Antiviral and antibacterial composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an antiviral and antibacterial composition as well as a preparation method and application thereof. The composition at least comprises the following components: rhizopus oryzae CICC 41440, rhizopus japonicus CICC 41297, a fermentation product of the rhizopus oryzae CICC 41440, a fermentation product of the rhizopus japonicus CICC 41297, and / or an organic solvent extract of the fermentation product of the rhizopus japonicus CICC 41297. Tests prove that the pharmaceutical composition can synergistically inhibit a plurality of pathogens such as rotavirus, streptococcus pneumoniae, adenovirus and klebsiella pneumoniae, meanwhile, the test verifies that the pharmaceutical composition is low in cytotoxicity and high in biological safety, the prevention and treatment level of intestinal diseases and respiratory diseases mediated by the pathogens can be effectively improved, and the pharmaceutical composition is suitable for clinical application. Therefore, the method has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms and biomedicine, and specifically relates to an antiviral and antibacterial composition and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Pathogens such as rotavirus, Streptococcus pneumoniae, Klebsiella pneumoniae, and adenovirus are important pathogens that cause human intestinal and respiratory infections. Among them, rotavirus infection is the main cause of severe diarrhea in infants and young children; Streptococcus pneumoniae and Klebsiella pneumoniae are typical opportunistic pathogens, and the emergence of multidrug-resistant strains increases the difficulty of clinical treatment of community-acquired pneumonia (CAP) and nosocomial infections; and respiratory infections caused by adenovirus are prone to develop into severe cases in immunocompromised populations.

[0004] However, current clinical prevention and treatment methods have limitations: for example, there is no specific antiviral drug for rotavirus, and the main treatment is fluid replacement support; at the same time, the overuse of antibiotics has led to a continuous increase in the resistance rate of Streptococcus pneumoniae and Klebsiella pneumoniae, and the WHO has listed them as key drug-resistant pathogens; existing broad-spectrum antiviral drugs have limited inhibitory effects on adenovirus and are prone to toxic side effects. At the same time, the increase in mixed infection cases has exposed the treatment defects of existing single-target drugs, and combined drug regimens often have the risk of drug interactions and the problem of superposition of toxic side effects.

[0005] The inventors noted that although some antibacterial and antiviral compositions exist in the prior art, their action spectrum is often narrow. In addition, although traditional Chinese medicine compound preparations have multi-target action characteristics, they have technical bottlenecks such as unclear effective ingredients and difficult quality control. Therefore, the development of drugs with broad-spectrum synergistic antipathogen activity and good biosafety is of great significance for improving the level of prevention and treatment of infectious diseases. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide an antiviral and antibacterial composition and its preparation method and application. Experiments have shown that the pharmaceutical composition can synergistically inhibit multiple pathogens such as rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae. At the same time, experiments have verified that it has low cytotoxicity and high biosafety, and can effectively improve the prevention and treatment of intestinal diseases and respiratory diseases mediated by the above-mentioned pathogens. Based on the above research results, the present invention is completed.

[0007] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0008] The present invention provides a way and method for preventing and / or treating diseases caused by rotavirus, Streptococcus pneumoniae, adenovirus, Klebsiella pneumoniae and the like.

[0009] The first aspect of the present invention provides an antiviral and antibacterial composition, wherein the composition comprises at least: Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC41297, their fermentation products and / or their fermented organic solvent extracts.

[0010] The mass ratio of Rhizopus oryzae CICC 41440 to Rhizopus japonicus CICC 41297, their fermentation products and / or their fermented organic solvent extracts is 1:0.5-5, preferably 1:1.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned composition, the preparation method comprising:

[0012] Rhizopus oryzae and Rhizopus japonicus are fermented to obtain fermentation broth, and then the fermentation broth is extracted with an organic solvent to obtain an extract.

[0013] The organic solvent is ethyl acetate.

[0014] The mass ratio of the Rhizopus oryzae fermentation extract to the Rhizopus japonicus fermentation extract is 1:0.5-5, preferably 1:1.

[0015] The present invention proves through experiments that the Rhizopus oryzae fermentation extract and the Rhizopus japonicus fermentation extract (i.e., the antiviral and antibacterial composition) prepared above have synergistic antiviral and antibacterial effects on four pathogens, namely, rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae.

[0016] The third aspect of the present invention provides the use of the above composition in the preparation of antiviral and antibacterial drugs.

[0017] In the present invention, the viruses include rotavirus and adenovirus, and the bacteria include Streptococcus pneumoniae and Klebsiella pneumoniae.

[0018] Therefore, the drug can be used to prevent and / or treat related diseases mediated by the above viruses and bacteria, without specific limitation herein.

[0019] A fourth aspect of the present invention provides an antiviral and / or antibacterial method, comprising administering the above composition to a subject.

[0020] Beneficial technical effects of one or more of the above technical solutions:

[0021] 1. The antiviral and antibacterial composition provided by the above technical solution can effectively prevent or treat diseases caused by rotavirus, Streptococcus pneumoniae, adenovirus, Klebsiella pneumoniae, etc.

[0022] 2. The composition obtained by the above technical solution has synergistic antiviral and antibacterial activity, and has a significant inhibitory effect on rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae.

[0023] 3. The preparation method of the above technical solution is simple, easy to operate and suitable for industrial production.

[0024] 4. The composition obtained by the above technical solution has good antibacterial and antiviral properties, low cytotoxicity and high biosafety, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are microscopic examinations of the toxic effects of rotavirus and adenovirus on cells of the present invention, A is the toxic effect of rotavirus on MA-104 cells, and B is the toxic effect of adenovirus on A549 cells.

[0026] Figure 2 The microscopic examination diagrams of drug cytotoxicity after applying 300 μg / mL of the composition of the present invention, A is MA-104 cells, and B is A549 cells. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0028] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0029] As mentioned above, although there are some antibacterial and antiviral compositions in the existing technology, their spectrum of action is often narrow. In addition, although traditional Chinese medicine compound preparations have multi-target action characteristics, they have technical bottlenecks such as unclear effective ingredients and difficult quality control.

[0030] In view of this, in a typical embodiment of the present invention, an antiviral and antibacterial composition is provided, wherein the composition comprises at least: Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297, their fermentation products and / or their fermented organic solvent extracts.

[0031] In another specific embodiment of the present invention, the organic solvent may be ethyl acetate or ethanol, among which ethyl acetate is preferred.

[0032] In another specific embodiment of the present invention, the composition may also contain other auxiliary ingredients commonly used in drugs, including but not limited to any one or more of sucrose, sodium citrate, xylitol, glutamic acid, glutathione, lecithin, flaxseed oil, corn oil, vitamin A, vitamin E, vitamin D3, vitamin K2, xylitol, zinc gluconate and magnesium gluconate, which are not specifically limited herein.

[0033] In another specific embodiment of the present invention, the mass ratio of Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297, their fermentation products and / or their fermented organic solvent extracts is 1:0.5-5, preferably 1:1.

[0034] Among them, Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297 were purchased from China Center of Industrial Culture Collection (CICC).

[0035] In another specific embodiment of the present invention, a method for preparing the above-mentioned composition is provided, and the preparation method comprises:

[0036] Rhizopus oryzae and Rhizopus japonicus are fermented to obtain fermentation broth, and then the fermentation broth is extracted with an organic solvent to obtain an extract.

[0037] Specifically, the preparation method comprises:

[0038] A potato glucose liquid culture medium is prepared, and activated Rhizopus oryzae and Rhizopus japonicus species are inoculated into the potato glucose liquid culture medium respectively, and shake culture is performed to obtain liquid cultures of Rhizopus oryzae and Rhizopus japonicus respectively; the liquid culture is filtered to separate mycelium and fermentation liquid, and the obtained fermentation liquid is extracted with an organic solvent to obtain a fermentation extract, and the Rhizopus oryzae fermentation extract and the Rhizopus japonicus fermentation extract are mixed to obtain the product.

[0039] The potato glucose liquid culture medium can be obtained commercially, or can be prepared by the following method: 200 g peeled potatoes, 20 g glucose and 1000 mL distilled water. Cut the potatoes into pieces, add them into distilled water and boil for 20 to 30 minutes, then filter out the potato residue with gauze. Then, add glucose and dissolve it, and finally add water to 1000 mL; pH 5.6±0.2, sterilize by high pressure steam, 121°C, 15 min for standby use.

[0040] The specific conditions of shaking culture are: culture at 100-160 rpm (preferably 120 rpm), 25-30° C. (preferably 28° C.) for 5-10 days (preferably 7 days).

[0041] The organic solvent is ethyl acetate.

[0042] The extraction conditions are as follows: the volume ratio of the fermentation liquid to ethyl acetate is 1:2-10, preferably 1:5.

[0043] In another specific embodiment of the present invention, the fermentation extract can be concentrated and dried to obtain a solid powder.

[0044] The mass ratio of the Rhizopus oryzae fermentation extract to the Rhizopus japonicus fermentation extract is 1:0.5-5, preferably 1:1.

[0045] The present invention tests various pathogens such as bacteria and viruses, and the results show that the Rhizopus oryzae fermentation extract and the Rhizopus japonicus fermentation extract (i.e., the composition) prepared above have synergistic antiviral and antibacterial effects on four pathogens including rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae. Therefore, the composition of the present invention can be used as a sanitary product, a disinfectant for environmental disinfection and / or a medicine.

[0046] When the composition is used as a medicine, the medicine has pharmacological activity of inhibiting diseases caused by rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae.

[0047] In the present invention, the composition can be administered in a unit dosage form. The dosage form can be a conventional dosage form, such as a liquid dosage form such as an emulsion dosage form, a colloid, a true solution, a microparticle dosage form, a vortex dosage form; such as other conventional dosage forms such as tablets, capsules, dripping pills, aerosols, pills, oral liquids, powders, injections, solutions, suspensions, emulsions, granules, inclusion compounds, landfills, etc. These dosage forms can be prepared according to conventional preparation methods in the pharmaceutical field, such as mixing, granulation, tableting, filling, dissolving or suspending and dispersing, etc., which are not specifically limited here.

[0048] In another specific embodiment of the present invention, there is provided use of the above composition in the preparation of antiviral and antibacterial drugs.

[0049] In the present invention, the viruses include rotavirus and adenovirus, and the bacteria include Streptococcus pneumoniae and Klebsiella pneumoniae.

[0050] Therefore, the drug can be used to prevent and / or treat related diseases mediated by the above viruses and bacteria, without specific limitation herein.

[0051] In another specific embodiment of the present invention, an antiviral and / or antibacterial method is provided, which comprises administering a therapeutically effective amount of the above composition to a subject.

[0052] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and interval of the active ingredients of the present invention are determined by their properties and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dosage of the compound over a specified period of time, can be determined by methods known in the art.

[0053] The present invention is further described below in conjunction with examples. The present invention is further described below by way of examples, but the present invention is not limited to the scope of the embodiments described. Based on the embodiments in the present invention, any variation of the present invention by those skilled in the art without making a creative premise all belongs to the protection scope of the present invention. Meanwhile, in the embodiments of the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0054] Example 1 Sample preparation

[0055] An antiviral and antibacterial composition, the preparation method of which is as follows:

[0056] Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297 were activated on potato dextrose solid (PDA) slant medium for 48 h, and spore suspensions (concentration 1×10 6 CFU / mL), inoculated into a potato glucose liquid culture medium at an inoculum amount of 5% (v / v), cultured at 120rpm and 28°C for 7 days, respectively obtaining liquid cultures of Rhizopus oryzae and Rhizopus japonicus; filtering the obtained Rhizopus oryzae liquid culture and Rhizopus japonicus liquid culture through four layers of gauze, extracting the obtained Rhizopus oryzae fermentation liquid and Rhizopus japonicus fermentation liquid with ethyl acetate at a volume ratio of 1:5, collecting the ethyl acetate layer to obtain the Rhizopus oryzae ethyl acetate fermentation extract and the Rhizopus japonicus ethyl acetate fermentation extract, and mixing the two at a mass ratio of 1:1 after concentration and drying to obtain a solid powder, which is the antiviral and antibacterial composition.

[0057] Effect verification

[0058] 1. Antibacterial activity test of the composition against Streptococcus pneumoniae and Klebsiella pneumoniae

[0059] 1.1 Experimental materials: Streptococcus pneumoniae (clinical isolates SP1, SP2 and quality control strains ATCC

[0060] 49619), Klebsiella pneumoniae (clinical isolates KP1, KP2 and quality control strain ATCC 700603).

[0061] Test samples: the ethyl acetate fermentation extract powder of Rhizopus oryzae prepared in Example 1 (hereinafter referred to as R), the ethyl acetate fermentation extract powder of Rhizopus japonicus prepared in Example 1 (hereinafter referred to as J), and the antiviral and antibacterial composition powder prepared in Example 1 (i.e., R+J, and R:J=1:1, w / w).

[0062] Positive control: levofloxacin (Sigma, purity ≥98%).

[0063] Culture medium: Mueller-Hinton broth (MHB, Qingdao Hibo Biotechnology).

[0064] 1.2 Experimental methods

[0065] (1) Minimum inhibitory concentration (MIC) determination

[0066] 1. Dilute R, J and R+J with MHB in the ratio of 512, 256, 128, 64, 32,

[0067] Gradient concentrations of 16, 8, 4, 2, 1, 0.5 μg / mL;

[0068] 2. Add 100 μL of the dilution to a 96-well plate, and inoculate 100 μL of a bacterial suspension at 5×10 5 CFU / mL into each well;

[0069] 3. Observe after culturing at 37°C for 24 h. The lowest concentration without visible turbidity to the naked eye is taken as the MIC value.

[0070] (2) Verification of synergistic effect

[0071] Calculate according to the FICI method:

[0072]

[0073] Among them, FICI ≤ 0.5 indicates a synergistic effect, 0.5 < FICI < 1 indicates a partial synergistic effect, FICI = 1 indicates an additive effect, 1 < FICI < 4 indicates an irrelevant effect, and FICI ≥ 4 indicates an antagonistic effect.

[0074] 1.3 Experimental results

[0075] The specific experimental results are shown in Table 1 and Table 2. It can be seen from Table 1 and Table 2 that the composition has good synergistic antibacterial effects against Streptococcus pneumoniae and Klebsiella pneumoniae, and its synergistic antibacterial effect against Klebsiella pneumoniae is better.

[0076] Table 1 Antibacterial activity test results of the composition against Streptococcus pneumoniae

[0077]

[0078] Table 2 Antibacterial activity test results of the composition against Klebsiella pneumoniae

[0079]

[0080] II. Antiviral activity test of the composition against rotavirus and adenovirus

[0081] 2.1 Experimental materials Viruses: Rotavirus SA11 strain (ATCC VR-2018), Adenovirus AdV5 type (ATCC VR-1516).

[0082] Cell lines: MA-104 rhesus monkey kidney cells (rotavirus), A549 cells (adenovirus). Positive control: Acyclovir (Sigma, purity ≥ 99%).

[0083] Cell maintenance medium: RPMI 1640 containing 10% fetal bovine serum (Gibco).

[0084] 2.2 Experimental methods

[0085] (1) Plaque reduction assay

[0086] 1. Pre-incubate R, J and R+J (10-300 μg / mL) with virus suspension (MOI = 0.1) at 37°C for 1 h;

[0087] 2. After 1 hour of infection of the monolayer cells, cover with cell maintenance medium;

[0088] 3. Cultivate for 48 hours (rotavirus) or 72 hours (adenovirus), fix and stain, count plaques, and calculate the inhibition rate:

[0089]

[0090] (2) Verification of synergistic effects

[0091] Determination of IC of R and J alone 50 The combined effect index (CI) was calculated:

[0092]

[0093] Among them, D R and D J are the concentrations of R and J when the inhibition rate is 50% when used in combination, IC 50,R and IC 50,J These are the concentrations of R and J when the inhibition rate is 50% when applied alone.

[0094] CI < 1 indicates a synergistic effect, CI = 1 indicates an additive effect, and CI > 1 indicates an antagonistic effect.

[0095] 2.3 Experimental Results

[0096] The results are shown in Table 3. It can be seen from Table 3 that it has a good synergistic antiviral effect on rotavirus and adenovirus.

[0097] Table 3 Antiviral activity test results of the composition against rotavirus and adenovirus

[0098]

[0099] 3. Composition cytotoxicity test

[0100] 3.1 Experimental methods

[0101] 1. MA-104 / A549 cells were seeded in 96-well plates (1×10 4 cells / well), cultured for 24 h;

[0102] 2. Add R+J (10-300 μg / mL) and continue culturing for 48 h;

[0103] 3. Detect cell viability using CCK-8 method and calculate CC 50 value (half cytotoxic concentration).

[0104] 3.2 Experimental Results

[0105] The R+J composition was diluted in proportion and added to MA-104 cells and A549 cells. The results showed that the cells at all dilutions grew densely and evenly, with good light transmittance and no obvious morphological changes. The experimental results are shown in Tables 4 and Figure 2 , indicating that the composition has low cytotoxicity and good biosafety, which is very beneficial for its practical application.

[0106] Table 4 Composition cytotoxicity test results

[0107] Cell lines sample <![CDATA[CC 50 (μg / mL)]]> <![CDATA[SI(CC 50 / IC 50 )]]> MA-104 R+J ＞300 ＞10.5 A549 R+J ＞300 ＞8.5

[0108] The above experiments have proved that the composition of the present invention can effectively prevent or treat diseases caused by rotavirus, Streptococcus pneumoniae, adenovirus, Klebsiella pneumoniae, etc., and has high biological safety and great value for promotion and application.

[0109] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An antiviral and antibacterial composition, characterized in that: The composition comprises at least: Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297, fermented products thereof and / or fermented organic solvent extracts thereof.

2. The composition according to claim 1, characterized in that The organic solvent is ethyl acetate or ethanol, preferably ethyl acetate.

3. The composition according to claim 1, characterized in that The mass ratio of Rhizopus oryzae CICC41440 and Rhizopus japonicus CICC 41297, their fermentation products and / or their fermented organic solvent extracts is 1:0.5-5, preferably 1:

1.

4. The composition according to claim 1, characterized in that The composition further comprises pharmaceutical excipient components.

5. A method for preparing the composition according to any one of claims 1 to 4, characterized in that: The preparation method comprises: Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297 are fermented to obtain fermentation broth, and then the fermentation broth is extracted with an organic solvent to obtain an extract.

6. The preparation method according to claim 5, characterized in that: A potato glucose liquid culture medium is prepared, and activated Rhizopus oryzae and Rhizopus japonicus species are inoculated into the potato glucose liquid culture medium respectively, and shake culture is performed to obtain liquid cultures of Rhizopus oryzae and Rhizopus japonicus respectively; the liquid culture is filtered to separate mycelium and fermentation liquid, and the obtained fermentation liquid is extracted with an organic solvent to obtain a fermentation extract, and the Rhizopus oryzae fermentation extract and the Rhizopus japonicus fermentation extract are mixed to obtain the product.

7. The preparation method according to claim 5, characterized in that: The specific conditions of shaking culture are: culture at 100-160rpm, 25-30°C for 5-10 days.

8. The preparation method according to claim 5, characterized in that: The organic solvent is ethyl acetate; The extraction conditions are as follows: the volume ratio of the fermentation liquid to ethyl acetate is 1:2-10, preferably 1:5; Furthermore, the fermentation extract can be concentrated and dried to obtain solid powder.

9. The preparation method according to claim 8, characterized in that: The mass ratio of the Rhizopus oryzae fermentation extract to the Rhizopus japonicus fermentation extract is 1:0.5-5, preferably 1:

1.

10. Use of the composition according to any one of claims 1 to 4 in the preparation of antiviral and antibacterial drugs; in, The viruses include rotavirus and adenovirus, and the bacteria include Streptococcus pneumoniae and Klebsiella pneumoniae.

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