An antiviral and antibacterial composition, its preparation method and use

Abstract

The present application belongs to the field of microbial technology, and particularly relates to an antiviral and antibacterial composition, a preparation method and application thereof. The composition comprises at least the following components: Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297, fermentation products thereof and / or organic solvent extracts thereof. The drug composition can synergistically inhibit rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae and other pathogens, and has low cytotoxicity and high biological safety. The drug composition can effectively improve the prevention and treatment level of intestinal diseases and respiratory diseases mediated by the above pathogens, and therefore has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms and biological medicine, and particularly relates to an antiviral and antibacterial composition, a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Rotavirus, Streptococcus pneumoniae, Klebsiella pneumoniae and Adenovirus are important pathogenic sources of 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, as typical opportunistic pathogens, have increased the clinical treatment difficulty of community-acquired pneumonia (CAP) and nosocomial infection due to the emergence of multiple drug-resistant strains; and respiratory infections caused by adenovirus are prone to develop into severe cases in immunocompromised populations.

[0004] However, the current clinical prevention and treatment methods have limitations: for example, there is no specific antiviral drug for rotavirus, and the main treatment relies on fluid replacement support; meanwhile, the overuse of antibiotics has led to a continuous increase in drug resistance of Streptococcus pneumoniae and Klebsiella pneumoniae, which has been listed by WHO as a key drug-resistant pathogen; existing broad-spectrum antiviral drugs have limited inhibitory effect on adenovirus and are prone to produce toxic side effects. At the same time, the increase in mixed infection cases exposes the treatment defects of existing single-target drugs, and the combination drug regimen often has the risk of drug interaction and the problem of superimposed toxic side effects.

[0005] The inventors have noticed that although there are some antibacterial and antiviral compositions in the prior art, they often have a narrow spectrum of action, in addition, traditional Chinese medicine compound preparations have the characteristics of multi-target action, but have technical bottlenecks such as unclear effective components and difficulty in quality control. Therefore, it is of great significance to develop a drug with broad-spectrum synergistic anti-pathogen activity and good biological safety for improving the prevention and treatment level of infectious diseases. SUMMARY

[0006] In view of the above deficiencies of the prior art, the present application aims to provide an antiviral and antibacterial composition, a preparation method and application thereof. The drug composition can synergistically inhibit rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae and other pathogens, and can effectively improve the prevention and treatment level of intestinal diseases and respiratory diseases mediated by the above pathogens. Based on the above research results, the present application is completed.

[0007] In order to achieve the above technical purpose, the technical scheme provided by the present application is as follows:

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

[0009] In a first aspect of the present application, an antiviral and antibacterial composition is provided, which comprises at least: Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297, fermentation products thereof and / or organic solvent extracts thereof.

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

[0011] In a second aspect of the present application, a preparation method of the above composition is provided, which comprises:

[0012] Rhizopus oryzae and Rhizopus japonicus are respectively 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 and the Rhizopus japonicus fermentation extract is 1:0.5-5, preferably 1:1.

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

[0016] In a third aspect of the present application, the above composition is provided for use in the preparation of an antiviral and antibacterial drug.

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

[0018] Therefore, the medicine can be used for preventing and / or treating diseases caused by the viruses and bacteria mentioned above, which are not limited herein.

[0019] In a fourth aspect of the present application, a method for preventing and / or treating viruses and bacteria is provided, which comprises administering the composition mentioned above to a subject.

[0020] The above technical solutions have the following beneficial technical effects:

[0021] 1. The anti-virus and anti-bacteria composition provided by the above technical solutions can effectively prevent or treat diseases caused by rotavirus, Streptococcus pneumoniae, adenovirus, Klebsiella pneumoniae, etc.

[0022] 2. The composition obtained by the above technical solutions has synergistic anti-virus and anti-bacteria activities, and has a significant inhibitory effect on rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae.

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

[0024] 4. The composition obtained by the above technical solutions has good anti-bacteria and anti-virus properties, low cytotoxicity, high biological safety, and therefore has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure 1 is a photomicrograph of the toxic effect of rotavirus and adenovirus on cells according to the present application, wherein 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 Figure 2 is a photomicrograph of drug cytotoxicity after applying 300 μg / mL of the composition according to the present application, wherein A is MA-104 cells, and B is A549 cells. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0028] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments consistent with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

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

[0030] Therefore, in one typical embodiment of the present application, an antiviral and antibacterial composition is provided, which comprises at least Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297, fermentation products thereof and / or organic solvent extracts thereof.

[0031] In another embodiment of the present application, the organic solvent can be ethyl acetate or ethanol, and ethyl acetate is preferred.

[0032] In another embodiment of the present application, the composition can further comprise other commonly used excipients for drugs, including but not limited to any one or more of sucrose, sodium citrate, xylitol, glutamic acid, glutathione, lecithin, linseed oil, corn oil, vitamin A, vitamin E, vitamin D3, vitamin K2, xylitol, zinc gluconate and magnesium gluconate, which are not limited herein.

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

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

[0035] In another embodiment of the present application, a preparation method of the above-mentioned composition is provided, which comprises:

[0036] R. oryzae and R. japonicum are fermented respectively to obtain fermentation liquor, and then the fermentation liquor is extracted with organic solvent to obtain extract.

[0037] Specifically, the preparation method comprises:

[0038] Potato glucose liquid medium is prepared, and activated R. oryzae and R. japonicum are inoculated into the potato glucose liquid medium respectively for shaking culture to obtain liquid culture of R. oryzae and R. japonicum respectively; the liquid culture is filtered to separate mycelium and fermentation liquor, the obtained fermentation liquor is extracted with organic solvent to obtain fermentation extract, and the fermentation extract of R. oryzae and the fermentation extract of R. japonicum are mixed to obtain the composition.

[0039] The potato glucose liquid medium can be obtained by market purchase or prepared by the following method: 200 g of peeled potato, 20 g of glucose and 1000 mL of distilled water. The potato is cut into pieces, boiled in the distilled water for 20 to 30 minutes, and then filtered with gauze to remove the potato residue. Then, the glucose is added and dissolved, and finally water is added to 1000 mL; the pH is 5.6±0.2, and the medium is sterilized by high-pressure steam at 121℃ for 15 min for standby use.

[0040] The specific conditions of the shaking culture are as follows: 100-160 rpm (preferably 120 rpm), 25-30℃ (preferably 28℃) 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 liquor to ethyl acetate is 1:2-10, preferably 1:5.

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

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

[0045] The present application tests various pathogens such as bacteria and viruses, and the results show that the fermentation extract of R. oryzae and the fermentation extract of R. japonicum (i.e. the composition) have synergistic antiviral and antibacterial effects on four kinds of pathogens including rotavirus, Streptococcus pneumoniae, adenovirus and Klebsiella pneumoniae. Therefore, the composition of the present application can be used as a health product, a disinfectant product 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 application, the composition can be administered in unit dosage form. The administration form can be a conventional form, such as a liquid form, for example, an emulsion form, a colloid form, a true solution form, a microparticle form, a mixed form; or other conventional forms, for example, a tablet, a capsule, a drop pill, an aerosol, a pill, an oral solution, a powder, an injection, a solution, a suspension, an emulsion, a granule, an inclusion compound, a landfill, and the like. These dosage forms can be prepared according to conventional methods in the pharmaceutical field, such as mixing, granulation, tabletting, filling, dissolution, or suspension dispersion, and the like, without specific limitation.

[0048] In another embodiment of the present application, the use of the above-mentioned composition in the preparation of an antiviral and antibacterial drug is provided.

[0049] In the present application, the virus includes rotavirus and adenovirus, and the bacteria include Streptococcus pneumoniae and Klebsiella pneumoniae.

[0050] Therefore, the drug can be used for preventing and / or treating diseases related to the above-mentioned viruses and bacteria, without specific limitation.

[0051] In another embodiment of the present application, an antiviral and / or antibacterial method is provided, which comprises administering a therapeutically effective amount of the above-mentioned 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 an active compound or pharmaceutical agent, including the compound of the present application, which can elicit the biological or medical response of a tissue system, animal, or human that is being sought by the researcher, veterinarian, medical doctor, or other medical person, which includes 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 administration of the active ingredients of the present application will be determined by such considerations as the characteristics of the compound, such as the form, route, and site of administration, and the particular mammal being treated, and this optimal dosage can be determined using routine techniques. It must also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a given period of time, can be determined using methods known in the art.

[0053] The present application is further illustrated by the following examples. The present application is further illustrated by the following examples, but is not limited to the examples described. Based on the examples in the present application, any changes to the present application made by those skilled in the art without creativity are within the scope of protection of the present application. Meanwhile, in the examples of the present application, all the raw materials for preparation are commercially available products known to those skilled in the art, unless otherwise specified.

[0054] Preparation of samples in Example 1

[0055] An antiviral and antibacterial composition is prepared by the following method:

[0056] Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297 were activated on potato dextrose solid (PDA) slant medium for 48 hours, and spore suspensions (concentration 1 x 10 6 CFU / mL) were prepared with sterile normal saline. The two strains were inoculated into potato dextrose liquid medium at a 5% (v / v) inoculation amount, respectively, and cultured at 120 rpm for 7 days at 28°C to obtain liquid cultures of Rhizopus oryzae and Rhizopus japonicus, respectively. The obtained liquid cultures of Rhizopus oryzae and Rhizopus japonicus were filtered through four layers of gauze, and the obtained Rhizopus oryzae fermentation broth and Rhizopus japonicus fermentation broth were extracted with ethyl acetate at a volume ratio of 1:5, respectively. The ethyl acetate layers were collected to obtain Rhizopus oryzae ethyl acetate fermentation extract and Rhizopus japonicus ethyl acetate fermentation extract. After concentration and drying, the two were mixed at a mass ratio of 1:1 to obtain the solid powder, which was the antiviral and antibacterial composition.

[0057] Effect verification

[0058] I. Test of antibacterial activity of the composition on Streptococcus pneumoniae and Klebsiella pneumoniae

[0059] 1.1 Experimental materials Strains: Streptococcus pneumoniae (clinical isolates SP1, SP2 and quality control strain ATCC 49619), Klebsiella pneumoniae (clinical isolates KP1, KP2 and quality control strain ATCC 700603).

[0060] 1.1 Experimental materials Strains: Streptococcus pneumoniae (clinical isolates SP1, SP2 and quality control strain ATCC 49619), Klebsiella pneumoniae (clinical isolates KP1, KP2 and quality control strain ATCC 700603).

[0061] Test samples: Rhizopus oryzae ethyl acetate fermentation extract powder prepared in Example 1 (hereinafter referred to as R), Rhizopus japonicus ethyl acetate fermentation extract powder 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 Haibo Biological).

[0064] 1.2 Experimental method

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

[0066] 1. R, J and R+J were diluted with MHB by a factor of 512, 256, 128, 64, 32,

[0067] 16, 8, 4, 2, 1, 0.5 μg / mL gradient concentration;

[0068] 2. Take 100 μL of the diluent into a 96-well plate, inoculate 5 x 10 5 CFU / mL bacterial suspension 100 μL;

[0069] 3. After 24 h of culture at 37°C, observe and take the lowest concentration without visible turbidity as the MIC value.

[0070] (2) Synergistic effect verification

[0071] According to the FICI method:

[0072]

[0073] Among them, FICI≤0.5 has synergistic effect, 0.5<FICI<1 has partial synergistic effect, FICI is 1 has additive effect, 1<FICI<4 has irrelevant effect, and FICI≥4 has antagonistic effect.

[0074] 1.3 Experimental results

[0075] The specific experimental results are shown in Table 1 and Table 2. As shown in Table 1 and Table 2, the composition has good synergistic antibacterial effect on Streptococcus pneumoniae and Klebsiella pneumoniae, and the synergistic antibacterial effect on Klebsiella pneumoniae is better.

[0076] Table 1 Test results of antibacterial activity of the composition on Streptococcus pneumoniae

[0077]

[0078] Table 2 Test results of antibacterial activity of the composition on Klebsiella pneumoniae

[0079]

[0080] II. Test of antiviral activity of the composition on rotavirus and adenovirus

[0081] 2.1 Experimental materials Virus: rotavirus SA11 strain (ATCC VR-2018), adenovirus AdV5 type (ATCC VR-1516).

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

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

[0084] 2.2 Experimental method

[0085] (1) Plaque reduction assay

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

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

[0088] 3. Culture 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 using the following formula:

[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%.

[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. As can be seen from Table 3, it has good synergistic antiviral effects on rotavirus and adenovirus.

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

[0098]

[0099] 3. Cytotoxicity test of the composition

[0100] 3.1 Experimental methods

[0101] 1. MA-104 / A549 cells were seeded into 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. Cell survival rate was detected by CCK-8 method, and CC50 (half cytotoxicity concentration) was calculated. 50

[0104] 3.2 Experimental results

[0105] After the R+J composition was diluted in proportion and added to MA-104 cells and A549 cells, it was found that the cell growth of all dilutions was relatively dense, uniform, good in transmittance, and no obvious morphological changes, and the experimental results were shown in Table 4 and Figure 2 , which indicated that the composition had low cytotoxicity and good biological safety, and was very beneficial to practical application.

[0106] Table 4 Cytotoxicity test results of the composition

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

[0108] It is proved by the above experiments that the composition of the application can effectively prevent or treat diseases caused by rotavirus, streptococcus pneumoniae, adenovirus, klebsiella pneumoniae and the like, and has high biological safety and great application value.

[0109] It should be noted that the above examples are only used to illustrate the technical solutions of the application but not to limit the same. Although the application has been described in detail with reference to the examples given, ordinary skilled person in the art can modify or equivalently replace the technical solutions of the application according to the needs without departing from the spirit and scope of the technical solutions of the application.​

Claims

1. An antiviral and antibacterial composition, characterized in that: The composition is composed of Rhizopus oryzae ( Rhizopus oryzae ) CICC 41440 and Rhizopus japonicus ( Rhizopus japonicus ) CICC 41297 fermentation broth ethyl acetate extract; the Rhizopus oryzae ( Rhizopus oryzae ) CICC 41440 and Rhizopus japonicus ( Rhizopus japonicus ) The mass ratio of the ethyl acetate extract of CICC 41297 fermentation broth is 1:0.5-5; The viruses include rotavirus and adenovirus, and the bacteria include Streptococcus pneumoniae and Klebsiella pneumoniae.

2. The composition according to claim 1, wherein The Rhizopus oryzae ( Rhizopus oryzae ) CICC41440 and Rhizopus japonicus ( Rhizopus japonicus ) The mass ratio of the ethyl acetate extract of CICC 41297 fermentation broth was 1:

1.

3. The composition according to claim 1, wherein The composition further comprises pharmaceutical excipient components.

4. The method for preparing the composition according to any one of claims 1 to 3, characterized in that: The preparation method comprises: Rhizopus oryzae CICC 41440 and Rhizopus japonicus CICC 41297 were fermented to obtain fermentation broths, and then the fermentation broths were extracted with ethyl acetate to obtain extracts.

5. The preparation method according to claim 4, wherein 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 cultures are filtered to separate mycelium and fermentation liquid, and the obtained fermentation liquid is extracted with ethyl acetate to obtain a fermentation extract, and the Rhizopus oryzae fermentation extract and the Rhizopus japonicus fermentation extract are mixed to obtain the product.

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

7. The preparation method according to claim 4, wherein The extraction conditions are as follows: the volume ratio of the fermentation liquid to ethyl acetate is 1:2-10.

8. The preparation method according to claim 7, wherein The extraction conditions are as follows: the volume ratio of the fermentation liquid to ethyl acetate is 1:

5.

9. The preparation method according to claim 4, wherein The fermentation extract can be concentrated and dried to obtain solid powder.

10. The preparation method according to any one of claims 7 to 9, characterized in that: The mass ratio of the Rhizopus oryzae fermentation extract to the Rhizopus japonicus fermentation extract is 1:0.5-5.

11. The preparation method according to claim 10, characterized in that The mass ratio of the Rhizopus oryzae fermentation extract to the Rhizopus japonicus fermentation extract is 1:

1.

12. Use of the composition according to any one of claims 1 to 3 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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