Bromelain hydrogel containing aminated tannic acid

Abstract

The invention belongs to the field of medicines, and particularly relates to bromelain hydrogel containing aminated tannic acid. According to the bromelain hydrogel and the preparation method thereof, the activity of bromelain at high temperature is remarkably protected by performing amination modification on tannic acid, and then the bromelain hydrogel containing aminated tannic acid, which is good in thermal stability and antibacterial and anti-inflammatory effects, is prepared from polyvinyl alcohol and sodium borate. The invention has the characteristics of simple preparation process, convenience in use, safety, effectiveness and the like, and is beneficial to industrial large-scale production and application.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a bromelain hydrogel containing amino tannic acid. Background Art

[0002] Bromelain is a thiol protease, a proteolytic enzyme extracted from pineapple plants, mainly found in the stems and fruits of pineapples. my country is rich in pineapple resources, but a large amount of pineapple peels and stems are wasted every year. The application of bromelain can recycle natural resources such as pineapple peels and stems. In the pharmaceutical industry, bromelain has important medicinal value. It can treat edema and various inflammations, is used for burn scab removal, and prevents and treats cardiovascular diseases. However, its activity is affected by many factors, including physical factors (temperature, humidity, light, radiation, high pressure), chemical factors (oxidation-reduction, organic solvents, metal ions, pH value), biological factors (enzyme modification, enzyme degradation), etc., which lead to a decrease in its activity during processing and storage.

[0003] Gel has many advantages in the pharmaceutical field, such as easy use, simple preparation process, environmental protection and economy, good controlled release effect, etc. It is particularly suitable for local application and drug delivery under specific conditions. At present, there are bromelain enteric-coated tablets and enteric-coated capsules on the market, but there are no topical preparations of bromelain. According to the anti-inflammatory properties of bromelain, it is prepared into bromelain hydrogel, which undoubtedly provides an efficient solution for wound healing. However, some studies have shown that gel has been proven to be the least ideal basis for adding bromelain as a topical preparation, because it is unstable and easily affected by temperature, etc., and it is impossible to maintain the integrity and activity of bromelain, thereby affecting the characteristics of the preparation. Therefore, it is crucial to protect the activity of bromelain at high temperatures and prepare bromelain hydrogels with good thermal stability and significant antibacterial and anti-inflammatory effects.

[0004] Tannic acid has antibacterial and antioxidant properties. Experiments have shown that tannic acid has a certain effect on protecting the activity of bromelain, but the effect is limited. Its molecules contain a large number of phenolic hydroxyl groups and ester bonds. These groups are highly active and can be introduced through reactions to improve their effects. Amino groups are one of the important functional groups in protein molecules. Amino groups help maintain the structure of enzymes by forming hydrogen bonds and electrostatic effects. Therefore, introducing amino groups is a good choice.

[0005] In the preparation of hydrogels, polyvinyl alcohol (PolyvinylAlcohol) is a commonly used gel material. It has excellent swelling properties, good biocompatibility, excellent gelling properties, excellent mechanical properties, and good thermal stability and chemical stability. Polyvinyl alcohol hydrogels show high stability in a variety of environments and can resist thermal and chemical degradation within a certain range. Polyvinyl alcohol is usually prepared by the freeze-thaw method, but freeze-thaw has a great effect on enzyme activity. Cross-linking agents such as glutaraldehyde will also affect enzyme activity. Literature research shows that sodium borate has a certain degree of protective effect on the thermal stability of bromelain, and the concentration change has little effect on the protective effect, so it is just used as a cross-linking agent.

[0006] At present, there are no reports that amino tannins can significantly protect the activity of bromelain at high temperatures, nor are there any reports on the preparation of amino tannin-containing bromelain hydrogels with good thermal stability, antibacterial and anti-inflammatory effects based on polyvinyl alcohol and sodium borate. Summary of the invention

[0007] The object of the present invention is to provide a bromelain hydrogel containing amino tannic acid: by aminated modification of tannic acid to significantly protect the activity of bromelain at high temperature, and then preparing the bromelain hydrogel containing amino tannic acid with good thermal stability, antibacterial and anti-inflammatory effects by polyvinyl alcohol and sodium borate. Specifically, the present invention is realized by the following technical scheme:

[0008] The present invention provides a bromelain hydrogel containing aminated tannic acid, characterized in that the aminated tannic acid is added during the preparation of the bromelain hydrogel to improve the thermal stability of the bromelain;

[0009] The bromelain hydrogel comprises the following components, in percentage by mass: 0.1-0.5% bromelain, 0.01-0.8% amino tannic acid, 5-10% polyvinyl alcohol-1799, 0.1-0.5% sodium tetraborate decahydrate, and the balance is water;

[0010] The specific preparation method of the bromelain hydrogel is as follows:

[0011] (1) Preparation of amino tannic acid: 1 mL of deionized water, 4 mL of anhydrous ethanol and 1 g of γ-aminopropyltriethoxysilane were added to a beaker and stirred with a glass rod for 5 min; then 1 g of tannic acid was added and stirred with a glass rod for 5 min, and the mixture was allowed to stand for 1 h to allow for a full reaction; the precipitate was washed alternately with anhydrous ethanol and deionized water to remove unreacted silane coupling agent and impurities; the mixture was dried in a vacuum oven at 45° C. to constant weight and then ground into powder to obtain 1.5 g of amino tannic acid;

[0012] (2) Preparation of bromelain hydrogel: Weigh an appropriate amount of polyvinyl alcohol-1799, add it to deionized water, and completely dissolve it under magnetic stirring at 90°C; after cooling to room temperature, add a certain amount of bromelain; weigh a certain amount of amino tannic acid, dissolve it in a certain amount of 0.5 mol / L sodium hydroxide aqueous solution, and add it to the above aqueous solution; after mixing the three, adjust the pH to 6.0 with 0.1 mol / L hydrochloric acid solution, and stir it magnetically at room temperature for 2 hours to obtain a uniform aqueous solution; place the aqueous solution in an ultrasonic cleaning machine for a period of time until the bubbles are completely removed, and then pour it into a mold; weigh an appropriate amount of sodium tetraborate decahydrate, prepare it into an aqueous solution of a certain concentration, and add it dropwise while stirring; let it stand for 1 hour to obtain bromelain hydrogel.

[0013] The mass concentration of the polyvinyl alcohol-1799 aqueous solution of the present invention is 10%, the mass concentration of the sodium tetraborate decahydrate aqueous solution is 2%, and the mass ratio of the polyvinyl alcohol-1799 to the sodium tetraborate decahydrate is 25:1.

[0014] The mass ratio of the amino tannic acid to bromelain of the present invention is 1:10-5:3, preferably 1:5.

[0015] The bromelain hydrogel containing amino tannic acid of the present invention is used as wound dressing.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The bromelain hydrogel containing aminated tannic acid of the present invention is used in combination with bromelain in polyvinyl alcohol / sodium borate hydrogel by aminated modification of tannic acid, thereby significantly improving the thermal stability of bromelain, having a high enzyme activity retention rate, and being resistant to storage, and having a significant anti-inflammatory effect, and having an obvious therapeutic effect when used as a wound dressing. The present invention has the characteristics of simple preparation process, convenient use, safety and effectiveness, and is conducive to industrial large-scale production and application.

[0018] Attached pictures:

[0019] Figure 1 : Infrared spectra of tannic acid and amino tannic acid;

[0020] Figure 2 :Results of wound healing in mice. DETAILED DESCRIPTION

[0021] The method of the present invention is further described in detail below through specific examples.

[0022] Example 1

[0023] Preparation of Aminated Tannic Acid

[0024] Take 1 mL of deionized water, 4 mL of anhydrous ethanol and 1 g of γ-aminopropyltriethoxysilane and add them to a beaker, and stir with a glass rod for 5 minutes; then add 1 g of tannic acid, stir with a glass rod for 5 minutes, and let it stand for 1 hour to allow it to react fully; use anhydrous ethanol and deionized water to alternately wash the precipitate to remove unreacted silane coupling agent and impurities; dry it in a vacuum drying oven at 45°C to constant weight, and then grind it into powder to obtain 1.5 g of amino tannic acid.

[0025] Figure 1 This is the infrared spectrum of tannic acid and amino tannic acid. Figure 1 The infrared spectrum of amino tannic acid is significantly different from that of tannic acid: 3423 cm -1 The absorption peak of C=O increases from 1712cm -1 Displacement to 1701cm -1 and weakened at 1254cm -1 A new CN characteristic peak was added at the bottom of the column, which proved that tannic acid had undergone amino modification.

[0026] Preparation of 0.1% Bromelain Hydrogel Containing Aminated Tannic Acid

[0027] 2.5 g of polyvinyl alcohol-1799 was weighed and added to 25 mL of deionized water, and completely dissolved under magnetic stirring at 90° C.; after cooling to room temperature, 0.035 g of bromelain (BR, 300 u / mg) was added; 4 mg of amino tannic acid was weighed and dissolved in 1 mL of 0.5 mol / L sodium hydroxide aqueous solution, and added to the above aqueous solution; after the three were mixed, the pH was adjusted to 6.0 with 0.1 mol / L hydrochloric acid solution, and magnetic stirring was performed at room temperature for 2 hours to obtain a uniform aqueous solution; the aqueous solution was placed in an ultrasonic cleaning machine for a period of time until the bubbles were completely removed, and then poured into a mold; 0.1 g of sodium tetraborate decahydrate was weighed, prepared into a 2% aqueous solution, and added dropwise while stirring; and the mixture was allowed to stand for 1 hour to obtain a 0.1% bromelain hydrogel.

[0028] Embodiment 2-5

[0029] 0.1% bromelain hydrogel containing aminated tannic acid at different mass ratios of aminated tannic acid to bromelain

[0030] The operation method of Examples 2-5 is the same as that of Example 1, except that the weight of the amino tannic acid is changed, as shown in Table 1.

[0031] Table 1 Example groups under different weights of aminated tannic acid

[0032] Group Aminated tannic acid (mg) Example 1 4 Example 2 5.5 Example 3 7 Example 4 8.5 Example 5 10

[0033] Comparative Example 1

[0034] 0.1% bromelain hydrogel containing tannic acid (tannic acid: bromelain = 1:5)

[0035] 2.5 g of polyvinyl alcohol-1799 is weighed and added to 25 mL of deionized water, and completely dissolved under magnetic stirring at 90° C.; 0.035 g of bromelain is added after cooling to room temperature; 7 mg of tannic acid is weighed and dissolved in 1 mL of 0.5 mol / L sodium hydroxide aqueous solution, and the mixture is added to the aqueous solution; after the three are mixed, the pH value is adjusted to 6.0 with 0.1 mol / L hydrochloric acid solution, and magnetic stirring is performed at room temperature for 2 hours to obtain a uniform aqueous solution; the aqueous solution is placed in an ultrasonic cleaning machine for a period of time until bubbles are completely removed, and then poured into a mold; 0.1 g of sodium tetraborate decahydrate is weighed and prepared into a 2% aqueous solution, and the mixture is added dropwise while stirring; and the mixture is allowed to stand for 1 hour to obtain a 0.1% bromelain hydrogel containing tannic acid.

[0036] Comparative Example 2

[0037] 0.1% Bromelain hydrogel containing neither aminated tannins nor tannic acid

[0038] 2.5 g of polyvinyl alcohol-1799 is weighed and added to 25 mL of deionized water, and completely dissolved under magnetic stirring at 90° C.; 0.035 g of bromelain is added after cooling to room temperature; the pH of the two is adjusted to 6.0 after mixing, and magnetic stirring is performed for 2 hours at room temperature to obtain a uniform aqueous solution; the aqueous solution is placed in an ultrasonic cleaning machine for a period of time until the bubbles are completely removed and then poured into a mold; 0.1 g of sodium tetraborate decahydrate is weighed, prepared into a 2% aqueous solution, and added dropwise while stirring; and the mixture is allowed to stand for 1 hour to obtain a 0.1% bromelain hydrogel containing neither amino tannic acid nor tannic acid.

[0039] Comparative Example 3

[0040] Blank polyvinyl alcohol / sodium borate hydrogel containing neither bromelain nor aminated tannic acid

[0041] 2.5 g of polyvinyl alcohol-1799 was weighed and added to 25 mL of deionized water, and completely dissolved under magnetic stirring at 90° C.; after cooling to room temperature, it was placed in an ultrasonic cleaner for a period of time until the bubbles were completely removed and then poured into a mold; 0.1 g of sodium tetraborate decahydrate was weighed, prepared into a 2% aqueous solution, and added dropwise while stirring; it was allowed to stand for 1 hour to obtain a polyvinyl alcohol / sodium borate blank hydrogel containing neither bromelain nor amino tannic acid.

[0042] Comparative Example 4

[0043] 0.1% Bromelain Liquid

[0044] Weigh 0.035 g of bromelain into a beaker, add deionized water to 35 mL, and stir until dissolved to obtain 0.1% bromelain solution.

[0045] Comparative Example 5

[0046] 0.1% bromelain / aminated tannic acid complex solution (aminated tannic acid: bromelain = 1:5)

[0047] Weigh 0.035g of bromelain into a beaker, add a small amount of deionized water and stir until dissolved; then weigh 7mg of amino tannic acid and dissolve it in 1mL, 0.5mol / L sodium hydroxide aqueous solution, and add it to the beaker; after mixing the two, adjust the pH to 6.0 with 0.1mol / L hydrochloric acid solution, add deionized water to 42mL, and stir evenly to obtain a 0.1% bromelain / amino tannic acid composite solution.

[0048] Test experiment

[0049] Experiment on the high temperature resistance of bromelain in hydrogel

[0050] The experimental group is the 0.1% bromelain hydrogel containing amino tannic acid of Examples 1-5, and the control group is the 0.1% bromelain hydrogel containing tannic acid of Comparative Example 1 and the 0.1% bromelain hydrogel containing neither amino tannic acid nor tannic acid of Comparative Example 2. They are opened and placed in suitable clean containers, placed at 40°C for 40 days, and samples are taken on the 5th day, the 10th day, the 20th day and the 40th day, respectively, to detect the residual enzyme activity. The determination method is the FIP method, with casein as the substrate, after being hydrolyzed by bromelain, the concentration of hydrolyzed tyrosine is determined at 275nm using an ultraviolet spectrophotometer, and the enzyme activity retention rate is calculated. The results are shown in Table 2.

[0051] Table 2 Experimental results of enzyme activity retention rate (%) of bromelain at 40°C

[0052] Group Day 5 Day 10 Day 20 Day 40 Example 1 74.0 57.1 38.4 20.5 Example 2 81.4 62.8 41.8 22.0 Example 3 86.1 66.5 45.4 23.8 Example 4 76.4 58.7 39.1 20.6 Example 5 62.2 47.4 31.7 17.3 Comparative Example 1 49.3 23.8 5.9 0.8 Comparative Example 2 30.6 6.2 0.6 0.0

[0053] The results show that the enzyme activity retention rate of comparative example 1 is slightly higher than that of comparative example 2, and the enzyme activity retention rates of examples 1-5 are significantly improved compared with comparative examples 1 and 2, among which the enzyme activity retention rate of example 3 is the highest, indicating that the addition of tannic acid has a certain protective effect on the enzyme activity of bromelain at high temperature, and the protective effect of the amino-modified tannic acid is significantly enhanced. The results show that the thermal stability of the bromelain hydrogel containing amino-tannic acid is good and the optimal ratio of amino-tannic acid to bromelain is 1:5.

[0054] Antibacterial experiment of bromelain hydrogel

[0055] Experimental bacteria: Escherichia coli, Staphylococcus aureus

[0056] Experimental method: The Oxford cup method was used for determination. The experimental group was the 0.1% bromelain hydrogel containing amino tannic acid in Example 3, and the control group was the blank polyvinyl alcohol / sodium borate hydrogel containing neither bromelain nor amino tannic acid in Comparative Example 3, the single 0.1% bromelain solution in Comparative Example 4, and the 0.1% bromelain / amino tannic acid composite solution in Comparative Example 5. Each group was set up for 3 parallels, and the test was repeated 3 times. The colony diameter of the plate culture medium was measured using the cross method, and the average value was calculated.

[0057] The standards for determining antibacterial activity are as follows: if the diameter of the inhibition zone is greater than 15 mm, it is highly inhibited; if the diameter of the inhibition zone is between 10-15 mm, it is moderately inhibited; if the diameter of the inhibition zone is between 5-10 mm, it is lowly inhibited; if the diameter of the inhibition zone is less than 5 mm, it is non-inhibitory.

[0058] The results are shown in Table 2.

[0059] Table 2 Inhibition zone test results

[0060] Group Escherichia coli (mm) Staphylococcus aureus (mm) Comparative Example 3 9.2 9.5 Comparative Example 4 10.1 9.7 Comparative Example 5 11.6 10.3 Example 3 17.3 16.2

[0061] The results show that the diameter of the inhibition zone of Escherichia coli and Staphylococcus aureus in Example 3 is significantly larger, and the antibacterial ability is significantly enhanced compared with Comparative Examples 3-5. At the same time, the diameter of the inhibition zone of Comparative Example 5 is greater than that of Comparative Example 4, indicating that the addition of amino tannic acid can also improve the antibacterial ability of bromelain. The results show that the bromelain hydrogel containing amino tannic acid has good antibacterial ability and can highly inhibit the growth of Escherichia coli and Staphylococcus aureus.

[0062] Bromelain hydrogel promotes wound healing in mice

[0063] (1) Establishment of mouse wound model

[0064] A total of 25 ICR mice weighing about 25g were taken, all male, and kept in a clean environment. One week later, 1% sodium pentobarbital was injected intraperitoneally to completely anesthetize the mice, and the depilatory cream was evenly applied to the back of the mice, and the hair of an appropriate area was shaved with a razor. After three minutes, the excess depilatory cream was wiped off, and then the back of the shaved mice was rinsed, wiped, and disinfected with iodine tincture. The full-thickness skin of the back of the mice with a diameter of about 1cm was cut with sterile scissors and tweezers, and the wound was infiltrated with Escherichia coli to prepare a wound animal model.

[0065] (2) Group treatment

[0066] The experiment randomly divided the mice into 5 groups, with 5 parallel samples in each group, including a blank control group without treatment, a polyvinyl alcohol / sodium borate blank hydrogel group containing neither bromelain nor amino tannic acid in comparative example 3, a single 0.1% bromelain liquid group in comparative example 4, a 0.1% bromelain / amino tannic acid composite solution group in comparative example 5, and a 0.1% bromelain hydrogel group containing amino tannic acid in example 3. The prepared samples were placed on the back wound surface, and the medication was administered every other day, with a dosage of 200 μL each time. The healing of the wound was observed on the 1st, 4th, 7th, 10th, and 14th days after surgery. The results are shown in Figure 2 .

[0067] The results showed that the wounds of mice in the blank control group healed slowly, and no scabs formed after 14 days; the blank polyvinyl alcohol / sodium borate hydrogel containing neither bromelain nor amino tannic acid in comparative example 3 and the single 0.1% bromelain solution in comparative example 4 had a certain effect on wound healing, and tiny scabs could be seen on the wounds of the two groups of mice; the 0.1% bromelain / amino tannic acid composite solution in comparative example 5 had a slightly improved effect on wound healing compared with the single 0.1% bromelain solution in comparative example 4, and the scabs of the wounds of mice were relatively complete; the scabs of mice in the 0.1% bromelain hydrogel group containing amino tannic acid in Example 3 fell off, the epidermis of the wound surface was re-epithelialized, and the new skin was covered and almost smooth and complete. Compared with other groups, the wound healing effect was significantly improved and the healing speed was fast. The results showed that the bromelain hydrogel containing amino tannic acid had good anti-inflammatory ability and had obvious therapeutic effects on wound healing.

[0068] In the above animal experiments, no obvious adverse reactions were observed in the bromelain hydrogel of the present invention, and the experimental animals had good tolerance to it.

[0069] The embodiments described above are only preferred solutions of the present invention and are not intended to limit the present invention in any form. Other modifications and variations are possible without exceeding the technical solutions described in the claims.

Claims

1. A bromelain hydrogel containing aminated tannic acid, characterized in that: Aminated tannic acid was added to the preparation of bromelain hydrogel to improve the thermal stability of bromelain; The bromelain hydrogel comprises the following components, in percentage by mass: 0.1-0.5% bromelain, 0.01-0.8% amino tannic acid, 5-10% polyvinyl alcohol-1799, 0.2-0.4% sodium tetraborate decahydrate, and the balance is water; The specific preparation method of the bromelain hydrogel is as follows: (1) Preparation of amino tannic acid: 1 mL of deionized water, 4 mL of anhydrous ethanol and 1 g of γ-aminopropyltriethoxysilane were added to a beaker and stirred with a glass rod for 5 min; then 1 g of tannic acid was added, and stirred with a glass rod for 5 min, and allowed to stand for 1 h to allow it to fully react; the precipitate was washed alternately with anhydrous ethanol and deionized water to remove unreacted silane coupling agent and impurities; the precipitate was dried in a vacuum drying oven at 45° C. to constant weight and then ground into powder to obtain amino tannic acid; (2) Preparation of bromelain hydrogel: Weigh an appropriate amount of polyvinyl alcohol-1799, add it to deionized water, and completely dissolve it under magnetic stirring at 90°C; after cooling to room temperature, add a certain amount of bromelain; weigh a certain amount of amino tannic acid, dissolve it in a certain amount of 0.5 mol / L sodium hydroxide aqueous solution, and add it to the above aqueous solution; after mixing the three, adjust the pH to 6.0 with 0.1 mol / L hydrochloric acid solution, and stir it magnetically at room temperature for 2 hours to obtain a uniform aqueous solution; place the aqueous solution in an ultrasonic cleaning machine for a period of time until the bubbles are completely removed, and then pour it into a mold; weigh an appropriate amount of sodium tetraborate decahydrate, prepare it into an aqueous solution of a certain concentration, and add it dropwise while stirring; let it stand for 1 hour to obtain bromelain hydrogel.

2. A bromelain hydrogel containing aminated tannic acid as claimed in claim 1, characterized in that: The mass concentration of the polyvinyl alcohol-1799 aqueous solution is 10%, the mass concentration of the sodium tetraborate decahydrate aqueous solution is 2%, and the mass ratio of the polyvinyl alcohol-1799 to the sodium tetraborate decahydrate is 25:

1.

3. A bromelain hydrogel containing aminated tannic acid as claimed in claim 1, characterized in that: The mass ratio of the amino tannic acid to bromelain is 1:10-5:

3.

4. A bromelain hydrogel containing aminated tannic acid as claimed in claim 1, characterized in that: The bromelain hydrogel is used as a wound dressing.

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