A preparation for enhancing the antibacterial property, encapsulation efficiency and drug loading rate of EGCG and its preparation method

Abstract

The present invention provides a preparation method of a preparation for enhancing the antibacterial property, encapsulation rate and drug loading rate of EGCG. The preparation method comprises the following steps: dissolving EGCG in water, adding mesoporous calcium silicate nanoparticles, stirring in the dark and then centrifuging, and then removing the supernatant and performing freeze-drying to obtain the product. The preparation of the present invention solves the technical problems existing in the application of EGCG in the prior art. The antibacterial rates of the obtained preparation against S. aureus and E. coli reach 95.57±0.54% and 93.41±1.04% respectively, and the encapsulation rate and drug loading rate of EGCG are as high as 72.03±0.49% and 58.46±0.41% respectively. The present invention has good application prospects in the preparation of antibacterial preparations and corresponding drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a preparation for enhancing the antibacterial property, encapsulation rate and drug loading rate of EGCG and a preparation method thereof. Background Art

[0002] Tea polyphenols are a general term for polyhydroxyphenolic substances in tea leaves. Due to their high-efficiency antioxidant and free radical scavenging functions, they are widely used in the fields of food, daily chemicals and medicine and hygiene.

[0003] Tea polyphenols are mainly catechins, accounting for about 65%-80% of the total tea polyphenols, including (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epicatechin (EC). Among them, the content of EGCG is the highest, accounting for about 80%.

[0004] As a natural antioxidant, tea polyphenols not only have immunomodulatory effects, but also have a broad-spectrum antibacterial function and can inhibit a variety of pathogenic bacteria. Staphylococcus is likely to cause skin and soft tissue infections, resulting in food poisoning, septicemia, etc. Among them, Staphylococcus aureus has the strongest virulence, and antibiotic-resistant Staphylococcus aureus has become the most common nosocomial infection pathogen. Research has proved that tea polyphenols can effectively inhibit Staphylococcus aureus and have a significant resistance to the α-toxin produced by it. In addition, tea polyphenols can also inhibit Peptostreptococcus, Enterococcus enteritidis, Streptococcus sanguinis, etc.

[0005] At present, the disadvantages of EGCG in the field of antibacterial applications are mainly as follows: (1) The antibacterial effect is not good; (2) It is difficult to balance high antibacterial property, high encapsulation rate and high drug loading rate when loading with biomaterials; (3) It is difficult to find substances or methods that can produce synergistic effects with it in terms of antibacterial. These disadvantages have led to many limitations in the practical application of EGCG.

[0006] To solve the above disadvantages, those skilled in the art have made attempts in multiple directions. For example, Jin Jiyue in her paper "Study on the Synthesis and Bactericidal Characteristics of EGCG-Cu Complex" [1] complexed EGCG with Cu 2+ to achieve the purpose of enhancing the antibacterial effect of EGCG and reducing the minimum inhibitory concentration to 200 mg / L. Fu Cheng et al. in their paper "Study on the Antibacterial Effect of Fmoc-Phenylalanine Hydrogel and Its Compound with EGCG" [2]It was found that the hydrogel formed by the complexation of Fmoc-DPhe and EGCG could synergistically inhibit the growth of Staphylococcus aureus. In the research on loading EGCG into biomaterials, Wang Qi in his paper "Physicochemical Properties of EGCG-Gelatin Composite Films and Their Mechanisms for Maintaining the Quality of Chilled Tilapia Fillets" [3] found that although loading EGCG into gelatin composite films could extend the shelf life of tilapia, it further reduced the antibacterial ability of EGCG, and its antibacterial ability was inferior to that of free EGCG. Chen Peiyu in her paper "Preparation and Sustained Release Performance of Tea Polyphenol / Poly (Lactic-Co-Glycolic Acid) Sustained Release Microspheres" [4] increased the encapsulation efficiency to over 80% in the paper, but the drug loading rate was only 4%; Lu Min et al. in their papers "Preparation and Antibacterial Activity of Sodium Alginate Nanomicrosphere Antibacterial Films" [5] and "Preparation of Tea Polyphenol / Chitosan / Sodium Alginate Nanomicrospheres" [6] increased the encapsulation efficiency and drug loading rate to 61.38% and 22.71% respectively in the papers, and obtained a certain antibacterial effect, but the encapsulation efficiency, drug loading rate and antibacterial property were still insufficient. In addition, researchers also studied the effect of grafting EGCG into biomaterials on antibacterial property, such as Moreno-Vásquez M J et al. [7] found that the antibacterial activity of the EGCG-grafted chitosan copolymer prepared by free radical-mediated grafting method was higher than that of unmodified chitosan, but Ji Tianchen in his paper "Synthesis and Properties of Bio-based Raw Materials and EGCG Graft Copolymers" [8] found that the antibacterial activity of the EGCG-grafted ε-PL copolymer was slightly lower than that of ε-PL. More importantly, in such technologies, the antibacterial property of EGCG was basically not effectively exerted. Most reports could only improve the antibacterial property of biomaterials, while the antibacterial property of general biomaterials was relatively low.

[0007] In summary, the above-mentioned disadvantages faced by EGCG in the field of antibacterial applications have not been well solved in the current field. It is mainly reflected in that the randomness of finding antibacterial active substances that have a synergistic effect with EGCG is large, making it difficult to provide guidance for other research; existing biomaterials either further inhibit the exertion of the antibacterial property of EGCG, or have low encapsulation efficiency and drug loading rate and the antibacterial property has not been significantly improved, making the practicality of the corresponding preparations not high.

[0008] References:

[0009] [1] Jin Jiyue. Study on the Synthesis and Bactericidal Characteristics of EGCG-Cu Complexes [D]. Beijing University of Civil Engineering and Architecture, 2018.

[0010] [2] Fu Cheng, Yuan Fahu, Hu Yunxia, Gao Lianjun, Wei Dongyu, Chen Chunlan. Study on the antibacterial effect of Fmoc-phenylalanine hydrogel and its compound with EGCG [J]. Food Research and Development, 2019, 40(10): 37-42.

[0011] [3] Wang Qi. Physicochemical properties of EGCG-gelatin composite film and its mechanism for maintaining the quality of refrigerated tilapia fillets [D]. Hainan University, 2019.

[0012] [4] Chen Peiyu. Preparation and sustained-release performance of tea polyphenols / polylactic-co-glycolic acid sustained-release microspheres [D]. Zhejiang University, 2017.

[0013] [5] Lu Min, Wang Liqiang. Preparation and antibacterial effect of sodium alginate nanospheres antibacterial film [J]. Journal of Functional Materials, 2018, 49(03): 3076-3081.

[0014] [6] Lu Min, Wang Liqiang. Preparation of tea polyphenols / chitosan / sodium alginate nanospheres [J]. Packaging Engineering, 2017, 38(19): 47-51.

[0015] [7] Moreno-Vásquez M J, Valenzuela-Buitimea E L, Plascencia-Jatomea M, et al. Functionalization of chitosan by a free radical reaction: Characterization, antioxidant and antibacterial potential [J]. Carbohydrate Polymers, 2017, 155: 117-127.

[0016] [8] Ji Tianchen. Synthesis and properties of bio-based raw materials graft copolymerized with EGCG [D]. Jiangnan University, 2021. Summary of the Invention

[0017] Aiming at the disadvantages of the prior art, the purpose of the present invention is to provide a preparation for enhancing the antibacterial activity of EGCG, which can effectively exert the antibacterial property of EGCG and obtain a significant improvement in antibacterial property; meanwhile, the encapsulation rate and drug loading rate of this preparation for EGCG can reach more than 70% and more than 55% respectively.

[0018] In order to achieve the above purpose, the technical solution provided by the present invention is as follows:

[0019] A preparation method of a preparation for enhancing the antibacterial property, encapsulation rate and drug loading rate of EGCG, the preparation method includes the following steps:

[0020] Dissolve EGCG in water, then add mesoporous calcium silicate nanospheres, stir in the dark and then centrifuge. After that, remove the supernatant and conduct freeze-drying to obtain the product.

[0021] As an embodiment of the present invention, the particle size of the mesoporous calcium silicate nanospheres is 150 - 250 nm.

[0022] As an alternative embodiment of the present invention, the preparation method of the mesoporous calcium silicate nanospheres is as follows: Dissolve CTAB in water, add ammonia water and then dropwise add TEOS. After sufficient reaction, add calcium nitrate to synthesize calcium silicate; stir, centrifuge to take the supernatant and control the pH of the supernatant to be 7.0, wash and then dry, and then conduct calcination to obtain the product. Preferably, the preparation method of the mesoporous calcium silicate nanospheres is as follows: Weigh 1.36 g of CTAB and dissolve it in 400 ml of deionized water, add 16 ml of 28% ammonia water, dropwise add 6.4 ml of TEOS, and synthesize silica through sufficient reaction. On this basis, weigh 6 g of calcium nitrate and add it to synthesize calcium silicate; stir overnight, collect it in a centrifuge tube, wash it with deionized water until the pH of the supernatant is 7.0; wash it three times with absolute ethanol and then dry it overnight at 40 °C, and calcine it at 650 °C for 3 hours.

[0023] As a preferred embodiment of the present invention, the steps of the preparation method are as follows: Dissolve 10 mg of EGCG in 5 ml of deionized water, add 5 mg of mesoporous calcium silicate nanospheres, stir in the dark at 37 °C overnight, then centrifuge at 8000 r / min for 15 min, remove the supernatant, add 5 ml of deionized water to wash once, centrifuge again to remove the supernatant, and finally conduct freeze-drying for 72 h to prepare the mesoporous calcium silicate nanospheres loaded with EGCG.

[0024] Another object of the present invention is to provide a preparation for enhancing the antibacterial property, encapsulation efficiency and drug loading rate of EGCG prepared by the above preparation method.

[0025] Another object of the present invention is to provide the application of the above preparation in the preparation of antibacterial drugs.

[0026] As shown in the examples of the present invention, the present invention can obtain very good antibacterial effects. At a concentration of 1 mg / ml, the antibacterial rates against S. aureus and E. coli reach 95.57 ± 0.54% and 93.41 ± 1.04% respectively. Compared with free EGCG, the antibacterial property has a significant increase.

[0027] It is not difficult to see that the present invention only relies on mesoporous calcium silicate nanospheres as carriers to achieve a significant improvement in antibacterial properties. Compared with finding active substances that can produce a synergistic antibacterial effect with EGCG and preparing corresponding preparations, the present invention is undoubtedly more convenient. At the same time, the encapsulation rate and drug loading rate of EGCG in the present invention are as high as 72.03±0.49% and 58.46±0.41% respectively, further solving the deficiencies of existing biomaterial carriers.

[0028] As shown in a comparative example of the present invention, when using the same type of nanospheres (mesoporous silica nanospheres), both the encapsulation rate and the drug loading rate are less than 50%.

[0029] Beneficial effects of the present invention:

[0030] The preparation of the present invention solves the technical problems of poor antibacterial properties, low encapsulation rate and low drug loading rate existing in the prior art in the application of EGCG. The antibacterial rates of the obtained preparation against S. aureus and E. coli reach 95.57±0.54% and 93.41±1.04% respectively, and the encapsulation rate and drug loading rate of EGCG are as high as 72.03±0.49% and 58.46±0.41% respectively; the present invention has good application prospects in the preparation of antibacterial preparations and corresponding drugs. Description of the drawings

[0031] Figure 1 It is the X-ray photoelectron spectroscopy (XPS) of MSN and MCS; where MSN is mesoporous silica nanospheres and MCS is mesoporous calcium silicate nanospheres;

[0032] Figure 2 It is the transmission electron microscopy (TEM) micrographs of MSN and MCS; where parts A-C are the TEM results of mesoporous silica nanospheres (MSN), and parts D-F are the TEM results of mesoporous calcium silicate nanospheres (MCS);

[0033] Figure 3 It is the N2 adsorption-desorption isotherm curves and BJH pore size distribution diagrams of MSN and MCS; where parts A and C are the N2 adsorption-desorption curves of mesoporous silica nanospheres / mesoporous calcium silicate nanospheres respectively, and parts B and D are the Barrett-Joyner-Halenda (BJH) pore size distribution results of mesoporous silica nanospheres / mesoporous calcium silicate nanospheres respectively;

[0034] Figure 4It is a comparison chart of the encapsulation efficiency and drug loading capacity of MSN and MCS for EGCG. Among them, MSN is mesoporous silica nanoparticles, the encapsulation efficiency is (41.63±0.71)%, and the drug loading capacity is (45.08±1.37)%. MCS is mesoporous calcium silicate nanoparticles, the encapsulation efficiency is (72.03±0.49)%, and the drug loading capacity is (58.46±0.41)%. The comparison between the two groups shows P<0.001 (Mean±S.D., n = 3; *P<0.05, **P<0.01, ***P<0.001).

[0035] Figure 5 It is the cumulative release curve of calcium ions / EGCG of MCS-EGCG. Among them, part A is the curve of the cumulative release amount of calcium ions, and part B is the curve of the cumulative release percentage of EGCG.

[0036] Figure 6 It is the qualitative evaluation of the antibacterial performance of MCS-EGCG against S. aureus and E. coli. Among them, MCS is mesoporous calcium silicate nanoparticles, and MCS-EGCG is mesoporous calcium silicate nanoparticles loaded with tea polyphenols. Part A is the qualitative photo of the number of surviving colonies after co-culturing MCS-EGCG with S. aureus and E. coli for 6 h. Parts B and C are the quantitative statistics of the number of surviving colonies after co-culturing MCS-EGCG with S. aureus and E. coli for 6 h. The comparison between groups shows P<0.001 (Mean±S.D., n = 3; *P<0.05, **P<0.01, ***P<0.001).

[0037] Figure 7 It is the result of the cck-8 experiment on cell proliferation of MCS-EGCG. Among them, MCS is mesoporous calcium silicate nanoparticles, and MCS-EGCG is mesoporous calcium silicate nanoparticles loaded with tea polyphenols. Detailed implementation mode

[0038] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above invention content still belong to the protection scope of the present invention.

[0039] Example 1

[0040] Materials and methods

[0041] Experimental content: Material preparation and characterization, in vitro antibacterial experiment, in vitro cytological experiment, one-way ANOVA.

[0042] Time and Place: The experiment was completed in the laboratory of the Institute of Tissue and Engineering Stem Cells, Nanchong Central Hospital from January 2020 to May 2022.

[0043] Materials: tetraethyl orthosilicate (TEOS, Sigma, USA), cetyltrimethylammonium bromide (CTAB, Sigma, USA), calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, Sigma, USA), ammonia water (NH4OH, Sigma, USA), epigallocatechin gallate (EGCG, Sigma, USA), CCK-8 kit (MCE, Shanghai Jiyi Biotechnology Co., Ltd.), Staphylococcus aureus (S. aureus) (ATCC25923), Escherichia coli (E. coli)(ATCC25922) (donated by North Sichuan Medical College), digital display constant temperature multi-head magnetic stirrer (Shanghai Haozhuang Instrument Co., Ltd.), electronic balance (Mettler-Toledo Instrument Co., Ltd.), X-ray photoelectron spectroscopy analyzer (XPS, Merck, USA), inductively coupled plasma mass spectrometer (ICP-MS, PerkinElmer, USA), ultraviolet spectrophotometer (Hitachi, Japan), BET specific surface area and pore size analyzer (Beijing Best Instrument Co., Ltd.) and field emission transmission electron microscope (TEM, Merck, USA).

[0044] Test method:

[0045] Preparation of EGCG-loaded MCS (mesoporous calcium silicate nanospheres)

[0046] Preparation of MCS: Weigh 1.36g CTAB and dissolve it in 400ml deionized water, add 16ml 28% ammonia water, add 6.4ml TEOS dropwise, react fully to synthesize silicon dioxide, and weigh 6g calcium nitrate to synthesize calcium silicate; stir overnight, collect into a centrifuge tube, wash with deionized water until the supernatant pH = 7.0. Wash three times with anhydrous ethanol, dry at 40℃ overnight, and calcine at 650℃ for 3h (heating rate 2℃ / min).

[0047] EGCG loading: 10 mg EGCG was dissolved in 5 ml deionized water, 5 mg MCS was added, stirred overnight at 37 °C in the dark, then centrifuged at 8000 r / min for 15 min, the supernatant was removed, 5 ml deionized water was added to wash once, centrifuged again to remove the supernatant, and finally freeze-dried for 72 h to prepare EGCG-loaded mesoporous calcium silicate nanospheres (MCS-EGCG).

[0048] Comparative Example 1

[0049] The raw materials of Example 1 were used to prepare EGCG-loaded MSNs (mesoporous silica nanospheres) as follows:

[0050] Preparation of MSN: Weigh 1.36 g of CTAB and dissolve it in 400 ml of deionized water. Add 16 ml of 28% ammonia water, and then gradually add 6.4 ml of TEOS. React fully to synthesize silica. Stir overnight, collect it in a centrifuge tube, and wash it with deionized water until the pH of the supernatant is 7.0. Wash it three times with absolute ethanol and then dry it at 40 °C overnight. Calcinate it at 650 °C for 3 h (heating rate: 2 °C / min).

[0051] Loading of EGCG: Dissolve 10 mg of EGCG in 5 ml of deionized water, add 5 mg of MCN, stir overnight in the dark at 37 °C, then centrifuge at 8000 r / min for 15 min. Discard the supernatant, add 5 ml of deionized water to wash once, centrifuge again to discard the supernatant, and finally freeze-dry for 72 h to prepare mesoporous silica nanoparticles loaded with EGCG (MCN-EGCG).

[0052] Experimental Examples

[0053] Conduct relevant experiments and characterizations on the products of Example 1 and Comparative Example 1

[0054] Characterization of sample performance:

[0055] Use X-ray photoelectron spectroscopy (XPS) to determine the chemical composition of the sample. Use a specific surface area and pore volume analyzer to measure the specific surface area and pore diameter of the sample. The specific surface area is calculated according to the Barrett-Emmett-Teller (BET) method. Use a transmission electron microscope (TEM) to characterize the particle size, morphology, and structure of the sample.

[0056] Determination of the encapsulation efficiency and drug loading of the sample

[0057] Determination of tea polyphenols by the ferrous tartrate method: Polyphenols in tea can form a purple-blue complex with ferrous ions, and then measure its absorbance at 540 nm with a UV spectrophotometer. Finally, calculate the content of tea polyphenols in the system according to the tea polyphenol standard curve.

[0058] Determination of encapsulation efficiency and drug loading: Dissolve 10 mg of EGCG in 5 ml of deionized water, add 5 mg of MSN and MCS respectively, stir overnight in the dark at low temperature, centrifuge at 8000 r / min for 15 min, collect the supernatant, wash it once with 5 ml of deionized water, and collect the supernatant again. Use a pipette to take 1 mL of the supernatant into a 50 mL centrifuge tube, add 4 mL of deionized water and 5 mL of ferrous tartrate solution, mix well, and make up the volume to 50 ml with phosphate buffer solution at pH 7.5. Measure the absorbance at a wavelength of 540 nm using an ultraviolet spectrophotometer.

[0059] According to the standard curve equation, calculate the corresponding concentration C, and calculate the content m of tea polyphenols in the supernatant according to the formula m = C × 10

[0060] Encapsulation efficiency = (10 - m) / 10 × 100%

[0061] Drug loading = (10 - m) / (10 - m) + 5 × 100%

[0062] Release determination of EGCG and calcium ion (Ca 2+ ):

[0063] Weigh 10 mg of MCS-EGCG and disperse it in 3 ml of PBS, place it in a shaker at 37 °C, take 1 ml of the centrifuged supernatant for testing at regular intervals, and then add 1 ml of fresh PBS. Measure the concentration of tea polyphenols in the collected PBS by the above-mentioned ferrous tartrate method for tea polyphenols. Determine the concentrations of calcium (Ca 2+ ) and silicate (SiO3 2- ) ions in the collected PBS by inductively coupled plasma mass spectrometry (ICP-MS), and plot the release curves of tea polyphenols, calcium (Ca 2+ ) and silicate (SiO3 2- ) ions.

[0064] Evaluation of antibacterial properties of the sample:

[0065] Plate coating method: Prepare a sample suspension with a concentration of 1 mg / ml, disperse it evenly, pipette 100 μl and place it in a 96-well plate and keep it at 37 °C. Then add the bacterial suspensions of Staphylococcus aureus (S. aureus) (ATCC 25923) and Escherichia coli (E. coli) (ATCC 25922) (10 μl, 1 × 10 6Add it to the well plate at (CFU / ml) and incubate it in a constant temperature shaker at 37°C for 6 hours. Use the bacteria cultured in 100 μl of PBS as a control. Thereafter, take 50 μl of the co-cultured bacterial suspension, inoculate it on a nutrient agar plate and culture it for 12 hours. The experiment is repeated 3 times, the results are photographed and recorded, and the number of colonies on the plate is counted using ImageJ software.

[0066] Evaluation of the cell biological properties of the samples:

[0067] The in vitro cell proliferation of the carrier was measured by the CCK-8 method. Inoculate bone marrow mesenchymal stem cells into a 96-well cell culture plate containing DMEM (containing 10% fetal bovine serum) (1×10 4 cells / well), and the final volume of the culture medium in each well is 100 μL. Culture the cells in an environment of 37°C and 5% CO2 for 24 hours. When the cells grow to about 80%, aspirate the culture medium, add serum-free culture medium, add the same mass of sterilized powder to the cell wells, set 5 replicates for each sample, and set a blank control group at the same time. At 1, 4, and 7 days respectively, add 10 μL of CCK-8 reagent to each well, continue to incubate in the incubator, and detect the absorbance value at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader after 2 hours.

[0068] Main observation indicators: The in vitro degradation, tea polyphenol release, antibacterial ability, and cell biological properties of mesoporous calcium silicate nanoparticles loaded with tea polyphenols.

[0069] Statistical analysis: The obtained experimental data were analyzed using SPSS 23.0 software. The experimental data were expressed as mean ± standard deviation (Mean ± S.D.). One-way analysis of variance was used for comparison among multiple groups, and Tukey's test was used for comparison between two groups. A P value < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).

[0070] Experimental results

[0071] Material characterization

[0072] The XPS detection results showed that the O peak (O1s) and Si peak (Si2p) were detected in both MNS and MCS, and the Ca peak (Ca2p) was also detected in MCS ( Figure 1 in parts A and B), indicating that the prepared MNS was SiO2, containing Si / O elements with contents of 29.67% and 64.91% respectively, and MCS was CaSiO3 containing Si / O / Ca elements with contents of 18.76%, 61.02%, and 6.92% respectively (Table 1).

[0073] From the TEM photos ( Figure 2) It can be seen that the microspheres prepared experimentally are mainly spherical in appearance, with good particle dispersion. The particle size is mainly distributed around 200 nm, and they are amorphous microspheres with low crystallinity. The color distribution is uneven under transmission electron microscopy ( Figure 2 parts B and E), indicating that the microspheres are not solid and have a mesoporous structure.

[0074] In addition, specific surface area analysis was performed on the prepared MSN / MCS. From the N2 adsorption-desorption curve ( Figure 3 parts A and C) and the Barrett-Joyner-Halenda (BJH) pore size distribution ( Figure 3 parts B and D), it can be seen that the adsorption and desorption isotherms do not coincide, and there is an obvious hysteresis loop. Therefore, the adsorption-desorption isotherm is a typical type IV isotherm, indicating that the product has a mesoporous structure. And the Barrett-Joyner-Halenda (BJH) pore size distribution ( Figure 3 parts B and D) results show that the product has a good mesoporous structure, and most of these pore sizes are micropores (pore size less than 10 nm). Table 2 shows that the specific surface area (BET), total pore volume, and average pore diameter of the prepared MCS are all greater than those of MSN, indicating that both MCS and MSN are mesoporous microspheres, and MCS may have better drug loading and adsorption properties than MSN.

[0075] Table 1 Atomic percentage of MSN / MCS

[0076]

[0077] Table 2 Comparison of specific surface area / total pore volume and average pore diameter of MSN / MCS

[0078]

[0079] Encapsulation efficiency and drug loading of MSN / MCS for EGCG:

[0080] As Figure 4 shown, when the prepared microspheres were used to carry tea polyphenols, the encapsulation efficiency of MCS was (72.03 ± 0.49)%, the drug loading was (58.46 ± 0.41)%, the encapsulation efficiency of MSN was (41.63 ± 0.71)%, and the drug loading was (45.08 ± 1.37)%, indicating that MCS has better drug-carrying ability than MSN (P < 0.001), which is consistent with the BET analysis results.

[0081] Antibacterial performance evaluation of MCS-EGCG:

[0082] The effect diagrams of the qualitative analysis test of the antibacterial performance of the sample against S. aureus and E. coli are as shown in Figure 6 part A. ByFigure 6 As can be seen from part A, compared with the control group, the number of colonies of S. aureus and E. coli decreased to a certain extent after being cultured with MCS and EGCG for 6 h, and the numbers of S. aureus and E. coli in the MCS-EGCG group were significantly reduced. The statistical results are as Figure 6 shown in parts B and C. The antibacterial rates of MCS against S. aureus and E. coli were (28.02±4.20)% and (27.52±6.29)% respectively, and the antibacterial rates of EGCG against S. aureus and E. coli were (67.44±2.35)% and (65.95±2.42)% respectively. The antibacterial rates of MCS-EGCG against S. aureus and E. coli reached (95.57±0.54)% and (93.41±1.04)% respectively.

[0083] The above test results show that MCS and EGCG have certain antibacterial properties, and the addition of EGCG can significantly improve the antibacterial properties of MCS.

[0084] Evaluation of the cell biological properties of MCS-EGCG:

[0085] The results of CCK-8 analysis are as Figure 7 shown. Compared with the blank control group, there was no significant difference on the first day of culturing with MCS and MCS-EGCG, indicating that the samples had no cytotoxicity. The cell proliferation rates on the 4th and 7th days were higher (P<0.05), proving that MCS and MCS-EGCG had good cell biocompatibility and the ability to promote cell proliferation.

Claims

1. A preparation method of a preparation for enhancing the antibacterial property, encapsulation rate and drug loading rate of EGCG, characterized in that, The preparation method comprises the following steps: Dissolve EGCG in water, add mesoporous calcium silicate nanospheres, stir in the dark and then centrifuge, and then remove the supernatant and perform freeze-drying to obtain the product. The particle size of the mesoporous calcium silicate nanospheres is 150 - 250 nm. The preparation method of the mesoporous calcium silicate nanospheres is as follows: Weigh 1.36 g of CTAB and dissolve it in 400 ml of deionized water, add 16 ml of 28% ammonia water, and dropwise add 6.4 ml of TEOS, and fully react to synthesize silica. On this basis, weigh 6 g of calcium nitrate and add it to synthesize calcium silicate; stir overnight, collect it in a centrifuge tube, and wash it with deionized water until the pH of the supernatant is 7.

0. Wash it three times with absolute ethanol, dry it overnight at 40 °C, and calcine it at 650 °C for 3 hours.

2. The preparation method according to claim 1, characterized in that, The steps of the preparation method are as follows: Dissolve 10 mg of EGCG in 5 ml of deionized water, add 5 mg of mesoporous calcium silicate nanospheres, stir in the dark at 37 °C overnight, then centrifuge at 8000 r / min for 15 min, remove the supernatant, add 5 ml of deionized water to wash once, centrifuge again to remove the supernatant, and finally perform freeze-drying for 72 h to prepare the mesoporous calcium silicate nanospheres loaded with EGCG.

3. A preparation for enhancing the antibacterial property, encapsulation rate and drug loading rate of EGCG, characterized in that, The preparation is obtained by the preparation method described in Claim 1 or 2.

4. Use of the preparation according to claim 3 in the preparation of antibacterial drugs.

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