阅读说明：本技术 表没食子儿茶素没食子酸酯的应用及手洗消毒剂 (Application of epigallocatechin gallate and hand washing disinfectant ) 是由 张其威 吴建国 谭秋萍 于 2021-09-26 设计创作，主要内容包括：本申请公开了表没食子儿茶素没食子酸酯在制备预防和/或治疗腺病毒引起疾病制剂中的应用及手洗消毒剂,涉及抗病毒应用技术领域。表没食子儿茶素没食子酸酯能够有效的抑制腺病毒的吸附、复制和组装,从而起到抑制腺病毒的感染,且其还能够直接对腺病毒进行灭活,具有用作制备预防和或治疗腺病毒引起疾病制剂的应用前景。(The application discloses application of epigallocatechin gallate in preparation of a preparation for preventing and/or treating diseases caused by adenovirus and a hand washing disinfectant, and relates to the technical field of antiviral application. The epigallocatechin gallate can effectively inhibit the adsorption, replication and assembly of the adenovirus so as to inhibit the infection of the adenovirus, can also directly inactivate the adenovirus, and has an application prospect of preparing a preparation for preventing and/or treating diseases caused by the adenovirus.)

1. Application of epigallocatechin gallate in preparing preparation for preventing and/or treating diseases caused by adenovirus is provided.

2. The use of claim 1, wherein the adenovirus is of a genotype comprising human adenovirus type 3 and human fluorescent green adenovirus type 3.

3. The use according to claim 1, wherein the adenovirus causes diseases including acute respiratory disease, acute bronchitis, epidemic conjunctivitis, gastroenteritis, keratoconjunctivitis, meningoencephalitis, cystitis, myocarditis, even severe pneumonia and morbid obesity.

4. Use according to claim 1, wherein the formulation comprises disinfectants, drugs and derivatives prepared on the basis of epigallocatechin gallate.

5. A hand sanitizer for adenovirus, comprising epigallocatechin gallate.

Technical Field

The application relates to the technical field of antiviral application, in particular to application of epigallocatechin gallate and a hand washing disinfectant.

Background

Adenoviruses are the main causative agent of acute respiratory infections, are widespread worldwide, and present epidemiological features of small seasonal fluctuations and major local distribution. People with low or defective immune function are most susceptible to HAdV infection, possibly from exogenous new infections, and also from activation of latent infection viruses. The types of adenovirus infected by human groups in the global range are mainly B (HAdV3, HAdV7, HAdV11, HAdV14 and HAdV55) and C (HAdV1, HAdV2 and HAdV5), the types of adenovirus infected by respiratory tract in China are B3 genotype (HAdV3 genotype) and B7 genotype, and different epidemic strains of the viruses of the genotypes have obvious regional limitation.

Diseases induced by adenovirus infection include acute respiratory diseases and acute bronchitis, epidemic conjunctivitis, gastroenteritis, keratoconjunctivitis, meningoencephalitis, cystitis, myocarditis and even severe pneumonia, and may cause non-inflammatory symptoms such as obesity. The mode of adenovirus transmission is mainly air droplets, direct contact with pollutants or infected tissues, drinking infected water sources, and can be transmitted through the fecal-oral route. However, there is currently no effective prophylactic or therapeutic agent against the virus, such as an effective hand sanitizer use, that can assist the vaccine in prophylaxis, thereby enhancing health management and medication.

Disclosure of Invention

In view of the above, the present application aims to provide an application of epigallocatechin gallate in preparing a preparation for preventing and/or treating adenovirus-induced diseases and a hand-washing disinfectant. The epigallocatechin gallate provided by the application can effectively inhibit adsorption, replication and assembly of adenovirus, so that the infection of adenovirus is inhibited, the adenovirus can be directly inactivated, and the epigallocatechin gallate has an application prospect of preparing a preparation for preventing and/or treating diseases caused by adenovirus.

In order to achieve the purpose of the application, the application provides the following technical scheme:

the application discloses application of epigallocatechin gallate in preparing a preparation for preventing and/or treating diseases caused by adenovirus.

In the technical scheme, the genotype of the adenovirus comprises human adenovirus type 3 and human adenovirus type 3 green fluorescent virus.

In the technical scheme, the diseases caused by the adenovirus comprise acute respiratory diseases, acute bronchitis, epidemic conjunctivitis, gastroenteritis, keratoconjunctivitis, meningoencephalitis, cystitis, myocarditis, even severe pneumonia and pathological obesity.

In the above technical scheme, the preparation comprises a disinfectant, a drug and a derivative prepared on the basis of epigallocatechin gallate.

The application also provides a hand sanitizer for adenovirus comprising epigallocatechin gallate.

Compared with the prior art, the application has at least the following beneficial effects:

the epigallocatechin gallate provided by the application can effectively inhibit adsorption, replication and assembly of adenovirus, so that the infection of adenovirus is inhibited, the adenovirus can be directly inactivated, and the epigallocatechin gallate has an application prospect of preparing a preparation for preventing and/or treating diseases caused by adenovirus.

Drawings

FIG. 1 is a graph showing the change in the sterilizing fluorescence of a disinfectant against HAdV-B3-GFP virus, with a treatment time of 60s, according to an example of the present application; the initial titer was 6.5X 10 on the top panel⁶Five titers of FFU/mL test virus suspension after 10-fold dilution; on the left, "1 to 3" represent test groups treated with disinfectants of type a, type B and type C, respectively, and "4" represents a positive control group not treated with disinfectants, and the virus titer corresponds to that of the test group.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

In the present application, the epigallocatechin gallate vinegar is also referred to as epigallocatechin-3-gallate a or epigallocatechin gallate. The source of epigallocatechin-3-gallate is not particularly limited, and conventional commercial products may be used, and preferably, epigallocatechin-3-gallate is commercially available from selelck.

The embodiment of the application provides application of epigallocatechin gallate in preparing a preparation for preventing and/or treating diseases caused by adenovirus.

In the above embodiments, the genotype of the adenovirus includes human adenovirus type 3 and human fluorescent green adenovirus type 3.

In the above embodiments, the adenovirus causes diseases including acute respiratory disease, acute bronchitis, epidemic conjunctivitis, gastroenteritis, keratoconjunctivitis, meningoencephalitis, cystitis, myocarditis, even severe pneumonia, and morbid obesity.

In the above embodiment, the preparation comprises a disinfectant, a drug and a derivative prepared on the basis of epigallocatechin gallate.

The embodiment of the application also provides a hand-washing disinfectant for adenovirus, which comprises epigallocatechin gallate.

In the present application, when the preparation is preferably a drug, the epigallocatechin gallate vinegar preferably accounts for l% to 99% of the total mass of the drug. The auxiliary materials of the medicine are not particularly limited, and the corresponding auxiliary materials are preferably selected through the medicine dosage form.

In the present application, when the preparation is preferably a disinfectant, the epigallocatechin gallate preferably accounts for l% to 99% of the total mass of the disinfectant. The auxiliary materials of the disinfectant are not particularly limited, and the auxiliary materials for preparing the disinfectant conventionally, such as palmitate, ethanol and the like, are preferably adopted.

The technical solutions in the present application will be clearly and completely described below with reference to the embodiments in the present application. It is to be understood that the embodiments described are only a few embodiments of the present application and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The following examples were conducted to verify the killing effect of EGCG-containing alcohol disinfectant on non-enveloped, commonly occurring human type 3 adenovirus causing respiratory infections in children, mainly by experiments.

Materials and methods

One) material

Epigallocatechin gallate palmitate (EGCG) and EGCG-palmitate (epigallocatechin gallate palmitate (EGCG)) are prepared by esterification of epigallocatechin gallate (EGCG) and palmitoyl chloride, so that the lipophilicity of EGCG is enhanced, the bioactivity of EGCG is maintained, the EGCG is provided by Sigma company, the mother liquid level is 20mg/mL, and the EGCG is sterilized, filtered and packaged.

Fetal bovine serum, DMEM cell culture medium and trypsin are all imported products. Analytically pure absolute ethyl alcohol is a product sold in China.

Model cells: human lung non-small cell lung cancer cells (a549), provided by the key laboratory in virology, guangdong province.

Strain: human type 3 Green fluorescent adenovirus (HAdV-B3-GFP), supplied by the major virology laboratory in Guangdong province.

Cell culture medium: 10% (v/v) fetal bovine serum FBS and 1% (v/v) penicillin-streptomycin antibiotic (100U/mL) are added into DMEM (high glucose medium); cell maintenance media were prepared using high glucose medium DMEM supplemented with 2% (v/v) fetal bovine serum FBS, 1% (v/v) penicillin-streptomycin antibiotic (100U/mL).

Cell culture: a549 cells were first cultured in a 2% cell maintenance medium to give a monolayer, and then inoculated with HAdV-B3-GFP virus to obtain a titer of 1.3X 10⁷FFU/mL of test virus suspension. Test virus suspension and 3% bovine serum albumin are subjected to double dilution to obtain 6.5 × 10⁶FFU/mL, ready for use.

A sample to be tested:

the A type disinfectant is an ethanol solution containing 2 per mill (v/v) of EGCG-palmitate;

type B disinfectant, 75% ethanol;

a mixture of type C disinfectant, iodine tincture and polyvinylpyrrolidone.

Two) method

1. Viral load detection

HAdV-B3-GFP virus was serially diluted in 5 gradients, each dilution being 6.5X 10⁵FFU/mL、6.5×10⁴FFU/mL、6.5×10³FFU/mL、6.5×10²FFU/mL and 65 FFU/mL.

A549 cells at 1 × 10⁴The density of each well was inoculated into a 96-well plate and cultured until the cells were fully grown, the culture medium supernatant was discarded, each dilution of the virus solution (100 ul/well) was added, 4 wells were repeated for each dilution, and the cells were placed in 5% CO₂Incubating at 37 deg.C for 2h in incubator for virus infection, discarding unadsorbed and infected virus solution, adding fresh cell maintenance culture medium into 96-well plate, and continuously placing in CO₂And after the culture is continued for 40 hours in the incubator, counting the quantity of green fluorescence of each hole, wherein the quantity of the fluorescence corresponds to the quantity of the virus, and calculating an inactivation logarithm value.

2. EGCG antiviral biosynthesis

6.5×10⁶FFU/mL virus 20 muL/hole infects and grows up to 80% monolayer A549 cells, after adsorbing 1h at 37 ℃, abandoning virus liquid, adding 100 muL/hole containing different concentration EGCG (0.5 mug/mL, 1 mug/mL, 1.5 mug/mLL, 5 mug/mL, 10 mug/mL) into culture hole, culturing at 37 ℃, observing CPE phenomenon, when more than 50% cells of virus control group show CPE phenomenon (control group is normal cell control group, virus control group with virus seed, and no EGCG is added), detecting virus inhibition rate according to the method. The virus inhibition rate (experimental fluorescence number-control fluorescence number)/(cell control fluorescence number-virus control fluorescence number) × 100%

3. Direct killing effect of EGCG on virus

Test groups: the mixture contained 50. mu.L of HAdV-B3-GFP virus suspension 6.5X 10⁶FFU/mL and 450 mu L of a sample to be tested (the sample to be tested is the type A disinfectant, the type B disinfectant and the type C disinfectant). The mixture was incubated at (20. + -. 1) ° C for 90 seconds and then virus titration was performed. In this case, the concentration of the virus suspension was diluted in a 10-fold gradient as shown in FIG. 1.

The number of fluorescence and the kill log value of the disinfectant to the virus are counted out from the three experimental groups, and the experiment is independently repeated for 3 times.

Three) results

1.EGCG antiviral biosynthesis

The EGCG has obvious inhibition effect on HAdV-B3-GFP virus within the range of 0.5-10 mug/mL, and the virus inhibition rate and the EGCG concentration are in a logarithmic straight line relationship within the range ((R)²＝0.927，P<0.05), half-effective concentration IC of antiviral biosynthesis of EUCU₅₀1.12. mu.g/mL and a Therapeutic Index (TI) of 9.23, indicating that EUCU has an anti-intracellular biosynthetic effect on HAdV-B3-GFP virus.

TABLE 1 rate of virus inhibition by EGCG

In Table 1, "+ + + +" indicates 50% to 75% presence of CPE, "+" indicates 25% to 50% presence of CPE, "+" indicates ≦ 25% presence of CPE, and "-" indicates no CPE.

2. Direct killing effect of EGCG on virus

The virus inactivation test result of the disinfectant containing EGCG-palmitate shows that the inactivation log value of the A-type disinfectant provided by the application on HAdV-B3-GFP in 1min is more than 4.00 as shown in figure 1, and the disinfection qualified requirement is met.

After the HAdV-B3-GFP virus is incubated with the type A disinfectant for 60s, the virus titer is reduced by 0; after the type B disinfectant is treated for 60s, the virus titer is 1.2 multiplied by 10³FFU/mL, type C is 3.2X 10³FFU/mL; both of them have limited virus inactivation effect and do not meet the requirements of disinfection technical code.

In addition, for the A-type disinfectant, when the incubation time is more than 30s, the average virus killing effect of the brand A disinfectant reaches the requirement of disinfection technical specification, and when the difference of the virus killing effect of the disinfectants with the incubation time of more than 60s has no statistical significance, namely the incubation time is more than 60s, the incubation time has no obvious influence on the reduction of the virus titer.

TABLE 2 Virus titer when three disinfectants were incubated for 60s

"-" indicates no disinfectant is added, corresponding to the fourth row in FIG. 1

There is still a need for alcohol-based hand washes that demonstrate activity against a wide range of non-enveloped viruses. The disinfectant provided by the application protects EGCG, and has a killing effect on HAdV-3-GF. The experimental result of the EGCG antiviral biosynthesis function speculates that EGCG can be competitively combined with a virus receptor of a cell, so that the adsorption of the virus is blocked, and the prevention effect is achieved.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.