Composite microsphere for enhancing antibacterial performance of baicalin as well as preparation method and application of composite microsphere

Abstract

The invention relates to a composite microsphere for enhancing the antibacterial performance of baicalin as well as a preparation method and application of the composite microsphere, and relates to the technical field of biological materials. The composite microsphere comprises zein and baicalin, wherein the baicalin is embedded into the hydrophobic interior of the zein. The composite microspheres can reduce the minimum inhibitory concentration of the baicalin and the cell membrane conductivity of the baicalin, and enhance the biological membrane clearance rate of the baicalin and the DNA inhibition effect of the baicalin, so that the antibacterial property of the baicalin is improved, and the composite microspheres have wide application prospects in the industries of biological materials and the like.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to composite microspheres for enhancing the antibacterial performance of baicalin, and a preparation method and application thereof. Background Art

[0002] Methicillin resistant Staphylococcus aureus (MRSA) is an important human pathogen and a zoonosis of public health and veterinary significance. MRSA causes severe disease in both humans and animals and is resistant to adverse environmental conditions such as sunlight and dryness. The bacterium can invade the skin, mucous membranes, and internal organs, causing severe disease in animals and humans, including purulent skin infections, acne, respiratory infections, and sepsis.

[0003] Baicalin is a flavonoid compound known as a "natural plant antibiotic" that has an inhibitory effect on drug-resistant Staphylococcus aureus. However, due to its low bioavailability and easy metabolism and excretion after oral administration, its antibacterial activity is unstable. Therefore, studying how to improve the inhibitory effect of baicalin on drug-resistant Staphylococcus aureus is a current research hotspot and difficulty. Summary of the invention

[0004] In view of the above problems, the present invention provides a composite microsphere for enhancing the antibacterial performance of baicalin. The composite microsphere can reduce the minimum inhibitory concentration of baicalin and its cell membrane conductivity, while enhancing the biofilm clearance rate of baicalin and its DNA inhibition effect, thereby improving the antibacterial performance of baicalin, and has broad application prospects in industries such as biomaterials.

[0005] In order to achieve the above object, the present invention provides a composite microsphere for enhancing the antibacterial property of baicalin, comprising zein and baicalin, wherein the baicalin is embedded in the hydrophobic interior of the zein.

[0006] Pure zein is colorless and tasteless, and its isoelectric point is 6.2, but because it is combined with lutein, most of it is a light yellow solid. In previous studies, it was found that zein contains a large number of α-helical structures, and the surface of the structure has ultra-high hydrophobicity and order, among which the three hydrophobic amino acids Val, Leu and Pro account for a large proportion, making it difficult to dissolve in water. During the research process, the inventor found that although zein is difficult to dissolve in water, it is easily soluble in alcohol-water mixed system solvents (especially 70%, 80% ethanol solutions) and other non-polar solvents. These characteristics make zein a carrier for loading bioactive substances. Therefore, the inventors proposed to use zein as a carrier to load baicalin, which not only solves the problems of low bioavailability of baicalin and easy metabolism and excretion after oral administration, but also reduces the minimum inhibitory concentration of baicalin and its cell membrane conductivity, while enhancing the biofilm clearance rate of baicalin and its DNA inhibition effect, thereby improving the antibacterial properties of Scutellaria baicalensis. The composite microspheres have broad application prospects in the pharmaceutical, biomaterials and other industries.

[0007] In one embodiment, the mass ratio of the zein to the baicalin is (8-12):1.

[0008] In one embodiment, the purity of the zein is 95-100%, and the purity of the baicalin is 95-100%.

[0009] In one embodiment, the baicalin is linked to the zein via hydrogen bonds and electrostatic interactions.

[0010] The present invention also provides a method for preparing the composite microspheres, comprising the following steps: dissolving zein and baicalin, embedding baicalin in zein by a pH-driven method, filtering, and drying to obtain the composite microspheres.

[0011] The present invention embeds baicalin into the hydrophobic interior of zein through the pH-driven method, and combines the two together through hydrogen bonds and electrostatic effects to form composite microspheres with specific microstructures.

[0012] In one embodiment, the drying temperature is 45-55° C., and the drying time is 20-30 hours.

[0013] In one embodiment, zein and baicalin are dissolved in alkaline solution, and the pH value of the alkaline solution is 10-12.

[0014] In one embodiment, the pH-driven method includes: adjusting the pH value to 6-8 with acid solution, and centrifuging at 700-800 rpm for 25-35 minutes.

[0015] In one embodiment, the alkaline solution includes sodium hydroxide, and the acid solution includes hydrochloric acid.

[0016] In one embodiment, the pH of the acid solution is 2-4.

[0017] The present invention also provides a medicine, comprising a medicine active ingredient and a pharmaceutically acceptable auxiliary material, wherein the medicine active ingredient comprises the composite microsphere.

[0018] The composite microspheres can enhance the antibacterial activity of baicalin against MRSA, and can 8 CFU / mL, 6.0×10 8 CFU / mL and 12.0×10 8 CFU / mL MRSA bacterial suspension has a good antagonistic effect.

[0019] The present invention also provides application of the composite microspheres in preparing antibacterial drugs or antibacterial materials.

[0020] The present invention also provides a biomaterial for inhibiting bacteria, comprising the composite microspheres.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention discloses a composite microsphere for enhancing the antibacterial performance of baicalin, a preparation method and an application thereof. Zein and baicalin form composite microspheres mainly through hydrogen bonds and electrostatic effects. The composite microspheres can reduce the minimum inhibitory concentration of baicalin and the conductivity of its cell membrane, while enhancing the biofilm clearance rate of baicalin and the inhibitory effect of its DNA, thereby improving the antibacterial performance of baicalin. The composite microspheres have broad application prospects in industries such as biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a flow chart of the preparation process of the composite microspheres for enhancing the antibacterial performance of baicalin of the present invention, wherein Zein@BA represents the zein microsphere particles loaded with baicalin;

[0024] Figure 2 is a characterization structure diagram of the microsphere particles in Example 2, wherein: Figure 2 (A) is the scanning electron microscope observation result of zein microsphere particles after the same treatment. Figure 2 (B) is the scanning electron microscope observation result of zein microsphere particles loaded with baicalin. Figure 2 (C) is the infrared spectrum of various microsphere particles. Figure 2 (D) is the particle size distribution result of microsphere particles. Figure 2 (E) is the turbidity result of the solution of microsphere particles. Figure 2 In the figure, Zein is zein microsphere particles, Zein@BA is zein microsphere particles loaded with baicalin, and BA is baicalin;

[0025] Figure 3 The figure shows the effect of different microsphere particles on the removal of MRSA biofilm, where PBS is normal saline, Zein is zein microsphere particles, Zein@BA is zein microsphere particles loaded with baicalin, and BA is baicalin;

[0026] Figure 4 The results of the influence of different microsphere particles on the cell membrane conductivity of MRSA, where BA is baicalin, and Zein@BA is zein microsphere particles loaded with baicalin;

[0027] Figure 5 The results of agarose gel electrophoresis after MRSA was treated with different microsphere particles, where 0: treatment with normal saline; 1: treatment with zein microsphere particles; 2: treatment with zein microsphere particles loaded with baicalin; 3: treatment with baicalin. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] source:

[0031] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0032] Example 1

[0033] A composite microsphere for enhancing the antibacterial property of baicalin and a preparation method thereof.

[0034] The preparation method is as follows:

[0035] 1. Mix zein and baicalin in a mass ratio of 10:1 and dissolve them in a sodium hydroxide solution with a pH of 12.0 to obtain a mixed solution.

[0036] 2. Stir the mixed solution at 750 rpm for 0.5 h to fully dissolve it, then add dilute hydrochloric acid with a pH of 3 to adjust the pH of the mixed solution to 7.0.

[0037] 3. The pH-adjusted mixed solution was centrifuged at 750 rpm for 30 min and then filtered to obtain a zein microsphere solution loaded with baicalin.

[0038] 4. The zein microsphere solution loaded with baicalin was placed in an oven and dried with hot air at a constant temperature of 50°C for 24 hours to obtain zein microsphere particles loaded with baicalin (i.e., composite microspheres with enhanced antibacterial properties of baicalin).

[0039] Example 2

[0040] 1. Under an accelerating voltage of 5 kV, the same mass of zein and zein microspheres loaded with baicalin were sprayed with platinum and then placed under a scanning electron microscope to observe their surface morphology.

[0041] 2. The infrared spectrum of baicalin, zein and zein microspheres loaded with baicalin was scanned 32 times at a wave number of 4000-400nm and a resolution of 4nm.

[0042] The particle size distribution of the sample was determined by dispersing the sample in deionized water using a laser particle size analyzer. The turbidity of the solution can be expressed by its transmittance, indicating the solubility and aggregation state of the sample in the solution. The same mass of samples were dissolved in sodium hydroxide solution (1 mg / mL) and measured by UV-visible spectrophotometry at 600 nm, with sodium hydroxide solution without sample as a blank background control.

[0043] Baicalin was dissolved in distilled water (1 mg / mL) and distilled water was used as a control, and the whole wavelength was scanned by UV-Vis spectrophotometer. Since the two characteristic absorption peaks of baicalin are located at 278 nm and 317 nm, the wavelength of 317 nm was finally selected to determine the content of baicalin to avoid the interference of the amino acid absorption peak and the standard curve was drawn with baicalin solutions of 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 25 mg / mL and 50 mg / mL, respectively.

[0044] 3. The results showed that there was a good linear relationship between the absorbance of baicalin in the range of 1-50 mg / mL and its mass concentration. The standard curve equation was y=0.00281x+0.0894, R 2=0.9912 (y represents the absorbance at 317 nm, x represents the concentration). Later, BA@Zein was ultrasonically treated for 30 minutes and then centrifuged at 1500 rpm for 10 minutes. The supernatant was taken and the total amount of drug loaded on BA@Zein was calculated by the above standard curve equation, which was expressed as W b The theoretical dose is W t The total amount of drug loading and the total mass of the carrier is W o The encapsulation efficiency (EE) can be calculated as follows:

[0045]

[0046] The test results are as follows:

[0047] Natural zein is processed by Figure 1 Methods After treatment (Zein), a large number of irregularly shaped and small-sized microspheres were observed under a scanning electron microscope. At the same time, as the pH value of the solution changed from alkaline to neutral, the solubility of zein and baicalin gradually decreased. Combined with the self-assembly behavior of biomacromolecules, zein microspheres loaded with baicalin (Zein@BA) were formed after hot air drying. Scanning electron microscope observation showed that Figure 2 (B) As shown. The interaction between zein and baicalin was analyzed by infrared spectroscopy. Figure 2 As shown in (C), baicalin exhibits a strong absorption peak at 3394.9 nm caused by the stretching vibration of intermolecular hydrogen bonds, and the peak shape is relatively broad. The absorption peak of zein at 3430.1 nm is related to the vibration of the OH bond.

[0048] When zein was loaded with baicalin, the hydrogen bond between zein and baicalin shifted to a wavelength of 3298.7 nm, and the absorption peaks of zein at 1631.2 nm and 1542.1 nm (related to amide II) shifted to higher wavenumbers, further proving that a hydrogen bond was formed between them. Due to the limitation of the carrier, the extension of the chemical group of baicalin was restricted, resulting in the characteristic absorption peak of baicalin at 1500 nm-1700 nm almost disappearing or overlapping with the absorption peak of zein, indicating that baicalin can be well embedded in the hydrophobic interior of zein.

[0049] According to formula (1) and (2), the encapsulation efficiency of the microsphere composite particles is (92.41% ± 0.06)%. In addition, the solution permeability is negatively correlated with its turbidity. The lower the permeability, the higher the turbidity. The order of the particle size distribution of the samples is Zein@BA>Zein( Figure 2 (D)), which may be because zein loaded with baicalin. The order of sample turbidity is Zein@BA>Zein( Figure 2(E)), which may be due to the fact that the larger the particle size, the greater the turbidity. The solution permeability of zein was the highest because its particle size was small and could be evenly dispersed in the solvent, but the turbidity and particle size increased after loading baicalin, which was consistent with the results of scanning electron microscopy observation.

[0050] Example 3

[0051] 1. Test method.

[0052] Preparation of MRSA bacterial suspension: Five different MRSA strains were selected and named MRSA-1, MRSA-2, MRSA-3, MRSA-4 and MRSA-5. After continuous passage, MRSA was taken out from the plate and diluted with nutrient broth liquid culture medium to a concentration of 1.5×10 8 CFU / mL.

[0053] The preparation formula of nutrient broth liquid culture medium is: 10g tryptone, 5g sodium chloride, 3g beef extract, and 1000mL distilled water.

[0054] Sample pretreatment: Zein, baicalin and zein microspheres loaded with baicalin were sterilized by ethanol vapor method (v / v, 75%) in a closed water bath for 24 hours and then ventilated and purged on a clean bench for two hours, and then dissolved in the same mass of sterilized PBS.

[0055] The sample solutions with antibacterial concentrations in the nutrient broth liquid culture medium were placed in the 1-10 columns of the 96-well plate, and then the MRSA bacterial solution in the logarithmic growth phase was added to the target concentration, so that the concentrations of the mixed sample solutions were 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 1 / 2μg / mL and 1 / 4μg / mL, and finally cultured in a 37°C constant temperature incubator for 18 hours. The positive control group was the same concentration of bacteria, while the negative control group was the same volume of PBS (pH 7.0). After 24 hours of culture, the minimum inhibitory concentration (MIC) measured at a wavelength of 600nm on the microplate reader was read. The parallel experiment was performed three times.

[0056] The sample was diluted to 2×MIC with antibacterial effect with sterile PBS and added to a 96-well plate numbered MRSA-1 for 72 hours. The volume of the sample solution in each well was 200 μL. The treatment time was 2 hours. After washing the sample solution in each well with sterile water, the residual amount of biofilm in the well plate was measured at a wavelength of 600 nm by crystal violet staining on an ELISA reader to evaluate the cleaning effect of the sample on MRSA biofilm. The control group was the same volume of PBS (pH 7.0).

[0057] The bacterial suspension of MRSA-1 in the logarithmic phase was inoculated into the nutrient broth liquid culture medium according to the inoculum amount to make the concentrations 2×MIC and 4×MIC respectively. After mixing evenly, the culture was cultured at 37°C for 12 hours and then centrifuged at 3000 rpm for 45 minutes. The supernatant was taken and the OD value was measured at 450 nm. This was repeated three times to obtain the characteristic conductivity.

[0058] The samples were inoculated with LB liquid medium into the logarithmic phase MRSA-1 and the final concentration was 4× MIC. -1 The cells were incubated on a shaker at a constant temperature for 24 h. An equal volume of PBS was added to the control group. 2 mL of the bacterial sample solution was taken and shaken at 2700 r·min. -1 After centrifugation for 10 min, the precipitate was suspended in 1 mL of sterile water to prepare a bacterial suspension. Then, the genomic DNA of MRSA was extracted using a DNA kit, and the mass concentration (48 μg / mL) and purity (OD 260nm / OD 280nm =1.89). Agarose gel (m / m, 80%) was mixed with 4S Red Plus (0.1 μL / mL). Next, DNA (12 μg / mL) was mixed with the sample, heated in a 37°C water bath for 30 minutes, and then agarose gel block electrophoresis was performed and photographed using a gel imager. The negative control was the same volume of PBS (pH=7.0).

[0059] 2. Test results.

[0060] Through three parallel broth dilution experiments, the results showed that PBS and Zein had no antibacterial activity against MRSA, while the MIC of BA against 5 strains of MRSA ranged from 2-4 mg / mL, with a mode of 2 mg / mL; and the MIC of Zein@BA against 5 strains of MRSA ranged from 1-2 mg / mL, with a mode of 1 mg / mL.

[0061] When zein loaded with baicalin to form composite microspheres, its residual rate on MRSA biofilm was lower than that of baicalin monomer ( Figure 3), indicating that it can enhance the biofilm clearance of MRSA by baicalin. In addition, at 2×MIC and 4×MIC concentrations, baicalin loaded with zein resulted in lower conductivity than baicalin monomer ( Figure 4 ), indicating that Zein@BA can reduce the cell membrane fluidity of MRSA and increase its permeability. At the same time, the inhibition rate of zein on MRSA DNA synthesis was also improved after baicalin was loaded ( Figure 5 ). Therefore, zein microspheres loaded with baicalin enhanced the antibacterial activity of baicalin against MRSA by reducing the minimum inhibitory concentration and cell membrane fluidity, improving the biofilm clearance rate and DNA synthesis inhibition rate.

[0062] In summary, the present invention can prepare a composite microsphere that can enhance the performance of baicalin in inhibiting drug-resistant staphylococci through hydrogen bonding and electrostatic interaction, and the particle size distribution is relatively uniform, the solution turbidity is relatively high, and the baicalin encapsulation rate and drug loading are relatively high. At the same time, by reducing the minimum inhibitory concentration and cell membrane fluidity, improving the biofilm clearance rate and DNA synthesis inhibition rate to enhance the antibacterial performance of baicalin against MRSA, it has a wide range of application prospects in related fields such as biomedicine and materials.

[0063] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0064] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A composite microsphere for enhancing the antibacterial property of baicalin, characterized in that: The invention comprises zein and baicalin, wherein the baicalin is embedded in the hydrophobic interior of the zein.

2. The composite microsphere according to claim 1, characterized in that: The mass ratio of the zein to the baicalin is (8-12):

1.

3. The composite microsphere according to claim 1, characterized in that: The baicalin is connected to the zein through hydrogen bonds and electrostatic interactions.

4. The method for preparing the composite microspheres according to any one of claims 1 to 3, characterized in that: The following steps are involved: Zein and baicalin are dissolved, baicalin is embedded in the zein by a pH-driven method, filtered, and dried to obtain composite microspheres.

5. The preparation method according to claim 4, characterized in that: Alkali solution is used to dissolve zein and baicalin, and the pH value of the alkali solution is 10-12.

6. The preparation method according to claim 4, characterized in that: The pH driving method comprises: adjusting the pH value to 6-8 with acid solution, and centrifuging at 700-800 rpm for 25-35 minutes.

7. The preparation method according to claim 6, characterized in that: The alkaline solution includes sodium hydroxide, and the acid solution includes hydrochloric acid.

8. A drug, characterized in that The invention comprises a pharmaceutically active ingredient and a pharmaceutically acceptable excipient, wherein the pharmaceutically active ingredient comprises the composite microsphere according to any one of claims 1 to 3.

9. Use of the composite microspheres according to any one of claims 1 to 3 in the preparation of antibacterial drugs or antibacterial materials.

10. A biomaterial for antibacterial use, characterized in that: The composite microsphere comprises the composite microspheres described in any one of claims 1 to 3.

Images