol microcapsules according to claim 2 in the preparation of drugs with antioxidant and / or antiallergic effects.

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