Preparation method of silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent

Abstract

The invention discloses a preparation method of a silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent, comprising: extracting Bletilla striata glucomannan; preparing oxidized Bletilla striata glucomannan; dissolving oxidized Bletilla striata glucomannan in deionized water, stirring in a water bath to form an oxidized Bletilla striata glucomannan solution, dropping an AgNO3 solution into the oxidized Bletilla striata glucomannan solution and stirring and mixing uniformly, stirring in a water bath in a dark environment and then naturally cooling to room temperature, centrifuging and collecting the suspension, and freeze-vacuum drying to obtain a silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent. The silver-loaded oxidized Bletilla striata glucomannan nano composite obtained by the invention has higher antibacterial activity than oxidized Bletilla striata glucomannan and silver nitrate, and in addition, improves the potential cytotoxicity of silver, shows higher cell compatibility than silver nitrate, and is expected to become a new type of silver-containing antibacterial agent.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial agent preparation, and more specifically, relates to a method for preparing a silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent. Background Art

[0002] Bletilla striata is the dried tuber of Bletilla striata of the orchid family. It has the effects of astringency, hemostasis, swelling and tissue regeneration. It is mainly used to treat pulmonary and gastric bleeding, traumatic bleeding, hematemesis due to tuberculosis, gastrointestinal ulcers, and skin chapped skin. The main active ingredients of Bletilla striata include polysaccharides, phenanthrene, anthraquinones, bibenzyls, etc. Among them, Bletilla striata glucomannan is a viscous polysaccharide obtained by water extraction and alcohol precipitation of Bletilla striata tubers. Its main chemical component is glucomannan. Studies have shown that external use of Bletilla striata glucomannan has a good hemostatic effect on solid organs, muscle blood vessel bleeding, etc. Its hemostatic mechanism is to enhance the activity of platelet factor III, shorten the thrombin generation time, inhibit the activity of fibrinase, and promote cell aggregation to form artificial thrombus, thereby achieving the effect of hemostasis. At the same time, Bletilla striata glucomannan also has certain antibacterial and anti-inflammatory effects and promotes wound healing. Bletilla striata glucomannan has good biodegradability, safety, no toxic side effects, no pyrogenicity, etc., and has been used as a safe and reliable biomedical material in the pharmaceutical and chemical industries.

[0003] Both oxidized Bletilla striata glucomannan and silver nitrate have antibacterial properties, but silver nitrate has poor cell compatibility and is toxic to cells. How to combine Bletilla striata glucomannan and silver nitrate to improve the toxicity of silver to cells and prepare an antibacterial agent with better antibacterial properties and better cell compatibility is an urgent problem to be solved. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] In order to achieve these purposes and other advantages according to the present invention, a method for preparing a silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent is provided, comprising the following steps:

[0006] Step 1, extracting glucomannan from Bletilla striata;

[0007] Step 2, preparing oxidized Bletilla striata glucomannan;

[0008] Step 3: dissolve oxidized Bletilla striata glucomannan in deionized water, stir in a water bath to form an oxidized Bletilla striata glucomannan solution, add AgNO3 solution dropwise to the oxidized Bletilla striata glucomannan solution and stir to mix evenly, then stir in a water bath in the dark and naturally cool to room temperature, collect the suspension by centrifugation, and finally obtain the silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent by freeze-vacuum drying.

[0009] Preferably, in the step 1, the method for extracting Bletilla striata glucomannan comprises: adding Bletilla striata tuber powder to deionized water, extracting by decoction, centrifuging, extracting the precipitate twice, collecting the supernatant, concentrating under vacuum with a rotary evaporator, slowly pouring the concentrated solution into anhydrous ethanol, precipitating in a refrigerator overnight, collecting the precipitate by centrifugation, and then freeze-vacuum drying to obtain Bletilla striata glucomannan.

[0010] Preferably, in the step one, the mass volume ratio of Bletilla striata tuber powder to deionized water is 1 g:40 mL; the decoction extraction time is 60 min, the centrifugal speed after decoction is 5000 rpm, and the centrifugation time is 10 min; the temperature for vacuum concentration using a rotary evaporator is 80°C, and the volume ratio of the concentrate to anhydrous ethanol is 1:3; the temperature of the refrigerator for alcohol precipitation overnight is 4°C.

[0011] Preferably, in the step 2, the method for preparing oxidized Bletilla striata glucomannan comprises: adding a certain amount of NaIO4 to a Bletilla striata glucomannan solution, reacting at room temperature in the dark, adding an ethylene glycol solution with an equal amount of NaIO4, continuing stirring for 2 hours to terminate the reaction, and centrifuging; the supernatant is dialyzed in distilled water for 48 hours through a dialysis bag with a cutoff flow of 3 kDa, and then freeze-vacuum dried to obtain oxidized Bletilla striata glucomannan.

[0012] Preferably, in the step 2, the concentration of the bletilla striata glucomannan solution is 2% (w / v); the molar ratio of NaIO4 to the bletilla striata glucomannan structural unit is 0.2:1; the reaction time at room temperature in the dark is 24h, and the stirring is carried out at a speed of 300r / min.

[0013] Preferably, in the step three, the concentration of the oxidized Bletilla striata glucomannan solution is 2.5% (w / v), and the water bath temperature is 80°C; the AgNO3 solution is added dropwise to the oxidized Bletilla striata glucomannan solution and stirred to mix evenly, and then stirred in a 90°C water bath in the dark for 60 minutes and then naturally cooled to room temperature; the volume ratio of the AgNO3 solution to the oxidized Bletilla striata glucomannan solution is 1:10, and the concentration of AgNO3 in the reaction system is 0.01~1.0% (w / v); the centrifugal speed for collecting the suspension by centrifugation is 10000rpm, and the centrifugal time is 15min; the freeze-vacuum drying temperature is -80°C, the vacuum degree is less than 10Pa, and the freeze-vacuum time is 48h.

[0014] Preferably, in the step three, after AgNO3 is added dropwise to the oxidized white and glucomannan solution, ultrasonic dispersion is first performed for 20 to 60 minutes at an ultrasonic frequency of 30 to 75 kHz; then the temperature is raised to 90°C at a rate of 2°C / min, stirred in a 90°C water bath in the dark for 60 minutes, and then the temperature is lowered to 25°C at a cooling rate of 5°C / min.

[0015] Preferably, the silver-loaded oxidized Bletilla striata glucomannan antibacterial agent is used to inhibit the growth and reproduction of Escherichia coli and Staphylococcus aureus.

[0016] The present invention at least includes the following beneficial effects: the present invention provides a method for preparing a silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent, wherein Bletilla striata glucomannan extracted from Bletilla striata tuber powder is oxidized and modified as a reducing agent and a stabilizer, and silver nitrate is used as a silver source, and the preparation is carried out under light-proof heating conditions. The obtained silver-loaded oxidized Bletilla striata glucomannan nanocomposite has higher antibacterial activity than oxidized Bletilla striata glucomannan and silver nitrate, and in addition, the potential cytotoxicity of silver is improved, and the cell compatibility is higher than that of silver nitrate, and it is expected to become a new type of silver-containing antibacterial agent.

[0017] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The UV-visible absorption spectra of the aqueous dispersion formed by AgNO3 solution and ORBP-Ag0.5 prepared in Example 4 at the same silver concentration (Ag concentration is 0.4 mg / mL);

[0019] Figure 2 The effects of different AgNO3 solution concentrations on the UV-visible absorption spectra of ORBP-Ag nanocomposites in Examples 1 to 5 are shown;

[0020] Figure 3 The infrared spectra of oxidized Bletilla striata glucomannan and ORBP-Ag0.5 prepared in Example 2;

[0021] Figure 4 The XRS spectrum of oxidized Bletilla striata glucomannan and ORBP-Ag0.5 prepared in Example 2;

[0022] Figure 5 C1s XPS high resolution spectrum of the oxidized Bletilla striata glucomannan prepared in Example 1-5;

[0023] Figure 6 Ag3d XPS high-resolution spectrum of ORBP-Ag prepared in Example 1-5;

[0024] Figure 7 This is the C1s XPS high-resolution spectrum of the ORBP-Ag prepared in Example 1-5;

[0025] Figure 8The cell viability of L929 cells after being treated with different silver concentration solutions for 24 hours using ORBP-Ag0.5 and AgNO3 of Example 2 as silver sources;

[0026] Fig. 9 The IC50 of ORBP-Ag0.5 prepared in Example 2 for L929 cells;

[0027] Fig.10 is the IC50 of AgNO3 on L929 cells. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0029] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0030] Example 1

[0031] This embodiment provides a method for preparing a silver-loaded oxidized leucopsis glucomannan nano antibacterial agent, comprising the following steps:

[0032] Step 1. Extraction of Rhizoma Bletillae striatae glucomannan (RBP): Add 10g of Rhizoma Bletillae striatae tuber powder to 400ml of deionized water, extract by decoction for 60min, centrifuge at 5000rpm for 10min, and extract the precipitate twice. Collect the supernatant and concentrate it under vacuum at 80℃ using a rotary evaporator; slowly pour the concentrate into 3 times the volume of anhydrous ethanol and precipitate it in a refrigerator at 4℃ overnight. Collect the precipitate by centrifugation and then freeze-dry it in a vacuum to obtain RBP;

[0033] Step 2, preparation of oxidized Bletilla striata glucomannan (ORBP): prepare a 2% (w / v) concentration of RBP solution, add a certain amount of NaIO4 (n(NaIO4):n(Glu units)=0.2:1), react for 24 hours (300r / min) at room temperature in the dark, add an ethylene glycol solution with the same amount of NaIO4, continue stirring for 2 hours to terminate the reaction, and centrifuge. The supernatant was dialyzed in distilled water for 48 hours through a dialysis bag with a cut-off flow rate of 3kDa, and then freeze-dried in a vacuum to obtain oxidized Bletilla striata glucomannan (ORBP);

[0034] Step 3, preparation of silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent (ORBP-Ag): dissolve oxidized Bletilla striata glucomannan (ORBP) in deionized water, stir evenly in a water bath at 80°C to form a 2.5% (w / v) ORBP solution. At room temperature, 2.5 mL of AgNO3 solution was added dropwise to 25 mL of ORBP (2.5%, w / v) solution and stirred evenly, and the final concentration (w / v) of AgNO3 in the reaction system was kept at 0.01%. Then, the above solution was stirred in a water bath at 90°C in the dark for 60 minutes and then naturally cooled to room temperature, and the suspension was collected by centrifugation at 10000 rpm for 15 minutes, and finally the ORBP-Ag nanocomposite was obtained by freeze-vacuum drying; the freeze-vacuum drying temperature was -80°C, the vacuum degree was less than 10Pa, and the freeze-vacuum time was 48 hours. The silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent prepared in this embodiment was named ORBP-Ag0.01.

[0035] Example 2

[0036] The present embodiment provides a method for preparing a silver-loaded oxybacterial nano-antibacterial agent of Bletilla striata and glucomannan, which is different from that of Example 1 in that in step three, 2.5 mL of AgNO3 solution is added dropwise to 25 mL of ORBP (2.5%, w / v) solution and stirred to mix evenly, and the final concentration (w / v) of AgNO3 in the reaction system is maintained at 0.05%. The rest of the preparation method is the same as that of Example 1. The silver-loaded oxybacterial nano-antibacterial agent of Bletilla striata and glucomannan prepared in this embodiment is recorded as ORBP-Ag0.05.

[0037] Example 3

[0038] The present embodiment provides a method for preparing a silver-loaded oxybacterial nano-antibacterial agent of Bletilla striata and glucomannan, which is different from that of Example 1 in that in step three, 2.5 mL of AgNO3 solution is added dropwise to 25 mL of ORBP (2.5%, w / v) solution and stirred to mix evenly, and the final concentration (w / v) of AgNO3 in the reaction system is maintained at 0.1%. The rest of the preparation method is the same as that of Example 1. The silver-loaded oxybacterial nano-antibacterial agent of Bletilla striata and glucomannan prepared in this embodiment is recorded as ORBP-Ag0.1.

[0039] Example 4

[0040] The present embodiment provides a method for preparing a silver-loaded oxybacterial nano-antibacterial agent of Bletilla striata and glucomannan, which is different from that of Example 1 in that in step three, 2.5 mL of AgNO3 solution is added dropwise to 25 mL of ORBP (2.5%, w / v) solution and stirred to mix evenly, and the final concentration (w / v) of AgNO3 in the reaction system is maintained at 0.5%. The rest of the preparation method is the same as that of Example 1. The silver-loaded oxybacterial nano-antibacterial agent of Bletilla striata and glucomannan prepared in this embodiment is recorded as ORBP-Ag0.5.

[0041] Example 5

[0042] The present embodiment provides a method for preparing a silver-loaded oxybacterial nano-antibacterial agent of Bletilla striata and glucomannan, which is different from that of Example 1 in that in step three, 2.5 mL of AgNO3 solution is added dropwise to 25 mL of ORBP (2.5%, w / v) solution and stirred to mix evenly, and the final concentration (w / v) of AgNO3 in the reaction system is maintained at 1.0%. The rest of the preparation method is the same as that of Example 1. The silver-loaded oxybacterial nano-antibacterial agent of Bletilla striata and glucomannan prepared in this embodiment is recorded as ORBP-Ag1.0.

[0043] Example 6

[0044] The present embodiment provides a method for preparing a silver-loaded oxidized white and glucomannan nano antibacterial agent, which is different from that of Example 4 in that, in step three, after AgNO3 is added dropwise to the oxidized white and glucomannan solution, ultrasonic dispersion is first performed for 20 minutes, and the ultrasonic frequency is 30kHz; then the temperature is raised to 90°C at a rate of 2°C / min, and after stirring in a 90°C water bath in the dark for 60 minutes, the temperature is lowered to 25°C at a cooling rate of 5°C / min. The rest of the preparation method is the same as that of Example 4. The silver-loaded oxidized white and glucomannan nano antibacterial agent prepared in this embodiment is recorded as ORBP-Ag0.5-1.

[0045] Example 7

[0046] The present embodiment provides a method for preparing a silver-loaded oxidized Bletilla striata and glucomannan nano antibacterial agent, which is different from that of Example 14 in that, in step three, after AgNO3 is added dropwise to the oxidized Bletilla striata and glucomannan solution, ultrasonic dispersion is first performed for 60 minutes, and the ultrasonic frequency is 75kHz; then the temperature is raised to 90°C at a rate of 2°C / min, and after stirring in a 90°C water bath in the dark for 60 minutes, the temperature is lowered to 25°C at a cooling rate of 5°C / min. The rest of the preparation method is the same as that of Example 4. The silver-loaded oxidized Bletilla striata and glucomannan nano antibacterial agent prepared in this embodiment is recorded as ORBP-Ag0.5-2.

[0047] 1. UV analysis

[0048] Analysis method: Using deionized water as blank, UV spectrophotometer was used to scan the ORBP solution, AgNO3 solution and ORBP-Ag nanocomposite suspension. Figure 1 and Figure 2 shown.

[0049] like Figure 1 As shown in the figure, the AgNO3 aqueous dispersion (Ag concentration of 0.4 mg / mL) has no absorption peak between 300-800 nm, while the ORBP-Ag0.5 aqueous dispersion (Ag concentration of 0.4 mg / mL) has a surface plasma absorption peak near 420 nm, which is caused by the excitation of free electrons in the silver nanoparticles. This confirms the existence of silver nanoparticles in ORBP-Ag. Figure 2 It can be seen that there is no absorption peak near 420nm in the ORBP solution (i.e., AgNO3 concentration is 0%). When the AgNO3 concentration is in the range of 0.01%-1.0%, the ORBP-Ag nanocomposite has an absorption peak belonging to nanosilver near 420nm. With the increase of AgNO3 concentration, the absorption peak of nanosilver first increases and then decreases, which shows that the AgNO3 concentration in the reaction system affects the content of nanosilver particles in the ORBP-Ag nanocomposite. When the AgNO3 concentration is 0.5%, the absorption peak of nanosilver in the ORBP-Ag nanocomposite is the strongest, indicating that the content of nanosilver particles is the highest.

[0050] (II) Infrared analysis

[0051] Analysis method: ORBP and ORBP-Ag nanocomposite powders were prepared by potassium bromide disc pressing method, and infrared spectra were obtained by scanning on a Fourier transform infrared spectrometer with a scanning range of 400-4000cm -1 , resolution 4cm -1 The test results are as follows: Figure 3 Shown

[0052] In the infrared spectrum of ORBP, 3434cm -1 and 2927cm -1 The peaks at 1736 cm-1 are the OH stretching vibration peak and the CH asymmetric stretching vibration peak. -1 and 1634cm -1 The peaks at 3425 cm-1 are the C=O stretching vibration peak and the band of bound water. Compared with ORBP, the OH peak (3425 cm-1) in ORBP-Ag0.5 -1 ) intensity weakened and moved to the low frequency direction, which indicates that the hydroxyl group in ORBP participated in the reduction of nanosilver and interacted with silver. Since the nanosilver solution is weakly alkaline, some acetyl groups in ORBP are hydrolyzed, so ORBP-Ag0.5 at 1736cm -1The C=O stretching vibration peak intensity also weakened.

[0053] 3. X-ray Photoelectron Spectrometer (XPS) Analysis

[0054] Analysis method: The elemental composition and chemical state of ORBP and ORBP-Ag nanocomposites were collected using X-ray photoelectron spectroscopy (XPS). Figure 4-Figure 7 shown.

[0055] The surface chemical compositions of ORBP and ORBP-Ag were characterized by XPS. Figure 4 Only C and O elements were detected in ORBP, while Ag was also detected in ORBP-Ag, which further proves the successful loading of silver. The Ag 3d XPS high-resolution spectrum of ORBP-Ag is shown in Figure 5 As shown, Ag 3d 5 / 2 and Ag 3d 3 / 2 The double peak spacing is 6.1 eV, indicating the metallic nature of silver, which also proves that Ag + Successfully reduced to Ag 0 The peak fitting results show that the silver in ORBP-Ag exists + and Ag 0 The C1s XPS high-resolution spectra of ORBP and ORBP-Ag are shown in Figure 2. Figure 6 and Figure 7 As shown in the figure, the binding energies of C=O, β-glycosidic bond, COC / COH and CC / CH in ORBP are 289.30 eV, 287.67 eV, 286.42 eV and 285.03 eV, respectively. Compared with ORBP, except for the CC / CH binding energy which has not changed, the corresponding peaks of C=O, β-glycosidic bond and COC / COH in ORBP-Ag shift to 289.38 eV, 287.87 eV and 286.51 eV, respectively, indicating that they play an important role in the chelation and stabilization of silver.

[0056] (IV) Particle size analysis

[0057] Analysis method: An appropriate amount of ORBP-Ag nanocomposite was dispersed in deionized water to form a 1 mg / mL dispersion, and then the particle size and distribution of the ORBP-Ag nanocomposite were analyzed using a laser particle size analyzer. The test results are shown in Table 1.

[0058] Table 1 Particle size and dispersion index of ORBP-Ag nanocomposites

[0059]

[0060] The particle size and dispersion index of the ORBP-Ag nanocomposites prepared at different AgNO3 concentrations are shown in Table 1. In the concentration range of 0.01%-1.0%, as the AgNO3 concentration in the reaction system increases, the particle size of the ORBP-Ag nanocomposites decreases first and then increases. When the AgNO3 concentration is 0.5%, the particle size of the prepared ORBP-Ag0.5 is the smallest and the dispersion index is the lowest, which indicates that the particle size of the ORBP-Ag0.5 nanocomposites is small and more uniform, and the particle size of the complexes of Example 6 and Example 7 is better than that of Example 4. When the AgNO3 concentration is further increased to 1.0%, the particle size of the prepared ORBP-Ag1.0 is the largest, about 315nm.

[0061] 3. Antibacterial properties of silver-loaded oxidized white and glucomannan nano-antibacterial agent

[0062] 1. Antibacterial performance test

[0063] Escherichia coli ( E. coli , ATCC 25922) and Staphylococcus aureus ( S. aureus , ATCC 6538) was used as the test bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ORBP-Ag nanocomposites against the test bacteria were tested to study the effect of AgNO3 concentration in the reaction system on the antibacterial activity of ORBP-Ag nanocomposites.

[0064] Preparation of LB medium: Dissolve 25g LB broth in 1000mL deionized water and sterilize at 121℃ for 15min to obtain LB liquid medium. Dissolve 25g LB broth and 15g agar powder in 1000mL deionized water and sterilize at 121℃ for 15min to obtain LB solid medium.

[0065] MIC test: The MIC test was performed using the microdilution method. First, appropriate amounts of sterilized ORBP and ORBP-Ag were dispersed in sterile water to form a 4 mg / mL sample solution. A series of sample solutions were obtained by performing a 2-fold gradient dilution in a 96-well culture plate. 100 μL of LB liquid culture medium was added to the wells of the 96-well culture plate, and then 100 μL of the 4 mg / mL sample solution was added. The mixture was repeatedly pipetted (more than 5 times) to form a 2 mg / L sample solution; 100 μL of the 2 mg / mL sample solution was pipetted into the next well (containing 100 μL of LB liquid culture medium), and the mixture was repeatedly pipetted (more than 5 times) to form a 1 mg / mL sample solution; this was repeated until the last concentration was 0.0156 mg / mL, and 100 μL of the liquid in the last concentration well was pipetted and discarded; each sample was repeated 3 times. Then, 100 μL of the diluted bacterial solution (10 5 -106 CFU / mL), at which time the sample concentration in each well, that is, the final sample concentration, is 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.8μg / mL. In addition, a negative control (only 200μL of blank LB liquid culture medium) and a positive control (100μL of bacterial liquid and LB liquid culture medium each) are made on the same culture plate. After the 96-well culture plate is placed in a 37℃ constant temperature incubator for 20-24h, the color of the culture medium is observed. The minimum sample concentration at which the color of the culture medium is consistent with that of the negative control group is the MIC.

[0066] MBC test: Pipette 50 μL of the mixed solution after MIC test onto the surface of LB solid culture medium, spread it evenly with a spreader, place it in a constant temperature incubator at 37°C for 24 hours, and observe the bacterial growth on the surface of the culture medium. The lowest concentration of ORBP-Ag nanocomplex when no bacteria grow on the LB culture plate is MBC.

[0067] ORBP and ORBP-Ag nanocomposites E.coil and S. aureus The MICs and MBCs are shown in Table 2.

[0068] Table 2 ORBP-Ag nanocomposites E.coil and S. aureus MIC and MBC

[0069]

[0070] ORBP can inhibit bacterial growth to a certain extent. E.coil and S. aureus The MIC of all the drugs was 2000 μg / mL. E.coil and S. aureus In terms of antibacterial ability, the ORBP-Ag nanocomposite is improved by about 8-125 times and 2-250 times compared with ORBP, respectively, which indicates that the ORBP-Ag nanocomposite has stronger antibacterial and bactericidal ability. The antibacterial ability of the ORBP-Ag nanocomposite does not increase with the increase of the AgNO3 concentration in the reaction system. The antibacterial ability of ORBP-Ag0.5 and ORBP-Ag1.0 is equivalent, and the silver content of ORBP-Ag1.0 is nearly 7.6% higher than that of ORBP-Ag0.5, which shows that the antibacterial ability of the ORBP-Ag nanocomposite is not only related to its silver content, but also to its particle size. From the perspective of antibacterial ability and cost, it is better to select a 0.5% AgNO3 concentration in the reaction system to prepare the ORBP-Ag nanocomposite.

[0071] (II) Comparison of antibacterial properties of silver nitrate and silver-loaded Bletilla striata glucomannan nano-antibacterial agents

[0072] We used AgNO3, ORBP-Ag0.5 and ORBP-Ag0.5-1 as silver sources and compared the antibacterial activity of different silver sources at the same silver concentration. Appropriate amounts of sterilized AgNO3, ORBP-Ag0.5 and ORBP-Ag0.5-1 were dispersed in sterile water to form a sample solution with a silver concentration of 0.4 mg / mL. A series of silver concentration samples were obtained by 2-fold dilution in a culture plate, and the final silver concentrations were 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 μg / mL, respectively. The rest of the steps were the same as the above MIC and MBC tests.

[0073] AgNO 3、 ORBP-Ag0.5 and ORBP-Ag0.5-1 E.coil and S. aureus The MICs and MBCs are shown in Table 3.

[0074] Table 3. Effect of AgNO3 and ORBP-Ag0.5 E.coil and S. aureus MIC and MBC (in terms of silver concentration)

[0075]

[0076] for E.coil For ORBP-Ag0.5, AgNO3 and ORBP-Ag0.5-1, they showed similar antibacterial ability, with MIC of 1.56 μg / mL. However, ORBP-Ag0.5-1 (MBC: 0.78 μg / mL) and ORBP-Ag0.5 (MBC: 3.13 μg / mL) showed stronger bactericidal ability than AgNO3 (MBC: 6.25 μg / mL). S. aureus Although the bactericidal abilities were similar, ORBP-Ag0.5-1 (MIC: 0.78 μg / mL) and ORBP-Ag0.5 (MIC: 1.56 μg / mL) had better antibacterial ability than AgNO3 (MIC: 3.13 μg / mL). E. coil and S. aureus The antibacterial effect of AgNO3 is better than that of AgNO3, and it is expected to become a substitute for silver nitrate antibacterial agent. (III) Comparison of cell compatibility of silver-loaded Bletilla striata glucomannan nano-antibacterial agent

[0077] Mouse fibroblasts (L929) were used as test cells to compare the cell compatibility of AgNO3 and ORBP-Ag0.5 as silver sources. 5The complete cell culture medium was DMEM containing 100 U / L penicillin and 100 mg / L streptomycin.

[0078] MTT method for quantitative testing of cell viability: ORBP-Ag0.5 and AgNO3 were dispersed in DMEM medium containing 10% fetal bovine serum, and the samples with silver concentrations of 12, 10, 8, 6, 4, 2, 1 and 0.5 μg / mL were obtained by gradient dilution and used as standby. L929 cells were cultured at 1.0×10 5 The cells were inoculated into a 96-well cell culture plate at a density of 100 μL and cultured in a humidified incubator containing 5% CO2 at 37°C for 24 hours. The culture medium was removed and 100 μL of sample solution was added to continue incubation. The control group was incubated with 100 μL of culture medium. The cell viability was tested by MTT method after 24 hours of incubation. After the time was reached, 10 μL of MTT (5 mg / mL) reagent was added to each well and incubated for 4 hours. Then the culture medium in the well was carefully removed and 150 μL of DMSO was added and incubated for another 15 minutes. After the incubation, the optical density value was read at 570 nm using a microplate reader. With silver concentration as the horizontal axis and cell viability as the vertical axis, the half-maximum inhibitory concentration (IC50) of AgNO3 and ORBP-Ag0.5 on L929 cells was calculated by curve fitting.

[0079] like Figure 8 As shown in the figure, when AgNO3 was used as the silver source, the cell viability was about (71.0±6.0)% when the silver concentration was 4μg / mL, while when ORBP-Ag0.5 was used as the silver source, the cell viability was still higher than 70% when the silver concentration reached 12μg / mL, and the cell viability was 73.4%. The curve was fitted with silver concentration as the horizontal axis and cell viability as the vertical axis, and the half-inhibitory concentration (IC50) of ORBP-Ag0.5 and AgNO3 as the silver source on L929 cells was calculated. The results are shown in the figure. Fig. 9 and Fig.10 As shown in Figure 2, the IC50 value (15.99 μg / mL) of ORBP-Ag0.5 as the silver source is about 3.2 times that of the IC50 value (4.93 μg / mL) of AgNO3 as the silver source. Therefore, at the same silver concentration, ORBP-Ag nanocomposite has better cell compatibility than AgNO3.

[0080] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0081] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing a silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent, characterized in that: The following steps are involved: Step 1, extracting glucomannan from Bletilla striata; Step 2, preparing oxidized Bletilla striata glucomannan; Step 3, dissolving oxidized Bletilla striata glucomannan in deionized water, stirring in a water bath to form an oxidized Bletilla striata glucomannan solution, dropping the AgNO3 solution into the oxidized Bletilla striata glucomannan solution and stirring to mix evenly, then stirring in a water bath in the dark and naturally cooling to room temperature, collecting the suspension by centrifugation, and finally freeze-vacuum drying to obtain a silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent; In the step three, the concentration of the oxidized Bletilla striata glucomannan solution is 2.5%, and the water bath temperature is 80°C; the AgNO3 solution is added dropwise to the oxidized Bletilla striata glucomannan solution and stirred to mix evenly, and then stirred in a water bath at 90°C in the dark for 60 minutes and then naturally cooled to room temperature, or the AgNO3 is added dropwise to the oxidized Bletilla striata glucomannan solution, and then ultrasonically dispersed for 20 to 60 minutes, with an ultrasonic frequency of 30 to 75kHz; then the temperature is raised to 90°C at a rate of 2°C / min, stirred in a water bath at 90°C in the dark for 60 minutes, and then cooled to 25°C at a cooling rate of 5°C / min; the volume ratio of the AgNO3 solution to the oxidized Bletilla striata glucomannan solution is 1:10, and the concentration of AgNO3 in the reaction system is 0.5 to 1.0%; the centrifugal speed for collecting the suspension by centrifugation is 10,000 rpm, and the centrifugal time is 15 minutes; the temperature of the freeze-vacuum drying is -80°C, the vacuum degree is less than 10Pa, and the freeze-vacuum time is 48 hours.

2. The method for preparing the silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent as claimed in claim 1, characterized in that: In the step 1, the method for extracting Bletilla striata glucomannan comprises: adding Bletilla striata tuber powder into deionized water, extracting by decoction, centrifuging, extracting the precipitate twice, collecting the supernatant, concentrating the supernatant under vacuum with a rotary evaporator, slowly pouring the concentrated solution into anhydrous ethanol, precipitating the solution in a refrigerator overnight, collecting the precipitate by centrifugation, and then freeze-drying the solution in a vacuum to obtain Bletilla striata glucomannan.

3. The method for preparing the silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent as claimed in claim 2, characterized in that: In the step 1, the mass volume ratio of the bletilla tuber powder to deionized water is 1 g:40 mL; the decoction extraction time is 60 min, the centrifugal speed after decoction is 5000 rpm, and the centrifugation time is 10 min; the temperature for vacuum concentration using a rotary evaporator is 80° C., and the volume ratio of the concentrate to anhydrous ethanol is 1:3; the temperature of the refrigerator for alcohol precipitation overnight is 4° C.

4. The method for preparing the silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent as claimed in claim 1, characterized in that: In the step 2, the method for preparing oxidized Bletilla striata glucomannan includes: adding a certain amount of NaIO4 to the Bletilla striata glucomannan solution, reacting at room temperature in the dark, adding an ethylene glycol solution with an equal amount of NaIO4, continuing stirring for 2 hours to terminate the reaction, and centrifuging; the supernatant is dialyzed in distilled water for 48 hours through a dialysis bag with a cutoff flow of 3 kDa, and then freeze-vacuum dried to obtain oxidized Bletilla striata glucomannan.

5. The method for preparing the silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent as claimed in claim 4, characterized in that: In the step 2, the concentration of the bletilla striata glucomannan solution is 2%; the molar ratio of NaIO4 to the bletilla striata glucomannan structural unit is 0.2:1; the reaction time is 24 hours at room temperature in the dark, and the stirring is carried out at a speed of 300r / min.

6. An application of the method for preparing the silver-loaded oxidized Bletilla striata glucomannan nano antibacterial agent according to any one of claims 1 to 5, characterized in that: The silver-loaded oxidized Bletilla striata glucomannan antibacterial agent prepared by the method is used for preparing a preparation for inhibiting the growth and reproduction of Escherichia coli and Staphylococcus aureus.

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