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

Abstract

The application discloses a kind of preparation and application of broad-spectrum antibacterial antiviral nanometer zinc and compound nanometer zinc, belongs to the technical field of medical materials.The ginkgo leaf extract is mixed with zinc salt aqueous solution, reduction reaction is carried out under the action of ultrasonic and stirring, and nanometer zinc solution is obtained.Vitamin C and N-acetyl cysteine are added to nanometer zinc solution, reduction reaction is continuously carried out under the action of ultrasonic and stirring, then mixed with equal volume of water solution containing anti-inflammatory antipyretic analgesic component uniformly, and compound nanometer zinc solution is obtained.Nanometer zinc solution or compound nanometer zinc solution obtained is mixed with equal mass of sodium hyaluronate hydrogel uniformly to obtain nanometer zinc gel or compound nanometer zinc gel.The method of the application avoids the toxic side effects caused by using chemical reducing agents such as hydrazine hydrate and sodium borohydride for preparing nanometer zinc.The nanometer zinc and compound nanometer zinc of the application have excellent characteristics of broad-spectrum and high-efficiency antibacterial antiviral, and have good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical materials, and particularly relates to preparation and application of broad-spectrum antibacterial and antiviral nano-zinc and compound nano-zinc. BACKGROUND

[0002] Zinc is an essential trace element for human body, and plays a very important role in important physiological processes such as human growth and development, reproduction and heredity, immunity, and neuroendocrine. Human body contains 2-3 grams of zinc, and zinc deficiency can lead to growth and development stagnation, low intelligence, and neuroendocrine dysfunction. Therefore, zinc is often used to supplement the lack of zinc elements in the human body. In recent years, the high-efficiency antibacterial and antiviral properties of nano-silver have attracted widespread attention. Relatively speaking, the preparation and antibacterial and antiviral properties of nano-zinc have not been widely concerned and deeply studied. Although the antibacterial and antiviral effect of nano-zinc is not as efficient as that of nano-silver, nano-zinc has many advantages that cannot be compared with nano-silver. The cytotoxicity of nano-zinc is far less than that of nano-silver, and nano-zinc can enhance the regeneration capacity of human tissues, promote protein synthesis, accelerate cell division and proliferation, and promote rapid wound healing. Nano-zinc can also provide immune function and enhance the body's resistance to bacterial and viral infections.

[0003] Nanoparticles refer to ultra-fine particles of 1-100 nm, and nanoparticles have some new and different physical and chemical properties, which are obviously different from the physical and chemical properties of macroscopic 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 prospect in the fields of new materials, biology and clinical medicine.

[0004] At present, the commonly used preparation method can obtain nano-zinc with a wide particle size range, and the particle size is generally between tens of nanometers and hundreds of nanometers. The large particle size can lead to unstable nano-zinc solution, particle deposition, and reduced antibacterial and antiviral effect. Therefore, it is urgent to develop a new preparation method of nano-zinc and compound nano-zinc.

[0005] Ginkgo biloba is a plant with high medicinal value, and has the effects of expanding blood vessels and promoting blood circulation to remove blood stasis. The natural active flavonoids contained in Ginkgo biloba have strong antioxidant effect. Ginkgo biloba extract, vitamin C and N-acetylcysteine all have strong reducing property. So far, there has been no report on the preparation of nano-zinc by reducing zinc ions with Ginkgo biloba extract, vitamin C and N-acetylcysteine. SUMMARY

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

[0007] The purpose of the present application is achieved by the following means:

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

[0009] (1) After the ginkgo biloba leaves are dried and crushed, the obtained ginkgo biloba powder is mixed with water to form a 0.1wt%-5wt% solution, which is extracted at 80-100℃ for 10-60min, and then placed at 1-5℃ for 10-24h, filtered and centrifuged, and the obtained supernatant is sterilized to obtain ginkgo biloba extract;

[0010] (2) The ginkgo biloba extract obtained in step (1) is mixed with an aqueous zinc salt solution, and a reduction reaction is carried out under the action of ultrasonic and stirring to prepare a stable nano-zinc solution;

[0011] Alternatively, the ginkgo biloba extract obtained in step (1) is mixed with an aqueous zinc salt solution, and a reduction reaction is carried out under the action of ultrasonic and stirring to prepare a stable nano-zinc solution, and then vitamin C and N-acetylcysteine are added to the obtained nano-zinc solution, and a reduction reaction is continued under the action of ultrasonic and stirring, and the obtained solution is mixed with an equal volume of an aqueous solution containing 0.1wt%-2wt% anti-inflammatory, antipyretic and analgesic components to obtain a compound nano-zinc solution.

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

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

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

[0015] Based on the above technical scheme, further, the volume ratio of the ginkgo leaf extract solution to the zinc salt aqueous solution in step (2) 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.

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

[0017] Based on the above technical scheme, further, the adding amount of vitamin C in step (2) is 1-30 g / L, and the adding amount of N-acetyl cysteine is 20-80 g / L.

[0018] Based on the above technical scheme, further, the anti-inflammatory, antipyretic and analgesic component in step (2) comprises acetylsalicylic acid, arginine aspirin and calcium carbaspirin.

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

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

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

[0022] Based on the above technical scheme, further, the preparation process of the sodium hyaluronate hydrogel is as follows: adding purified water into sodium hyaluronate with a molecular weight of 1.2 million, uniformly stirring, placing at room temperature for 10-20 h, fully dissolving, and then stirring again for 5-30 min to prepare the sodium hyaluronate hydrogel.

[0023] Based on the above technical scheme, further, the stirring speed is 100-1000 rpm.

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

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

[0026] In a fifth aspect, the application provides use of the above-mentioned nano-zinc solution, compound nano-zinc solution, nano-zinc gel and compound nano-zinc gel in preparation of antibacterial and antiviral materials or drugs.

[0027] Based on the above technical solution, 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 application has the following beneficial effects relative to the prior art:

[0029] 1. The nano-zinc prepared by the ginkgo leaf extract is a green and environmentally-friendly product, and has simple preparation process, low equipment investment, and no pollution in the whole preparation process, and does not contain any harmful chemical components; the nano-zinc prepared by the ginkgo leaf extract is safe and non-toxic, has high efficient and broad spectrum antibacterial and antiviral properties, and has a killing rate of more than 99.95% on gram-positive bacteria, gram-negative bacteria and fungi, and has obvious inhibitory effect on clinically common drug-resistant bacteria; has high efficient and broad spectrum killing effect on various human viruses and animal viruses; the nano-zinc prepared by the ginkgo leaf extract has important development value, can develop high efficient and broad spectrum antibacterial preparations and antiviral preparations, and has wide application prospect in the fields of clinical medicine and agricultural and animal husbandry for preventing and treating early bacterial infection and viral infection.

[0030] 2. The compound nano-zinc solution prepared by the ginkgo leaf extract combined with vitamin C, N-acetylcysteine and acetylsalicylic acid is safe and non-toxic, has high efficient and broad spectrum antibacterial and antiviral properties, and has a killing rate of 99.95%-99.99% on gram-positive bacteria, gram-negative bacteria and fungi, also has obvious inhibitory effect on clinically common drug-resistant bacteria, has high efficient and broad spectrum killing effect on various human viruses and animal viruses, and has anti-inflammatory and analgesic effects; the compound nano-zinc solution prepared by the application can be further developed into a preparation or drug for treating respiratory viruses.

[0031] 3. The nano-zinc gel prepared by the ginkgo biloba extract and the compound nano-zinc gel prepared by combining vitamin C, N-acetyl cysteine, acetylsalicylic acid and sodium hyaluronate are safe and non-toxic, have high efficient and broad spectrum antibacterial and antiviral properties, and have a killing rate of more than 99.95% on gram-positive bacteria, gram-negative bacteria and fungi, and have obvious inhibitory effect on clinically common drug-resistant bacteria. The nano-zinc gel and the compound nano-zinc gel prepared by the application have a high efficient killing effect on human papilloma virus, and have anti-inflammatory and analgesic effects. The nano-zinc and the compound nano-zinc gel prepared by the application can be further developed into urogenital antibacterial and antiviral preparations or drugs for preventing and treating human papilloma virus, herpes simplex virus and HIV infection. The nano-zinc and the compound nano-zinc gel prepared by the application can also be further developed into antibacterial preparations for promoting wound and wound healing and preparations or drugs for treating hemorrhoids. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

[0035] Figure 3 The broad spectrum antibacterial effect detection result diagram of the nano-zinc, wherein A is Escherichia coli, B is Staphylococcus aureus, C is white Candida beads, 0 is zinc oxide ointment, 1 is nano-zinc solution, 2 is compound nano-zinc solution, 3 is nano-zinc gel, and 4 is compound nano-zinc gel.

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

[0037] Figure 5 The broad spectrum antiviral effect detection diagram of the nano-zinc solution and the compound nano-zinc solution. DETAILED DESCRIPTION

[0038] The application will be described in detail below with reference to the embodiments, but the embodiments of the application are not limited thereto. It is obvious that the embodiments described below are only some of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0039] Example 1: Preparation of ginkgo leaf extract

[0040] Fresh ginkgo leaves were fully air-dried, washed with water, and dried at 80°C for 6 hours. The ginkgo leaves were broken and ground into powder with a grinder for 10 minutes, and then the ginkgo leaf powder was dissolved in purified water to prepare a 1wt% solution. The solution was heated at 100°C for 30 minutes with a small fire, and then was placed at 4°C for 12 hours. The solution was filtered with double-layer filter paper, and centrifuged at 4000rpm for 15 minutes. The supernatant was sterilized with a 0.25μm filter membrane and was ready for use.

[0041] Example 2: Preparation of nano-zinc solution with ginkgo leaf extract

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

[0043] Example 3: Preparation of compound nano-zinc solution

[0044] 1) Preparation of A liquid: Zinc sulfate was dissolved in purified water to prepare a 1000ml zinc sulfate solution containing 10mg / ml zinc. Under the conditions of ultrasonic wave (frequency 40KHz, power 1080W) and magnetic stirring (600rpm), 50ml of 1wt% ginkgo leaf extract was slowly added to the zinc sulfate solution, and the reduction was continued for 15 minutes. Then, 10g of vitamin C was added and dissolved, and 50g of N-acetylcysteine was added. The reduction was continued for 15 minutes under the conditions of ultrasonic wave and magnetic stirring to prepare nano-zinc A liquid.

[0045] 2) Preparation of B liquid: 0.5wt% acetylsalicylic acid was dissolved in purified water. Under the conditions of ultrasonic wave (frequency 40KHz, power 1080W) and magnetic stirring (600rpm) at 45°C, the acetylsalicylic acid was fully dissolved for 30 minutes.

[0046] 3) A liquid and B liquid were mixed in equal volume to prepare a compound nano-zinc solution.

[0047] The compound nano-zinc solution has pH 5.0 and is placed at room temperature for 12 months without obvious deposition.

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

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

[0050] 2) Preparation of sodium hyaluronate gel: 30 g of sodium hyaluronate with a molecular weight of 1.2 million was placed in 1000 ml of purified water, and fully stirred and uniformly placed at room temperature for 12 hours to fully dissolve, and fully stirred for 15 minutes again after 12 hours to prepare 3% sodium hyaluronate gel.

[0051] 3) A liquid and 3% sodium hyaluronate gel were mixed in equal quality, and fully stirred for 15 minutes to prepare nano-zinc gel.

[0052] The nano-zinc gel has a viscosity of 65000 mPa.s, pH 6.5, and is placed at room temperature for 12 months, and the gel is stable without liquefaction.

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

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

[0055] 2) Preparation of B liquid: 0.5% acetylsalicylic acid was prepared by using purified water, and acetylsalicylic acid was fully dissolved under the conditions of ultrasonic wave (frequency 40 KHz, power 1080 W) and magnetic stirring (600 rpm) at 45°C for 30 minutes.

[0056] 3) A liquid and B liquid were fully mixed in equal volume to prepare 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 place it at room temperature for 12 hours to allow it to dissolve completely. After 12 hours, stir again for 15 minutes to prepare a 3% sodium hyaluronate gel.

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

[0059] The viscosity of the compound nano-zinc gel is 64,000 mPa.s, the pH is 5.0, and the gel remains stable without liquefaction after being placed at room temperature for 12 months.

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

[0061] The nano-zinc solution prepared in Example 2 and the compound nano-zinc solution prepared in Example 3 were detected for particle size, distribution, and Zeta potential using a BeNano laser particle size analyzer.

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

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

[0064] 1. Bacteriostatic circle test

[0065] Dilute the logarithmic growth phase E. coli and S. aureus broth cultures and C. albicans liquid into 10 6 CFU / ml, respectively, and evenly spread 100 μL of each onto the surface of nutrient agar medium and Sabouraud medium, then use a sterile puncher 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 complex solution, nano-zinc gel, and compound nano-zinc gel prepared in Examples 2, 3, 4, and 5, respectively, and 15% zinc oxide ointment (a quasi-drug) to fill each hole, 80 μL or 80 μg per hole. Then place the medium at 37°C overnight, remove the medium the next day, and measure the diameter of the bacteriostatic circle of each hole.

[0066] The results of the bacteriostatic circle test are shown in Table 1 and Figure 3 ​​​​The results show that the nano-zinc solution, the compound nano-zinc solution, the nano-zinc gel and the compound nano-zinc gel have very obvious inhibiting effect on the E. coli, the Staphylococcus aureus and the Candida albicans. In the test, the zinc content of the zinc oxide ointment is 24 times or 48 times higher than that of the nano-zinc, but it has not shown obvious inhibiting effect, which may be related to the particle size and the dosage form of the zinc oxide.

[0067] Table 1 Broad-spectrum bacteriostatic effect of the nano-zinc and the compound nano-zinc

[0068]

[0069] 2. Bacteriostatic rate determination

[0070] 1. Test basis: GB 15979-2002 "Hygienic Standard for Disposable Hygienic Products" Appendix C.

[0071] 2. Test method:

[0072] The test was carried out in a constant temperature water bath at 20℃±1℃. 5.0 mL of the nano-zinc solution, the compound nano-zinc solution, 5 grams of the nano-zinc gel and the compound nano-zinc gel prepared in Example 2-5 were respectively added into sterilized test tubes, and 100 uL of the bacterial suspension (bacterial solution concentration: 1×10 4 CFU / mL~9×10 4 CFU / mL) of the E. coli 8099, the Staphylococcus aureus ATCC 6538 or the Candida albicans was respectively added into each tube. After the action for the predetermined time (5 min, 10 min and 20 min), 0.5 mL of the bacterial and drug suspension was taken and added into 4.5 mL of neutralizing agent (D / E neutralizing broth), and after 10 min of neutralization, 1 mL of the sample was taken and inoculated on a nutrient agar medium plate, which was cultured at 37℃. The residual viable bacteria were detected. The positive and negative controls were set in the test, and the test was repeated for 3 times.

[0073] 3. Results:

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

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

[0076]

[0077] Note: ①No bacterial colony growth in the negative control; ②The test was repeated for 3 times and the average value was taken.

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

[0079]

[0080] Note: ① negative control no colony growth; ② test repeated 3 times to take the mean.

[0081] Table 4 Bactericidal effect of nano-zinc gel on bacteria and fungi

[0082]

[0083] Note: ① negative control no colony growth; ② test repeated 3 times to take the mean.

[0084] Table 5 Bactericidal effect of compound nano-zinc gel on bacteria and fungi

[0085]

[0086] Note: ① negative control no colony growth; ② test repeated 3 times to take the mean.

[0087] Example 8: Inhibition effect detection of nano-zinc and compound nano-zinc on clinically drug-resistant bacteria

[0088] Bacteriostatic circle test

[0089] 1. Test strains: methicillin-resistant Staphylococcus aureus, ESBLs-producing Escherichia coli, ESBLs-producing Klebsiella pneumoniae, multi-drug resistant Pseudomonas aeruginosa, and multi-drug 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 compound solution (zinc content 0.5%), nano-zinc gel (zinc content 0.25%), and nano-zinc compound gel (zinc content 0.25%).

[0091] 3. Test method: the logarithmic growth phase of methicillin-resistant Staphylococcus aureus, ESBLs-producing Escherichia coli, ESBLs-producing Klebsiella pneumoniae, multi-drug resistant Pseudomonas aeruginosa, and multi-drug resistant Acinetobacter baumannii nutrient broth culture were diluted to 10 6 CFU / ml, respectively, 100 μL of each was uniformly coated on the nutrient agar medium, then a sterile puncher was used to punch on the surface of the medium and pick out the medium in the hole. 80 microliters or 80 micrograms of nano-zinc solution, compound nano-zinc solution, nano-zinc gel, and compound nano-zinc gel prepared in Examples 2, 3, 4, and 5 were added to each hole. Then the medium was cultured at 37°C overnight, the next day the medium was taken out, and the diameter of the bacteriostatic circle of each hole was measured.

[0092] The test results are shown in Tables 6 and Figure 4The results show that the nano-zinc solution, the compound nano-zinc solution, the nano-zinc gel and the compound nano-zinc gel have obvious bacteriostatic effects on the five common clinically resistant bacteria (methicillin-resistant Staphylococcus aureus, ESBLs-producing Escherichia coli, ESBLs-producing Klebsiella pneumoniae, multi-drug resistant Pseudomonas aeruginosa and multi-drug resistant Acinetobacter baumannii).

[0093] Table 6 Inhibition effect of nano-zinc on five clinically resistant bacteria

[0094]

[0095] Example 9: Effect detection of nano-zinc and compound nano-zinc on broad-spectrum antiviral

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

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

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

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

[0100] 3. Test method: the nano-zinc solution and the compound nano-zinc solution were respectively mixed 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) for 2 hours at room temperature, and then the above mixtures were respectively diluted 1:10 with sterile normal saline, and 0.1 mL was injected into the allantoic cavity of the chick embryo. The virus control group of chick embryos was injected with 0.1 mL of the same dilution of influenza virus H3N2, parainfluenza virus and Newcastle disease virus, respectively, and incubated at 37°C for 72 hours. Then, the allantoic fluid was harvested according to the conventional method, and the harvested allantoic fluid of the chick embryo was diluted 10 times with sterile normal saline, and then the hemagglutination test was performed according to the conventional method.

[0101] The test results are shown in Table 7. Figure 5 The results show that the nano-zinc solution and the compound nano-zinc solution have obvious inhibition effects on influenza virus H3N2, parainfluenza virus and Newcastle disease virus, and the hemagglutination titers are all lower than 1:20. The hemagglutination titers of the three virus control groups are between 1:640 and 1:1280. The P values of the comparison of the hemagglutination titers between the test group and the virus control group are all less than 0.01. The test results show that the nano-zinc solution and the compound nano-zinc solution have obvious inactivation effects on influenza virus H3N2, parainfluenza virus and Newcastle disease virus.

[0102] II. Effect detection of nano-zinc gel and compound nano-zinc gel in inactivating HPV type 16 virus

[0103] 1. Experimental materials

[0104] 1.1 Sample: 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 Roche Biopharma Co., Ltd.

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

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

[0108] 2. Experimental principle and method

[0109] 2.1 Experimental principle

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

[0111] 2.2 Experimental steps

[0112] 1) 293FT cells were inoculated in a 96-well cell culture plate, and the cells were attached for standby use.

[0113] 2) Take 10 μl HPV type 16 pseudovirus solution and add it to 90 μl sample (nano-zinc gel or compound nano-zinc gel) or 90 μl PBS, stir well and incubate at 37°C for 2 hours, then dilute with 900 μl medium as the first concentration, and then dilute by 10 times and 3 times. Take 100 μl of gradient-diluted virus solution and add it to the cells to determine the residual virus titer. Set 6 holes for each concentration gradient to detect, and incubate in a 37°C CO2 incubator. After 72 hours of culture, use a fluorescence microscope to count the number of fluorescent cells and calculate the FFU (fluorescent forming unit) of the virus. (Operation according to: "Disinfection Technical Specification" 2002 edition).

[0114] The detection results of nano-zinc gel and compound nano-zinc gel in inactivating HPV activity 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] This experiment detected the inactivation effect of nano-zinc gel and compound nano-zinc gel on HPV 16 pseudovirus at the cellular level. The test results showed that the virus titer of the virus control group was 4.05 x 10 6 FFU / ml, the virus titers of the nano-zinc gel and compound nano-zinc gel samples were less than 1.67 x 10 3 FFU / ml, compared with the virus control group, the nano-zinc gel and compound nano-zinc gel samples reduced the HPV 16 pseudovirus titer by more than 3.38 log values at 37°C water bath for 2 hours, with an inactivation rate of more 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 were provided by the Experimental Animal Center of Dalian Medical University, and the experimental animal production license number was SCXK (Liaoning) 2022-0002.

[0125] II. Methods

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

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

[0128] 3. Test method:

[0129] The maximum limit test method was used, and the KM mice were orally gavaged with 5000 mg / kg body weight. Forty KM mice, half male and half female, weighing 19.4 ± 0.7 (g), were fasted overnight. An appropriate amount of test substance was dissolved in pure water to the required concentration solution, and the mice were given a single oral gavage of 0.2 ml / 10 g body weight. The animals were observed for 14 days after administration for signs of poisoning and death.

[0130] III. Results

[0131] No obvious toxic symptoms and death were observed in the test animals within 14 days after oral administration at a dose of 5000 mg / kg body weight, and no abnormality was found in body weight. Macroscopic examination was performed on the animals at the end of the observation period, and no abnormal changes were found in the main organs under naked eye observation. The results are shown in Table 8. According to the classification standard for acute oral toxicity test in the Technical Standard for Disinfection (2002 edition), the sample of nano-zinc solution and the compound nano-zinc solution stock solution have no acute oral toxicity to KM mice, and the LD50 is greater than 5000 mg / kg body weight. 50 The sample is practically non-toxic.

[0132] Table 8 Results of acute oral toxicity test

[0133]

[0134] It should be finally pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a broad-spectrum antibacterial and antiviral compound nano-zinc, characterized in that, The preparation method comprises the following steps: (1) drying and crushing ginkgo leaves, and then adding the obtained ginkgo leaf powder into water to prepare a 0.1wt%-5wt% solution, extracting the solution at 80-100 ℃ for 10-60 min, and then placing the solution at 1-5 ℃ for 10-24 h, filtering and centrifuging the solution, and then 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 then performing a reduction reaction under the action of ultrasonic waves and stirring to obtain a stable nano-zinc solution, adding vitamin C and N-acetylcysteine into the nano-zinc solution, and then continuing the reduction reaction under the action of ultrasonic waves and stirring, mixing the obtained solution with an equal volume of a 0.1wt%-2wt% anti-inflammatory, antipyretic and analgesic component-containing aqueous solution to obtain a compound nano-zinc solution; in step (2), the zinc salt 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; and the zinc ion concentration in the zinc salt aqueous solution is 1-100 mg / ml; in step (2), the addition amount of vitamin C is 1-30 g / L, and the addition amount of N-acetylcysteine is 20-80 g / L; and the anti-inflammatory, antipyretic and analgesic component comprises acetylsalicylic acid, arginine aspirin and calcium carbaspirin; in step (2), the ultrasonic wave frequency is 25-80 KHz, and the ultrasonic power is 54-1080 W.

2. The production method according to claim 1, characterized by, in step (1), the content of the ginkgo leaf powder in the solution is 0.5wt%-2wt%; and the centrifugation is performed at a speed of 2000-6000 rpm for 5-30 min.

3. The preparation method according to claim 1, characterized in that, in step (2), the zinc ion concentration in the zinc salt aqueous solution is 1-10 mg / ml.

4. The method of claim 1, wherein, in step (2), the stirring speed is 100-1000 rpm; and the reduction reaction time is 10-60 min.

5. The compound nano-zinc solution prepared by the preparation method in any one of claims 1-4.

6. A method for preparing a compound nano-zinc gel, characterized in that, The compound nano-zinc gel is prepared by mixing the compound nano-zinc solution in claim 5 with an equal amount of a 1wt%-5wt% sodium hyaluronate hydrogel.

7. The production method according to claim 6, wherein The preparation process of the sodium hyaluronate hydrogel is as follows: adding purified water into sodium hyaluronate with a molecular weight of 1.2 million, stirring uniformly, placing at room temperature for 10-20 h, stirring again for 5-30 min after fully dissolving, and then obtaining the sodium hyaluronate hydrogel.

8. The compound nano-zinc gel prepared by the preparation method in claim 6 or 7.

9. The compound nano-zinc solution in claim 5 or the compound nano-zinc gel in claim 8 is used in the preparation of antibacterial and antiviral materials or drugs.

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