Preparation and application of broad-spectrum antibacterial and antiviral nano-zinc and compound nano-zinc

Abstract

The invention discloses preparation and application of broad-spectrum antibacterial and antiviral nano-zinc and compound nano-zinc, and belongs to the technical field of medical materials. The method comprises the following steps: mixing a ginkgo leaf extracting solution with a zinc salt aqueous solution, and carrying out reduction reaction under ultrasonic and stirring actions to obtain a nano zinc solution. Adding vitamin C and N-acetylcysteine into the nano-zinc solution, continuously carrying out reduction reaction under the ultrasonic and stirring action, and then uniformly mixing with an isometric aqueous solution containing anti-inflammatory, antipyretic and analgesic components to obtain the compound nano-zinc solution. And uniformly mixing the obtained nano-zinc solution or compound nano-zinc solution with sodium hyaluronate hydrogel with the same mass as the nano-zinc solution or compound nano-zinc solution to obtain nano-zinc gel or compound nano-zinc gel. The method provided by the invention avoids toxic and side effects caused by using chemical reducing agents such as hydrazine hydrate, sodium borohydride and the like to prepare nano-zinc. The nano-zinc and the compound nano-zinc have excellent characteristics of broad spectrum, high efficiency, antibacterial property and antiviral property, and have a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical materials, and particularly relates to the preparation and application of a broad-spectrum antibacterial and antiviral nano-zinc and a compound nano-zinc. Background Art

[0002] Zinc is an essential trace element in the human body and plays a very important role in important physiological processes such as human growth and development, reproduction and genetics, immunity, and neuroendocrine. The human body contains 2-3 grams of zinc. Zinc deficiency can lead to growth retardation and mental retardation. Zinc deficiency can also lead to neuroendocrine dysfunction. Therefore, zinc is often used to supplement the lack of zinc in the human body. In recent years, the highly efficient antibacterial and antiviral properties of nano-silver have received extensive attention. Relatively speaking, the preparation and antibacterial and antiviral properties of nano-zinc have not received extensive attention and in-depth research. Although the antibacterial and antiviral effect of nano-zinc is not as efficient as that of nano-silver, nano-zinc has many advantages that nano-silver cannot match. The cytotoxicity of nano-zinc is much smaller than that of nano-silver. Nano-zinc has the ability to enhance the regeneration ability of human tissues, promote protein synthesis, accelerate cell division and proliferation, and promote the rapid healing of wounds. Nano-zinc can also provide the body's immune function and enhance the body's resistance to bacterial and viral infections.

[0003] Nanoparticles refer to ultra-fine particles with a size of 1-100 nm. Nanoparticles have some novel physical and chemical properties, which are significantly different from the physical and chemical properties of macroscopic object particles. Nanoparticles have volume effect, surface effect, quantum size effect and macroscopic quantum tunneling effect. These special physical and chemical effects of nanoparticles make them have important research value and application prospects in the fields of new materials, biology and clinical medicine.

[0004] The nano-zinc obtained by the currently common preparation methods has a wide particle size range, generally between dozens of nanometers and hundreds of nanometers. The too large particles can lead to the instability of the nano-zinc solution, cause particle deposition, and reduce the antibacterial and antiviral effects. Therefore, it is urgent to research and develop new preparation methods for nano-zinc and compound nano-zinc.

[0005] Ginkgo biloba is a plant with high medicinal value, which has the effects of dilating blood vessels and promoting blood circulation to remove blood stasis. The natural active flavonoids contained in Ginkgo biloba have strong antioxidant effects. Ginkgo biloba extract, vitamin C and N-acetylcysteine all have strong reducing properties. So far, there has been no report on the related research of using Ginkgo biloba extract, vitamin C and N-acetylcysteine to jointly reduce zinc ions to prepare nano-zinc. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a preparation method and application of broad-spectrum antibacterial and antiviral nano-zinc and compound nano-zinc. The present invention utilizes the reducing properties of ginkgo leaf extract, vitamin C and N-acetylcysteine, and combines ultrasonic technology to prepare nano-zinc solution and compound nano-zinc solution with smaller and more stable nano-zinc particles. The method of the present invention avoids the toxic and side effects brought by using chemical reducing agents such as hydrazine hydrate and sodium borohydride to prepare nano-zinc. The nano-zinc and compound nano-zinc prepared by the present invention have excellent properties of broad-spectrum and high-efficiency antibacterial and antiviral, and have good application prospects.

[0007] The object of the present invention is achieved in the following ways:

[0008] In the first aspect, the present invention provides a preparation method of broad-spectrum antibacterial and antiviral nano-zinc and compound nano-zinc, comprising the following steps:

[0009] (1) Dry and crush ginkgo leaves, and make the obtained ginkgo leaf powder into a 0.1wt% - 5wt% solution with water. Extract at 80 - 100 °C for 10 - 60 min, then place at 1 - 5 °C for 10 - 24 hours, filter and centrifuge. The obtained supernatant is sterilized to obtain ginkgo leaf extract;

[0010] (2) Mix the ginkgo leaf extract obtained in step (1) with zinc salt aqueous solution, and carry out a reduction reaction under the action of ultrasonic and stirring to prepare a stable nano-zinc solution;

[0011] Alternatively, mix the ginkgo leaf extract obtained in step (1) with zinc salt aqueous solution, and carry out a reduction reaction under the action of ultrasonic and stirring to prepare a stable nano-zinc solution. Add vitamin C and N-acetylcysteine to the obtained nano-zinc solution, and continue to carry out a reduction reaction under the action of ultrasonic and stirring. Mix the obtained solution evenly with an equal volume of aqueous solution containing 0.1wt% - 2wt% anti-inflammatory and antipyretic analgesic components to obtain a compound nano-zinc solution.

[0012] Based on the above technical solution, further, the content of ginkgo leaf powder in the solution described in step (1) is 0.5wt% - 2wt%.

[0013] Based on the above technical solution, further, the centrifugation conditions in step (1) are: the rotation speed is 2000 - 6000 rpm, and the centrifugation time is 5 - 30 min.

[0014] Based on the above technical solution, further, the zinc salt described in step (2) is at least one of zinc sulfate, zinc acetate, zinc nitrate, zinc chloride and zinc phenolsulfonate.

[0015] Based on the above technical solution, further, the volume ratio of the ginkgo leaf extract to the zinc salt aqueous solution in step (2) is 100:1 to 10:1; the zinc ion concentration in the zinc salt aqueous solution is 1 to 100 mg / ml, preferably 1 to 10 mg / ml.

[0016] Based on the above technical solution, further, the conditions of the ultrasonic wave in step (2) are that the ultrasonic wave frequency is 25 to 80 KHz, the ultrasonic power is 54 to 1080 W, the rotation speed of the stirring is 100 to 1000 rpm; the reduction reaction time is 10 to 60 min.

[0017] Based on the above technical solution, further, the addition amount of vitamin C in step (2) is 1 to 30 g / L, and the addition amount of N-acetylcysteine is 20 to 80 g / L.

[0018] Based on the above technical solution, further, the anti-inflammatory, antipyretic and analgesic components in step (2) include acetylsalicylic acid, arginine aspirin and carbasalate calcium.

[0019] In the second aspect, the present invention provides the nano zinc solution and the compound nano zinc solution prepared by the above preparation method.

[0020] Based on the above technical solution, further, the average particle size of the nano zinc and the compound nano zinc is 5 to 40 nm, and the Zeta potential is -2.0 to -9.9 mV.

[0021] In the third aspect, the present invention provides a preparation method of a nano zinc gel and a compound nano zinc gel, which is prepared by uniformly mixing the above nano zinc solution or compound nano zinc solution with an equal mass of 1 wt% to 5 wt% sodium hyaluronate hydrogel to obtain the nano zinc gel or the compound nano zinc gel.

[0022] Based on the above technical solution, further, the preparation process of the sodium hyaluronate hydrogel is as follows: add purified water to sodium hyaluronate with a molecular weight of 1.2 million, stir evenly, place at room temperature for 10 to 20 h, and stir again for 5 to 30 min after complete dissolution to obtain the sodium hyaluronate hydrogel.

[0023] Based on the above technical solution, further, the rotation speed of the stirring is 100 to 1000 rpm.

[0024] In the fourth aspect, the present invention provides the nano zinc gel and the compound nano zinc gel prepared by the above preparation method.

[0025] Based on the above technical solution, further, the pH of the nano zinc gel and the compound nano zinc gel is 4 to 7, and the viscosity is 20000 to 150000 mPa·s.

[0026] Fifth aspect, the present invention provides the application of the above-mentioned nano zinc solution, compound nano zinc solution, nano zinc gel and compound nano zinc gel in the preparation of antibacterial and antiviral materials or drugs.

[0027] Based on the above technical solutions, further, the bacteria include Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans, and the viruses include influenza virus H3N2, parainfluenza virus, Newcastle disease virus and HPV virus.

[0028] The beneficial effects of the present invention compared with the prior art are as follows:

[0029] 1. The nano zinc prepared by the present invention using ginkgo leaf extract is a green and environment-friendly product. The preparation process is simple, with less equipment investment. The whole preparation process is pollution-free and does not contain any harmful chemical components. The nano zinc prepared by ginkgo leaf extract is safe and non-toxic, and has high-efficiency and broad-spectrum antibacterial and antiviral properties. The killing rates of Gram-positive bacteria, Gram-negative bacteria and fungi are all above 99.95%, and it has an obvious inhibitory effect on clinically common drug-resistant bacteria. It has a high-efficiency and broad-spectrum killing effect on various human viruses and animal viruses. The nano zinc prepared by ginkgo leaf extract has important development value, and can be developed into high-efficiency and broad-spectrum antibacterial and antiviral preparations, and has a wide application prospect in the prevention and treatment of early bacterial infections and virus infections in the fields of clinical medicine and agriculture and animal husbandry.

[0030] 2. The compound nano zinc solution prepared by the present invention using ginkgo leaf extract combined with vitamin C, N-acetylcysteine and acetylsalicylic acid is safe and non-toxic, and has high-efficiency and broad-spectrum antibacterial and antiviral properties. The killing rates of Gram-positive bacteria, Gram-negative bacteria and fungi are between 99.95% and 99.99%, and it also has an obvious inhibitory effect on clinically common drug-resistant bacteria. It has a high-efficiency and broad-spectrum killing effect on various human viruses and animal viruses, and at the same time has anti-inflammatory and analgesic effects. The compound nano zinc solution prepared by the present invention can be further developed into a preparation or drug for treating respiratory viruses.

[0031] 3. The nano-zinc gel prepared from the ginkgo leaf extract in the present invention and the compound nano-zinc gel prepared by combining vitamin C, N-acetylcysteine, acetylsalicylic acid and sodium hyaluronate are safe and non-toxic, and have highly efficient and broad-spectrum antibacterial and antiviral properties. The killing rates against Gram-positive bacteria, Gram-negative bacteria and fungi are all greater than 99.95%, and there is also an obvious inhibitory effect on clinically common drug-resistant bacteria. The nano-zinc gel and the compound nano-zinc gel prepared in the present invention have a highly efficient killing effect on human papillomavirus, and at the same time have anti-inflammatory and analgesic effects. The nano-zinc and the compound nano-zinc gel prepared in the present invention can be further developed into urogenital antibacterial and antiviral preparations or drugs for preventing and treating human papillomavirus, herpes simplex virus and HIV infections. The nano-zinc and the compound nano-zinc gel prepared in the present invention can also be further developed into antibacterial preparations for promoting wound and wound surface healing and preparations or drugs for treating hemorrhoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention, the drawings involved in the embodiments will be briefly introduced below.

[0033] Figure 1 Particle size (A) and Zeta potential (B) of nano-zinc.

[0034] Figure 2 Particle size (A) and Zeta potential (B) of compound nano-zinc.

[0035] Figure 3 It is a detection result diagram of the broad-spectrum antibacterial effect of nano-zinc. Among them, A is Escherichia coli; B is Staphylococcus aureus; C is Candida albicans; 0: zinc oxide ointment, 1: nano-zinc solution, 2: compound nano-zinc solution, 3: nano-zinc gel, 4: compound nano-zinc gel.

[0036] Figure 4 It is a detection result diagram of the inhibitory effect of nano-zinc on clinically drug-resistant bacteria. Among them, A is methicillin-resistant Staphylococcus aureus, B is Escherichia coli producing ESBLs, C is multi-drug resistant Pseudomonas aeruginosa, D is Klebsiella pneumoniae producing ESBLs, E is multi-drug resistant Acinetobacter baumannii; 1: nano-zinc solution, 2: compound nano-zinc solution, 3: nano-zinc gel, 4: compound nano-zinc gel.

[0037] Figure 5 It is a detection diagram of the broad-spectrum anti-respiratory virus effect of nano-zinc solution and compound nano-zinc solution. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The present invention will be described in detail below in conjunction with embodiments. However, the implementation manners of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, without creative efforts, obtaining other similar embodiments will fall within the protection scope of the present invention.

[0039] Example 1: Preparation of Ginkgo biloba Extract

[0040] After the fresh Ginkgo biloba leaves are fully dried in the air and then fully rinsed with water, they are dried at 80 °C for 6 hours. The Ginkgo biloba leaves are crushed by a grinder for 10 minutes to obtain Ginkgo biloba dry powder. Then, the Ginkgo biloba dry powder is made into a 1 wt% solution with purified water, and under the condition of 100 °C, it is heated gently for 30 minutes. Then, the Ginkgo biloba extract is placed at 4 °C for 12 hours, and then filtered through a double-layer filter paper. After centrifuging at 4000 rpm for 15 minutes, the supernatant is taken and sterilized with a 0.25 μm filter membrane for standby.

[0041] Example 2: Preparation of Nano-Zinc Solution Using Ginkgo biloba Extract

[0042] 1000 ml of zinc sulfate solution containing 5 mg / ml of zinc is prepared by dissolving zinc sulfate in purified water. Under the conditions of ultrasonic wave (frequency 40 KHz, power 1080 W) and magnetic stirring (600 rpm), 50 ml of 1 wt% Ginkgo biloba extract is slowly added to the zinc sulfate solution, and the reduction is continued for 30 minutes to prepare a nano-zinc solution. The pH of this nano-zinc solution is 6.5, and it is placed at room temperature for 12 months without obvious deposition.

[0043] Example 3: Preparation of Compound Nano-Zinc Solution

[0044] 1) Preparation of Solution A: 1000 ml of zinc sulfate solution containing 10 mg / ml of zinc is prepared by dissolving zinc sulfate in purified water. Under the conditions of ultrasonic wave (frequency 40 KHz, power 1080 W) and magnetic stirring (600 rpm), 50 ml of 1 wt% Ginkgo biloba extract is slowly added to the zinc sulfate solution. After continuous reduction for 15 minutes, 10 grams of vitamin C is added. After it is fully dissolved, 50 grams of N-acetylcysteine is added. Under the conditions of ultrasonic wave and magnetic stirring, the reduction is continued for 15 minutes to prepare nano-zinc Solution A.

[0045] 2) Preparation of Solution B: 0.5 wt% acetylsalicylic acid is prepared with purified water. Under the condition of 45 °C, with the aid of ultrasonic wave (frequency 40 KHz, power 1080 W) and magnetic stirring (600 rpm), the action is continued for 30 minutes to make acetylsalicylic acid fully dissolve.

[0046] 3) Solution A and Solution B are fully mixed in equal volume to obtain a compound nano-zinc solution.

[0047] The pH of this compound nano-zinc solution is 5.0. After being placed at room temperature for 12 months, there is no obvious deposition.

[0048] Example 4: Preparation of nano-zinc gel

[0049] 1) Preparation of Solution A: 1000 ml of zinc sulfate solution containing 5 mg / ml of zinc was prepared with purified water. Under the conditions of ultrasonic wave (frequency 40 KHz, power 1080 W) and magnetic stirring (600 rpm), 50 ml of 1 wt% ginkgo biloba extract was slowly added to the zinc sulfate solution, and reduction was continued for 30 minutes to prepare nano-zinc Solution A.

[0050] 2) Preparation of sodium hyaluronate gel: 30 grams of sodium hyaluronate with a molecular weight of 1.2 million was put into 1000 ml of purified water, stirred evenly, and left at room temperature for 12 hours to fully dissolve. After 12 hours, it was stirred again for 15 minutes to prepare 3% sodium hyaluronate gel.

[0051] 3) Solution A and 3% sodium hyaluronate gel were mixed in equal mass and stirred evenly for 15 minutes to obtain nano-zinc gel.

[0052] The viscosity of this nano-zinc gel is 65000 mPa.s, pH 6.5. After being placed at room temperature for 12 months, the gel property is stable and there is no liquefaction.

[0053] Example 5: Preparation of compound nano-zinc gel

[0054] 1) Preparation of Solution A: 1000 ml of zinc sulfate solution containing 10 mg / ml of zinc was prepared with purified water. Under the conditions of ultrasonic wave (frequency 40 KHz, power 1080 W) and magnetic stirring (600 rpm), 50 ml of 1 wt% ginkgo biloba extract was slowly added to the zinc sulfate solution. After reduction for 15 minutes, 10 grams of vitamin C was added. After it was fully dissolved, 50 grams of N-acetylcysteine was added. Under the conditions of ultrasonic wave and magnetic stirring, reduction was continued for 15 minutes to prepare nano-zinc Solution A.

[0055] 2) Preparation of Solution B: 0.5% acetylsalicylic acid was prepared with purified water. Under the conditions of ultrasonic wave (frequency 40 KHz, power 1080 W) and magnetic stirring (600 rpm) at 45°C, it was continuously acted for 30 minutes to fully dissolve acetylsalicylic acid.

[0056] 3) Solution A and Solution B were fully mixed in equal volume to obtain compound nano-zinc solution.

[0057] 4) Preparation of sodium hyaluronate gel: Take 30 grams of sodium hyaluronate with a molecular weight of 1.2 million, put it into 1000 ml of purified water, stir well, and let it stand at room temperature for 12 hours to fully dissolve. After 12 hours, stir well again for 15 minutes to prepare a 3% sodium hyaluronate gel.

[0058] 5) Mix the compound nano-zinc solution and 3% sodium hyaluronate gel in equal mass, and stir well for 15 minutes to obtain the compound nano-zinc gel.

[0059] The viscosity of the compound nano-zinc gel is 64000 mPa·s, pH is 5.0, and it is placed at room temperature for 12 months. The gel property is stable and there is no liquefaction.

[0060] Example 6: Detection of particle size and Zeta potential of nano-zinc and compound nano-zinc

[0061] Use a BeNano laser particle size analyzer to detect the particle size, distribution and Zeta potential of the nano-zinc solution prepared in Example 2 and the compound nano-zinc solution prepared in Example 3.

[0062] The test results are as Figure 1-2 shown. The results show that the particle size of the nano-zinc solution is mainly distributed between 5 and 40 nm, the average particle size is 12.54 nm, and the Zeta potential is -9.6302 mV ( Figure 1 A and Figure 1 B). The particle size of the compound nano-zinc is mainly distributed between 10 and 20 nm, the average particle size is 18.12 nm, and the Zeta potential is -2.2532 mV ( Figure 2 A and Figure 2 B).

[0063] Example 7: Detection of broad-spectrum antibacterial effect of nano-zinc and compound nano-zinc

[0064] 1. Inhibitory zone test

[0065] Dilute the broth cultures of Escherichia coli and Staphylococcus aureus and the Candida albicans solution in the logarithmic growth phase to 10 6 CFU / ml respectively. Take 100 μL of each and spread it evenly on the surface of nutrient agar medium and Sabouraud medium. Then use a sterile punch to punch holes on the surface of the medium and pick out the medium in the holes. Take the nano-zinc solution, compound nano-zinc composite solution, nano-zinc gel, compound nano-zinc gel and 15% zinc oxide ointment (drug with approval number) prepared in Example 2, Example 3, Example 4 and Example 5 respectively to fill each hole, 80 μL or 80 μg per hole. Then place the medium in an incubator at 37°C overnight. Take out the medium the next day and measure the diameter of the inhibitory zone of each hole.

[0066] The results of the inhibitory zone test are shown in Table 1 and Figure 3, The results showed that nano-zinc solution, compound nano-zinc solution, nano-zinc gel and compound nano-zinc gel all had very obvious inhibitory effects on Escherichia coli, Staphylococcus aureus and Candida albicans. In the experiment, the zinc content of zinc oxide ointment was 24 times or 48 times higher than that of nano-zinc, but no obvious antibacterial effect was shown, which might be related to the particle size and dosage form of zinc oxide.

[0067] Table 1 Broad-spectrum antibacterial effects of nano-zinc and compound nano-zinc

[0068]

[0069] 2. Determination of antibacterial rate

[0070] 1. Detection basis: Appendix C of GB15979-2002 Hygiene Standard for Disposable Sanitary Products.

[0071] 2. Test method:

[0072] The test was carried out in an electrothermal constant temperature water bath at 20°C ± 1°C. Respectively take 5.0 mL of the nano-zinc solution, compound nano-zinc solution prepared in Examples 2-5, or 5 g of nano-zinc gel and compound nano-zinc gel, add them into a sterilized test tube, and then add 100 μL of bacterial suspension (bacterial suspension concentration 1×10 4 CFU / mL~9×10 4 CFU / mL) of Escherichia coli 8099, Staphylococcus aureus ATCC 6538 or Candida albicans to each tube. After acting for the predetermined time (5 min, 10 min and 20 min), take 0.5 mL of the bacterial drug suspension and add it to 4.5 mL of neutralizer (D / E neutralizing broth). After neutralizing for 10 min, take 1 mL of the sample, pour it onto a nutrient agar medium, and after culturing at 37°C, check the number of residual viable bacteria. Positive and negative controls were set simultaneously in the test; the test was repeated 3 times.

[0073] 3. Results:

[0074] The specific test results are shown in Tables 2 to 5. Under the condition of 20°C ± 1°C, the average bactericidal rates of nano-zinc solution, compound nano-zinc solution, nano-zinc gel and compound nano-zinc gel acting on Escherichia coli, Staphylococcus aureus and Candida albicans for 5 min, 10 min and 20 min were all >99.95%.

[0075] Table 2 Bactericidal effects of nano-zinc solution on bacteria and fungi

[0076]

[0077] Note: ① No colonies grew in the negative control; ② The average value was taken after repeating the test 3 times.

[0078] Table 3 Bactericidal effects of compound nano-zinc solution on bacteria and fungi

[0079]

[0080] Note: ① No colonies grew in the negative control; ② The experiment was repeated 3 times and the average value was taken.

[0081] Table 4 Bactericidal effects of nano-zinc gel against bacteria and fungi

[0082]

[0083] Note: ① No colonies grew in the negative control; ② The experiment was repeated 3 times and the average value was taken.

[0084] Table 5 Bactericidal effects of compound nano-zinc gel against bacteria and fungi

[0085]

[0086] Note: ① No colonies grew in the negative control; ② The experiment was repeated 3 times and the average value was taken.

[0087] Example 8: Detection of inhibitory effects of nano-zinc and compound nano-zinc on clinical drug-resistant bacteria

[0088] Inhibitory zone test

[0089] 1. Test strains: Methicillin-resistant Staphylococcus aureus, ESBLs-producing Escherichia coli, ESBLs-producing Klebsiella pneumoniae, multidrug-resistant Pseudomonas aeruginosa, and multidrug-resistant Acinetobacter baumannii were provided by Xinhua Hospital Affiliated to Dalian University.

[0090] 2. Test samples: Nano-zinc solution (zinc content 0.5%), nano-zinc composite solution (zinc content 0.5%), nano-zinc gel (zinc content 0.25%), and nano-zinc composite gel (zinc content 0.25%).

[0091] 3. Test method: The nutrient broth cultures of Methicillin-resistant Staphylococcus aureus, ESBLs-producing Escherichia coli, ESBLs-producing Klebsiella pneumoniae, multidrug-resistant Pseudomonas aeruginosa, and multidrug-resistant Acinetobacter baumannii in the logarithmic growth phase were respectively diluted to 10 6 CFU / ml. 100 μL was respectively taken and evenly spread on the nutrient agar medium. Then, a sterile puncher was used to punch holes on the surface of the medium and the medium in the holes was picked out. 80 μL or 80 μg of the nano-zinc solution, compound nano-zinc solution, nano-zinc gel, and compound nano-zinc gel prepared in Example 2, Example 3, Example 4, and Example 5 were respectively taken and filled into each hole. Then, the medium was cultured overnight at 37°C. The next day, the medium was taken out and the diameter of the inhibitory zone of each hole was measured.

[0092] The test results are shown in Table 6 and Figure 4, The results showed that nano-zinc solution, compound nano-zinc solution, nano-zinc gel and compound nano-zinc gel all had obvious antibacterial effects against five common clinically drug-resistant bacteria (methicillin-resistant Staphylococcus aureus, ESBLs-producing Escherichia coli, ESBLs-producing Klebsiella pneumoniae, multi-drug resistant Pseudomonas aeruginosa, multi-drug resistant Acinetobacter baumannii).

[0093] Table 6 Inhibitory effects of nano-zinc on five clinically drug-resistant bacteria

[0094]

[0095] Example 9: Detection of broad-spectrum antiviral effects of nano-zinc and compound nano-zinc

[0096] I. Detection of broad-spectrum inhibitory effects of nano-zinc and compound nano-zinc on respiratory viruses

[0097] 1. Test virus strains: Influenza virus H3N2 and parainfluenza virus were provided by Wuhan Institute of Virology, Chinese Academy of Sciences;

[0098] Newcastle disease virus (low virulence strain) was provided by the Department of Pathogenic Biology, Dalian University School of Medicine.

[0099] 2. Test samples: Nano-zinc solution (zinc content 0.5%) and compound nano-zinc solution (zinc content 0.5%).

[0100] 3. Test method: Nano-zinc solution and compound nano-zinc solution were respectively mixed thoroughly with an equal volume of influenza virus H3N2 (hemagglutination titer 1:1024), parainfluenza virus (hemagglutination titer 1:1024) and Newcastle disease virus (hemagglutination titer 1:1024). After acting at room temperature for 2 h, the above mixtures were then diluted 1:10 with sterile normal saline. 0.1 mL was taken and injected into the allantoic cavity of chicken embryos. Chicken embryos in the virus control group were respectively injected with 0.1 mL of influenza virus H3N2, parainfluenza virus, and Newcastle disease virus with the same dilution multiple, and incubated at 37 °C for 72 h. Then, allantoic fluid was harvested by the conventional method. The harvested allantoic fluid of chicken embryos was diluted 10-fold with sterile normal saline, and then hemagglutination test was carried out by the conventional method.

[0101] The test results are shown in Figure 5 , and the results showed that nano-zinc solution and compound nano-zinc solution both had obvious inhibitory effects on influenza virus H3N2, parainfluenza virus and Newcastle disease virus, and the hemagglutination titers were all lower than 1:20. The hemagglutination titers of the three virus control groups were all between 1:640 and 1:1280. When comparing the hemagglutination titers of the test group and the virus control group, the P values were all less than 0.01. The test results showed that nano-zinc solution and compound nano-zinc solution had obvious inactivating effects on influenza virus H3N2, parainfluenza virus and Newcastle disease virus.

[0102] II. Detection of the Effect of Nano-Zinc Gel and Compound Nano-Zinc Gel on Inactivating Human Papillomavirus Type 16

[0103] 1. Experimental Materials

[0104] 1.1 Samples: Nano-zinc gel (zinc content 0.25%) and compound nano-zinc gel (zinc content 0.25%).

[0105] 1.2 Cells: 293FT cells were provided by Wuhan Welllead Biopharmaceutical Co., Ltd.

[0106] 1.3 Virus: Human papillomavirus type 16 pseudovirus (HPV16pp) was provided by Wuhan Welllead Biopharmaceutical Co., Ltd.

[0107] 1.4 Experimental reagents: DMEM medium, fetal bovine serum and other experimental reagents were provided by our laboratory.

[0108] 2. Experimental Principles and Methods

[0109] 2.1 Experimental Principle

[0110] After the HPV pseudovirus particles containing the reporter gene GFP infect 293FT cells, the infected cells will express green fluorescent protein. By observing the number of cells expressing GFP green fluorescence under a fluorescence microscope, the infection situation of the HPV virus can be reflected.

[0111] 2.2 Experimental Procedures

[0112] 1) 293FT cells were seeded in a 96-well cell culture plate and reserved after cell attachment.

[0113] 2) Take 10 μl of HPV16 pseudovirus solution and add it to 90 μl of sample (nano-zinc gel or compound nano-zinc gel) or 90 μl of PBS. After stirring and mixing evenly, incubate in a water bath at 37°C for 2 hours, then dilute with 900 μl of medium as the first concentration, and then perform 10-fold and 3-fold serial dilutions. Take 100 μl of the serially diluted virus solution and add it to the cells to measure the residual virus titer. Six wells were set for detection at each concentration gradient and cultured in a CO2 incubator at 37°C. After culturing for 72 h, use a fluorescence microscope to count the number of fluorescent cells and calculate the FFU (fluorescent focus unit) of the virus. (Operating basis: "Disinfection Technical Specification" 2002 Edition).

[0114] The detection results of the inactivation of HPV activity by nano-zinc gel and compound nano-zinc gel are shown in Table 7.

[0115] Table 7 Inactivation of HPV by Nano-Zinc Gel and Compound Nano-Zinc Gel

[0116]

[0117]

[0118] Note: “+” indicates the presence of fluorescent spots, “-” indicates the absence of fluorescent spots, and the numerical value represents the number of fluorescent spots counted per well.

[0119] 3. Test Results

[0120] In this experiment, the inactivating effect of nano-zinc gel and compound nano-zinc gel on HPV type 16 pseudovirus was detected at the cellular level. The test results showed that the virus titer of the virus control group was 4.05×10 6 FFU / ml, and the virus titers of the tested samples, nano-zinc gel and compound nano-zinc gel groups, were both less than 1.67×10 3 FFU / ml. Compared with the virus control group, the reduction of the titer of HPV type 16 pseudovirus by the tested samples, nano-zinc gel and compound nano-zinc gel, was greater than 3.38 log values under the condition of 37°C water bath for 2 hours, and the inactivation rate was greater than 99.96%.

[0121] Example 10: Acute Oral Toxicity Test of Nano-Zinc Solution and Nano-Zinc Composite Solution

[0122] I. Materials and Animals

[0123] 1. Samples: Nano-zinc solution (zinc content 0.5%) and compound nano-zinc solution (zinc content 0.5%).

[0124] 2. Animals: KM mice, provided by the Experimental Animal Center of Dalian Medical University, experimental animal production license number: SCXK(Liao)2022-0002.

[0125] II. Methods

[0126] 1. Test basis: "Disinfection Technical Specification" (2002 edition) 2.3.1.

[0127] 2. Detection concentration: Test with the original sample solution.

[0128] 3. Test methods:

[0129] The single maximum dose test method was adopted, and the animals were gavaged orally with the test substance at a dose of 5000 mg / kg body weight. Forty KM mice were selected, with 20 males and 20 females, weighing 19.4±0.7 (g), and fasted overnight. An appropriate amount of the test substance was prepared into a solution with the required concentration using pure water, and the mice were given a single oral gavage at a dose of 0.2 ml / 10 g body weight. The poisoning symptoms and death conditions of the animals were observed within 14 days after administration.

[0130] III. Results

[0131] After oral administration at a dose of 5000 mg / kg body weight, no obvious poisoning symptoms or deaths were observed in the test animals within the 14-day observation period, and no abnormalities were found in their body weights. At the end of the observation period, the animals were subjected to gross anatomical examination, and their main organs were observed macroscopically, and no abnormal changes were found. The specific results are shown in Table 8. According to the acute oral toxicity test classification standard in the "Disinfection Technical Specification" (2002 edition), the acute oral LD 50 of the sample nano-zinc solution and the original solution of the compound nano-zinc solution for KM mice was greater than 5000 mg / kg body weight, and the test samples were actually non-toxic.

[0132] Table 8 Results of acute oral toxicity test

[0133]

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing broad-spectrum antibacterial and antiviral nano zinc and compound nano zinc, characterized in that: The steps include: (1) drying and then crushing the ginkgo leaves, adding water to the obtained ginkgo leaf powder to prepare a 0.1 wt% to 5 wt% solution, extracting at 80 to 100° C. for 10 to 60 minutes, then leaving at 1 to 5° C. for 10 to 24 hours, filtering, centrifuging, and sterilizing the obtained supernatant to obtain a ginkgo leaf extract; (2) mixing the ginkgo leaf extract obtained in step (1) with a zinc salt aqueous solution, and performing a reduction reaction under the action of ultrasound and stirring to prepare a stable nano zinc solution; Alternatively, the ginkgo leaf extract obtained in step (1) is mixed with a zinc salt aqueous solution, and a reduction reaction is carried out under the action of ultrasound and stirring to prepare a stable nano zinc solution, vitamin C and N-acetylcysteine ​​are added to the obtained nano zinc solution, and the reduction reaction is continued under the action of ultrasound and stirring, and the obtained solution is mixed evenly with an equal volume of an aqueous solution containing 0.1wt% to 2wt% of an anti-inflammatory, antipyretic and analgesic component to obtain a composite nano zinc solution.

2. The preparation method according to claim 1, characterized in that: The content of ginkgo leaf powder in the solution in step (1) is 0.5wt% to 2wt%; the centrifugal conditions are: a rotation speed of 2000 to 6000 rpm and a centrifugal time of 5 to 30 minutes.

3. The preparation method according to claim 1, characterized in that: The zinc salt described in step (2) is at least one of zinc sulfate, zinc acetate, zinc nitrate, zinc chloride and zinc phenolsulfonate; the volume ratio of the ginkgo leaf extract to the zinc salt aqueous solution is 100:1-10:1; the zinc ion concentration in the zinc salt aqueous solution is 1-100 mg / ml, preferably 1-10 mg / ml.

4. The preparation method according to claim 1, characterized in that: The ultrasonic conditions in step (2) are as follows: ultrasonic frequency of 25 to 80 KHz, ultrasonic power of 54 to 1080 W, stirring speed of 100 to 1000 rpm; and reduction reaction time of 10 to 60 min.

5. The preparation method according to claim 1, characterized in that: The amount of vitamin C added in step (2) is 1-30 g / L, and the amount of N-acetylcysteine ​​added is 20-80 g / L; the anti-inflammatory, antipyretic and analgesic components include acetylsalicylic acid, arginine aspirin and carbazine calcium.

6. The nano zinc solution and compound nano zinc solution prepared by the preparation method according to any one of claims 1 to 5.

7. A method for preparing nano zinc gel and compound nano zinc gel, characterized in that: The nano zinc gel or the compound nano zinc gel is prepared by uniformly mixing the nano zinc solution or the compound nano zinc solution according to claim 6 with an equal mass of 1wt% to 5wt% sodium hyaluronate hydrogel.

8. The preparation method according to claim 7, characterized in that: The preparation process of the sodium hyaluronate hydrogel is as follows: adding purified water to sodium hyaluronate with a molecular weight of 1.2 million, stirring evenly, leaving at room temperature for 10 to 20 hours, stirring again for 5 to 30 minutes after being fully dissolved, and preparing the sodium hyaluronate hydrogel.

9. Nano zinc gel and compound nano zinc gel prepared by the preparation method according to claim 7 or 8.

10. Use of the nano zinc solution and compound nano zinc solution according to claim 6, and the nano zinc gel and compound nano zinc gel according to claim 9 in the preparation of antibacterial and antiviral materials or drugs.

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