CN106957826B

CN106957826B - Virus inactivating agent and application thereof

Info

Publication number: CN106957826B
Application number: CN201710148704.7A
Authority: CN
Inventors: 张建文; 李莹; 黄茜; 王萍; 任超; 李宁; 丛茜; 杨兆祥
Original assignee: KPC Pharmaceuticals Inc
Current assignee: KPC Pharmaceuticals Inc
Priority date: 2017-03-14
Filing date: 2017-03-14
Publication date: 2020-02-07
Anticipated expiration: 2037-03-14
Also published as: CN106957826A

Abstract

The invention discloses a virus inactivator and application thereof. The virus inactivator is a cinnamon extract with a final concentration of 0.5-1 mg/ml. The application is the application of the virus inactivator in the preparation of drugs for inactivating paramyxovirus, herpes virus, retrovirus and influenza virus. The invention discloses a virus inactivation effect of a cinnamon extract (KPC-rg 1), and researches show that the extract can be rapidly and strongly combined with envelope protein of a virus shell at normal temperature, so that the virus is inactivated, loses infection and amplification capabilities, and has antigenicity.

Description

Virus inactivating agent and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a virus inactivating agent and application thereof.

Background

At present, the main vaccines applied at home and abroad comprise attenuated vaccines and inactivated vaccines. Because the production process of the inactivated vaccine is simple, safe, convenient to use and easy to store, and can stimulate animals to generate high-titer antibodies and remarkable vaccine protection effect, the development of the high-quality and high-efficiency inactivated vaccine is the most effective means for preventing and controlling various virus infection diseases. Inactivated vaccines, as the name implies, are vaccines made by inactivating infectious live pathogens by suitable physical or chemical means such that the pathogens are rendered non-pathogenic, but remain immunogenic.

The traditional virus inactivator formaldehyde is a carcinogen with strong irritation. It can act on the amino-containing nucleotide base of virus and also on the virus coat protein. When formaldehyde acts on virus capsid protein, protein cross-linking or virus particle aggregation is easy to occur, so that the neutralizing immunogenicity of pathogen protein is seriously damaged. The formaldehyde is used for long inactivation time, and the treatment is generally carried out at 37-39 ℃ for more than 24 hours or longer; the residual free formaldehyde can produce an irritant reaction after being injected into the body along with the vaccine. Therefore, on the premise of ensuring absolute safety of inactivation, the method improves inactivation speed, shortens inactivation time, reduces loss of effective antigen, and is a development target of a novel virus inactivator.

Cinnamon is a common Chinese medicinal material, is listed as the superior product from Shen nong Ben Cao Jing, and has been recorded in important herbal medicines of all generations. Cinnamon and its contained ingredients have various pharmacological actions, and commonly reported activities include antibacterial, antioxidant, hypoglycemic, aldose reductase resistant, anti-inflammatory, anticomplementary, antitumor activities, and regulating action on digestive system, cardiovascular system, immune system, central nervous system, and endocrine system. Cinnamon has been used for the prevention and treatment of viral infections in very few reports.

Disclosure of Invention

The first object of the present invention is to provide a virus inactivating agent; the second purpose is to provide the application of the virus inactivator.

The first purpose of the invention is realized by that the virus inactivator is cinnamon extract with the final concentration of 0.5-1 mg/ml.

The second object of the present invention is achieved by the use of the virus inactivating agent for preparing a medicament for inactivating paramyxovirus, herpesvirus, retrovirus, and influenza virus.

The invention discloses a virus inactivation effect of a cinnamon extract (KPC-rg 1), and researches show that the extract can be rapidly and strongly combined with envelope protein of a virus shell at normal temperature, so that the virus is inactivated, loses infection and amplification capabilities, and has antigenicity.

The cinnamon provided by the invention comprises: cortex Cinnamomi, Cinnamomum zeylanicum, HUANANGGUI, ramulus Cinnamomi Blumeae Balsamiferae, YINXIANG, cortex Cinnamomi Japonici, flos Lonicerae, cortex Cinnamomi Japonici, and cortex Cinnamomi; preferably, the cinnamon comprises: cinnamon, cinnamon bark of large leaf and cinnamon bark of Ceylon blue; more preferably, the cinnamon comprises: cinnamon and cassia twig, cortex cinnamomi japonica thunb.

The plant part of cinnamon is bark.

The method for inactivating viruses by the inactivator is to inactivate virus liquid to be inactivated by the virus inactivator at 4-37 ℃.

Drawings

FIG. 1 is a schematic diagram showing the inhibitory effect of cinnamon extract (KPC-rg 1) as a virus inactivator on vero cell proliferation;

FIG. 2 is a schematic diagram showing the effect of HSV-1 and KPC-rg1 on cell morphology following infection of cells with HSV-1 virus;

FIG. 3 is a schematic representation of the change in HSV-1 viral titer following KPC-rg1 treatment;

FIG. 4 is a graph showing the body weight changes of H1N1 after challenge;

FIG. 5 is a schematic representation of hemagglutination inhibition titers against H1N1 virus in mouse sera;

FIG. 6 is a graph showing RNA copy number of H1N1 virus M gene in lung of mice at day 3 after infection and at experimental end point (day 28).

Detailed Description

The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.

The virus inactivator is a cinnamon extract with a final concentration of 0.5-1 mg/ml.

The virus inactivator also comprises cinnamaldehyde with the weight percentage of 1-5% of cinnamon extract;

the cinnamon extract is cinnamon water extract prepared under the condition that the PH value is 1-5.

The cinnamon extract is prepared by taking cinnamon as a raw material through the steps of pretreatment, extraction and post-treatment, and specifically comprises the following steps:

A. pretreatment: crushing and sieving a material a of a raw material cinnamon for later use;

B. extraction: adding water which is 3-30 times of the weight of the material a into the material a, extracting for 1-3 times at the temperature of 4-80 ℃ for 1-24 hours each time, and combining the extracting solutions to obtain an extracting solution b;

C. and (3) post-treatment: concentrating the extracting solution b until the relative density is 1.01-1.05 to obtain a concentrated solution c, adding the concentrated solution c into an acidic phosphate buffer solution, adjusting the pH value to 1-5 by hydrochloric acid, standing for 8-24 h, and taking a precipitate as the cinnamon extract; adding cinnamaldehyde with the weight percentage of 1-5% of the precipitate part, and uniformly mixing to obtain the target virus inactivator.

B. extraction: adding water which is 3-12 times of the weight of the material a into the material a, extracting for 1-3 times at the temperature of 4-40 ℃ for 1-8 hours each time, and combining the extracting solutions to obtain an extracting solution b;

C. and (3) post-treatment: concentrating the extracting solution b to a relative density of 1.01-1.05 to obtain a concentrated solution c, adding the concentrated solution c into an acidic phosphate buffer solution with a pH value of 3-6, adjusting the pH value to 2-4 with hydrochloric acid, standing for 8-24 h, and taking a precipitate as the cinnamon extract; adding cinnamaldehyde with the weight percentage of 1-5% of the precipitate part, and uniformly mixing to obtain the target virus inactivator.

B. extraction: adding 10 times of water to the material a, extracting at 20 deg.C for 3 times (2 hr each time), and mixing extractive solutions to obtain extractive solution b;

C. and (3) post-treatment: concentrating the extracting solution b to a relative density of 1.01-1.05 to obtain a concentrated solution c, adding the concentrated solution c into an acidic phosphate buffer solution with a pH value of 6, adjusting the pH value to 3 with hydrochloric acid, standing for 12h, and taking a precipitate part, namely the cinnamon extract; adding cinnamaldehyde with the weight percentage of 1-5% of the precipitate part, and uniformly mixing to obtain the target virus inactivator.

The application of the virus inactivating agent is the application of the virus inactivating agent in the preparation of medicaments for inactivating paramyxovirus, herpes virus, retrovirus and influenza virus.

The virus inactivator is applied to preparing vaccines for inactivating paramyxovirus, herpes virus, retrovirus and influenza virus.

The preparation method of the inactivated paramyxovirus, herpesvirus, retrovirus and influenza virus vaccine comprises the following steps:

A. preparing an antigen solution;

B. adding a cinnamon extract with the concentration of 0.5-1 mg/ml into an antigen solution, fully mixing uniformly, and placing the mixed solution at the temperature of 20-30 ℃ for shake incubation for 5-10 min to obtain a target inactivated vaccine; wherein the proportion of the cinnamon extract to the antigen liquid is 1: 2.5 (wherein cinnamon extract is in mass units and virus is in hemagglutination units).

The antigen solution is prepared by inoculating the virus into host cells according to the inoculation amount of 1-3% of the volume percentage, replacing the culture solution with a maintenance solution when the host cells grow to 40-90%, and collecting the virus after maintaining and observing for 48-72 hours to obtain the antigen solution.

The culture solution is selected correspondingly according to different virus host cells (different virus host cells and different culture media); the maintenance liquid is a culture liquid containing 2% fetal calf serum.

The invention is further illustrated by the following specific examples:

EXAMPLE 1 preparation of Virus inactivating agent

The method comprises the following steps: pulverizing cortex Cinnamomi, soaking in 3 times of water at 4 deg.C for 1 time, and extracting for 1 hr. Step two: filtering, and collecting filtrate and residue. Step three: adding ethanol into the filtrate until the ethanol content reaches 65%, and standing. And step four, centrifuging, and reserving the supernatant and storing the precipitate. Step five: and taking the supernatant obtained in the fourth step, concentrating under reduced pressure, and drying in vacuum. Step six: dissolving in water, and filtering. Step seven: the filtrate was adjusted to pH 5 with dilute hydrochloric acid. Step eight: filtering, taking the precipitate and drying. Mixing the fourth step, the eighth step and commercial cinnamaldehyde according to the proportion of 10:10:0.5 to obtain the virus inactivator.

EXAMPLE 2 preparation of Virus inactivating agent

The method comprises the following steps: pulverizing cortex Cinnamomi, and stirring with 8 times of water at room temperature (20 deg.C) for 3 times, each for two hours. Step two: filtering, and collecting filtrate and residue. Step three: adding ethanol into the filtrate until the ethanol content reaches 80%, and standing. And step four, centrifuging, reserving the supernatant and reserving the precipitate. Step five: and 4, concentrating the supernatant obtained in the fourth step under reduced pressure, and drying in vacuum. Step six: dissolving in water, and filtering. Step seven: the filtrate was adjusted to pH 2 with dilute hydrochloric acid. Step eight: filtering, taking the precipitate and drying. Mixing the fourth step, the eighth step and commercial cinnamaldehyde according to the proportion of 10:10:0.5 to obtain the virus inactivator.

Example 3 preparation of total polysaccharide and total polyphenol of Cinnamomum cassia bark

Preparing the cinnamon bark total polysaccharide: the method comprises the following steps: pulverizing cortex Cinnamomi, extracting with 30 times of water at 80 deg.C under stirring for 3 times, each for 24 hr. Step two: filtering, and collecting filtrate and residue. Step three: adding ethanol into the filtrate until the ethanol content reaches 90%, and standing. And step four, centrifuging, reserving the supernatant and reserving the precipitate. Step five: and 4, concentrating the supernatant obtained in the fourth step under reduced pressure, and drying in vacuum. Step six: dissolving in water, and filtering. Step seven: the filtrate was adjusted to pH 1 with dilute hydrochloric acid. Step eight: filtering, taking the precipitate and drying. Mixing the fourth step, the eighth step and commercial cinnamaldehyde according to the proportion of 10:10:0.5 to obtain the virus inactivator.

Example 6

Inhibitory Effect of KPC-rg1 on vero cell proliferation

Vero cells plated in 96-well plates at 37 ℃ with 5% C0₂Adding cortex Cinnamomi extract (kpc-rg 1) with different concentrations after the incubator is overnight full of monolayer, respectively setting experimental group, cell control group, and blank control group, detecting OD value with CCK8 kit after 48 hr, and determining the OD value according to formulaAnd calculating the cell survival rate. The results show that there is no cytotoxic effect on vero cells when the concentration of kpc-rg1 is less than 0.05 mg/ml. See figure 1 for specific data.

Example 7

Inhibition of HSV-1 viral activity by KPC-rg1

40ug/ml of KPC-rg1 and Mol =0.1 HSV-1 virus solution were incubated at 4 ℃ for 24 hours, added to a 96-well plate plated with vero cells, and after 24 hours of incubation, cell morphology was observed under a microscope with virus control and cell control (no virus, medium only), and the TCID50 value of the virus suspension was calculated. The result shows that KPC-rg1 has obvious inactivation effect on HSV-1 virus and plays a good role in protecting vero cells. The specific experimental results are shown in fig. 2 and 3.

Example 8

In vitro antiviral Activity test of KPC-rg1 against RSV and HIV-1 viruses

Sample preparation: KPC-rg1 was dissolved in sterile water to 8 mg/ml and heated in a water bath at 80 ℃ for 15 minutes. After centrifugation of the solution at 2000 RPM for 2 minutes, the supernatant was stored at 4 ℃ as a KPC-rg1 stock. In the EC50 assay experiments, KPC-rg1 and the reference compound were diluted 3-fold to test 8 concentration points, each concentration tested in duplicate wells. The highest concentration tested was 1000. mu.g/ml. The final concentration of DMSO in the cell culture medium was 0.5%. And antiviral activity was tested according to the following method:

TABLE 1 antiviral Experimental methods List

RSV Along virus cytopathic experiment: cells were seeded at a certain density (table 1) in a microplate and cultured overnight at 37 ℃ in a 5% CO2 incubator. The following day, pre-mixes of varying concentrations of compound and virus (table 1) were added to the cell plates and incubated at 37 ℃ with 5% CO2 for 3-5 days until the cytopathic effect in the virus control wells was complete. Cell viability was then measured using the CCK8 cell viability assay kit as indicated for the test reagents. The antiviral activity at different concentrations of the compound is determined by the inhibitory effect on the cytopathic effect caused by the virus. All data were calibrated against a blank infection control. EC50 was calculated analytically by GraphPadPrism software.

HIV-1 virus cytopathic experiment: the test compound and the reference compound were diluted in two fold and mixed with the virus (table 1), and then the mixture of the compound and the virus and MT-4 cells were added to the microplate and incubated in a 5% CO2 incubator at 33 ℃ or 37 ℃ for 2-6 days until the cytopathic effect in the virus control wells was complete. Cell viability was then measured using CCK8 or CellTiter Glo cell viability assay kit as indicated for the detection reagents. The antiviral activity of the compounds at different concentrations is determined by the inhibitory effect on the cytopathic effect caused by the virus. EC50 was calculated analytically by GraphPad Prism software.

The results show that the antiviral activity of all reference compounds of the antiviral activity test in the test is in accordance with the expectation, and the test result is reliable. The antiviral activity of KPC-rg1 is similar to that of positive control drug, and has better activity inhibition effect on two viruses, and the result is shown in Table 2.

TABLE 2 list of antiviral activities of cinnamon extract

Example 9

The anti-influenza virus effect of KPC-rg1 and the animal protection effect generated by KPC-rg1 induced "in situ solidification immune effect" are verified

Grouping of Experimental materials with animals

Test samples: KPC-rg1

Experimental virus strains: H1N1 (A/Puerto Rico/8/1934). The virus was propagated by dog kidney cells. The HA titer (HAU) of the virus strain was found to be 256 prior to storage at-70 ℃; the experimental virus was 250 HAU.

Experimental animals and groups: mice of 28-30 days of age.

A) PBS control group: 6 mice.

B) Virome (PR 8 challenge group): 8 mice (250 HAU/mouse).

C) KPC-rg1 administration group, total 8 mice.

Experimental methods

The indicated doses of KPC-rg1 (100 ug/tube) were pre-incubated with appropriate amounts of PBS for 5-10 min at room temperature with the virus (250 HAU/tube). Mice of 28-30 days of age were inoculated with either the pre-mixed KPC-rg 1/virus solution or virus solution. The total volume of KPC-rg 1/virus in PBS was 50 ul/mouse. Anesthesia was performed prior to inoculation. PBS was used as blank control. Mice were observed for 28-day survival levels. And mouse sera were collected from the eyeball blood collection at the end of the experiment (28 days) and tested for hemagglutination inhibition titer against H1N1 virus (HI titer) in mouse sera by the erythrocyte agglutination inhibition experiment (HI). The coagulation inhibition result is ≧ 20, which is the positive effective HI titer. n = 3. Meanwhile, obtaining mouse lungs on the 3 rd day after infection and at the experimental end point (28 days), extracting total lung RNA, and detecting the copy number of the virus M gene RNA of H1N1 virus in the lungs of each group of mice by RT-PCR.

Results of the experiment

Survival of mice on day 28

TABLE 3 survival level of groups of mice for 28 days

The mice in the challenge group died on the third day after challenge, and died by half within one week after challenge, with a survival rate of 37.5% (5 mice died in total of 8 mice). The KPC-rg1 group did not die after challenge, and survival rate was one hundred percent. The body weight and survival results reveal that the drug has significant protective effects on mice.

The change of body weight of the mice is shown in FIG. 4

The body weight of mice in the control PBS group steadily increased, while the body weight of mice in the challenge group involved in virus challenge, KPC-rg1 group, decreased after challenge, and gradually recovered and increased after one week.

The hemagglutination inhibition titers (antibody levels) of the mice are shown in FIG. 5

After 28 days of infection, 128 hemagglutination inhibition titers can be detected in the serum of mice in the challenge group and the KPC-rg1 group, which shows that the challenge of the virus to the mice leads the immune system of the mice to generate corresponding antibodies and protectiveness.

The viral load in the lungs of mice was measured as shown in FIG. 6

And (3) detecting the lung infection condition of the virus 3 days after the virus attack, wherein the lung virus load of each group of mice reaches 7 times of 10 and every 50mg of the lung, and the virus infection and proliferation condition in the mouse lung is shown. At the end of the experiment, 28 days, the lung viral load of the mice in the challenge group can not be detected, which indicates that the virus is cleared by the immune system of the mice. The above results reveal the progression of the virus from infection to clearance of the lung.

In combination with the above results, the virus had infected the lungs of all groups of mice after challenge, resulting in over half of the death of the challenge group within one week after infection. And the mice in the KPC-rg1 group do not die, which shows that the medicament has obvious protective effect on the mice attacked by the H1N1 influenza virus. Mice in the challenge group and the drug group which survive 28 days after infection have significant antibody production, and the virus is also eliminated by the immune system of the body.

Claims

1. A virus inactivator is characterized in that the preparation method of the virus inactivator comprises the following steps:

the method comprises the following steps: pulverizing cortex Cinnamomi, soaking and extracting with 3 times of water at 4 deg.C for 1 time for 1 hr; filtering, collecting filtrate, and collecting residue; adding ethanol into the filtrate until the ethanol content reaches 65%, and standing; centrifuging, reserving supernatant, and reserving a precipitate; step two: taking the supernatant obtained in the step one, concentrating under reduced pressure, and drying in vacuum; dissolving in water, and filtering; adjusting the pH value of the filtrate to 5 by using dilute hydrochloric acid; filtering, taking the precipitate, and drying; mixing the precipitates obtained in the first step and the second step with commercially available cinnamaldehyde according to the ratio of 10:10:0.5 to obtain the virus inactivator;

or, the first step: pulverizing cortex Cinnamomi, stirring with 8 times of water at 20 deg.C for 3 times, each for 2 hr; filtering, collecting filtrate, and collecting residue; adding ethanol into the filtrate until the ethanol content reaches 80%, and standing; centrifuging, reserving supernatant, and reserving a precipitate; step two: concentrating the supernatant in the step one under reduced pressure, and drying in vacuum; dissolving in water, and filtering; adjusting the pH value of the filtrate to 2 by using dilute hydrochloric acid; filtering, taking the precipitate, and drying; mixing the precipitates obtained in the first step and the second step with commercially available cinnamaldehyde according to the ratio of 10:10:0.5 to obtain the virus inactivator;

or, the first step: pulverizing cortex Cinnamomi, extracting with 30 times of water at 80 deg.C under stirring for 3 times, each for 24 hr; filtering, collecting filtrate, and collecting residue; adding ethanol into the filtrate until the ethanol content reaches 90%, and standing; centrifuging, reserving supernatant, and reserving a precipitate; step two: concentrating the supernatant in the step one under reduced pressure, and drying in vacuum; dissolving in water, and filtering; adjusting the pH value of the filtrate to 1 by using dilute hydrochloric acid; filtering, taking the precipitate, and drying; and (3) mixing the precipitates obtained in the first step and the second step with commercially available cinnamaldehyde according to the ratio of 10:10:0.5 to obtain the virus inactivator.

2. Use of the virus inactivating agent of claim 1, for the preparation of a medicament for inactivating paramyxovirus, herpesvirus, retrovirus or influenza virus.