CN108379280B

CN108379280B - Application of baicalin in preparation of anti-rotavirus drugs

Info

Publication number: CN108379280B
Application number: CN201810327123.4A
Authority: CN
Inventors: 赵文昌; 宋丽军; 周若夏
Original assignee: Guangdong Medical University
Current assignee: Guangdong Medical University
Priority date: 2018-04-12
Filing date: 2018-04-12
Publication date: 2020-11-06
Anticipated expiration: 2038-04-12
Also published as: CN108379280A

Abstract

The invention relates to the technical field of baicalin, in particular to application of baicalin in preparation of a rotavirus resisting medicine. The application of baicalin in preparing anti-rotavirus medicaments is the first research of an applicant to discover that the baicalin has the anti-rotavirus effect, fills the blank of anti-rotavirus medicaments, and has profound significance and value. Wherein, the research shows that the molar concentration of the baicalin in the anti-rotavirus medicament is 3.125 umol/L-400 umol/L.

Description

Application of baicalin in preparation of anti-rotavirus drugs

Technical Field

The invention relates to the technical field of baicalin, in particular to application of baicalin in preparation of a rotavirus resisting medicine.

Background

Rotaviruses (RV) are the main causative agent of acute diarrhea in infants, about 60 million infants die of RV infection every year, and are mainly epidemic in autumn and winter, and the occurrence is not affected by sanitary conditions. The incidence of disease in developed countries is comparable to that in developing countries, but the mortality rate in developing countries is much higher than that in developed countries. RV is generally transmitted through a fecal-oral route, and the incubation period is 2-4 days. RV is thought to mainly invade villi of small intestinal cells, the most main symptom of a patient is diarrhea, the patient is emergent, the diarrhea is caused more than ten times to dozens of times a day, fever can be caused, specific lgM and lgG antibodies can quickly appear in blood after infection, secretory lgA can locally appear in intestinal tracts, the virus can be neutralized, and the RV has an effect on homotype virus infection. The course of the disease is 3-5 days or about a week. Recent studies have shown that RV infections are not restricted to the gut, but can also invade other systems, causing many complications such as shock, encephalopathy, cardiac damage and extrahepatic bile duct obstruction.

At present, no specific medicine for treating RV infection exists. In the aspect of RV vaccines, the RV vaccines are still in the research stage at home, the RV vaccines are expensive to market abroad, and the RV strains have limited prevention range due to the diversity. At present, oral rehydration recommended by WHO is mostly adopted clinically to relieve symptoms. Therefore, it is very important to discover and develop safe and effective drugs for preventing and treating rotavirus infection.

Baicalin (structural formula shown below) is a flavonoid compound obtained by extracting and separating roots of Scutellaria baicalensis Georgi (Scutellaria baicalensis Georgi) which is a perennial herb of Scutellaria of Labiatae, has the effects of tranquilizing, protecting liver, benefiting gallbladder, resisting bacteria, diminishing inflammation and the like, and has been proved by researches to have the effect of inhibiting influenza viruses, herpes simplex viruses, Coxsackie viruses, respiratory syncytial viruses, hepatitis C viruses and the like, but whether the baicalin has the effect on RV is not reported.

Disclosure of Invention

The invention aims to provide the application of baicalin in preparing anti-rotavirus medicaments aiming at the defects of the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

provides the application of baicalin in preparing anti-rotavirus medicaments.

In the rotavirus resisting medicine, the molar concentration of the baicalin is 3.125 to 400 mu mol/L.

In the rotavirus resisting medicine, the molar concentration of the baicalin generating toxicity on cells is more than 800 mu mol/L.

The rotavirus resisting medicine is a tablet, a granule, powder, a capsule, a pellet, a liquid preparation, a semi-solid preparation or a liposome preparation.

The rotavirus resisting medicine includes composition or compound preparation with baicalin as component.

Compared with the prior art, the invention has the beneficial effects that:

the application of the baicalin in preparing the anti-rotavirus medicament is that the applicant finds that the baicalin has the anti-rotavirus effect for the first time, fills the blank of the anti-rotavirus medicament, and has profound significance and value.

Drawings

FIG. 1 is a graph showing the results of the cytotoxicity test of baicalin.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1.

The application of baicalin in preparing anti-rotavirus medicaments is the first research of the applicant to discover that the baicalin has the anti-rotavirus effect, fills the gap of anti-rotavirus medicaments, and has profound significance and value.

Example 2.

The application of baicalin in preparing anti-rotavirus medicines is disclosed, wherein the molar concentration of the baicalin in the anti-rotavirus medicines is 3.125-400 mu mol/L.

Example 3.

The application of baicalin in preparing anti-rotavirus medicines is disclosed, wherein in the anti-rotavirus medicines, the molar concentration of the baicalin generating toxicity on cells is more than 800 mu mol/L.

Example 4.

The application of baicalin in preparing anti-rotavirus medicaments is disclosed. Wherein the rotavirus resisting medicine is tablet, granule, powder, capsule, pellet, liquid preparation, semisolid preparation or liposome preparation.

Example 5.

The application of baicalin in preparing anti-rotavirus medicaments is disclosed. Wherein the rotavirus resisting medicine comprises a composition or a compound preparation taking baicalin as a component.

Experiment:

first, experimental material

Experimental drugs:

baicalin (Shanghai-sourced leaf Biotechnology Co., Ltd., purity 98%, batch No.: P16S8F 44143).

Experimental reagent:

DMEM culture solution and fetal bovine serum are provided by GIBCO company;

MTT (4, 5, dimethylthiazole-2, 5, diphenyltetrazolium bromide), available from M1124Amresco Inc.;

the mixed solution of penicillin and streptomycin, trypsin, PBS and dimethyl sulfoxide were all provided by solarbio.

Preparing and subpackaging reagents:

(1) fetal bovine serum was dispensed into sterile 50ml centrifuge tubes and stored at-20 ℃.

(2) Trypsin was dispensed into sterile 10ml centrifuge tubes and stored at-20 ℃.

(3) The mixed solution of streptomycin and streptomycin is subpackaged in sterilized 10ml centrifuge tubes and preserved at the temperature of minus 20 ℃.

(4) MTT application liquid:

50mg of MTT powder and 10 mg of PBS10ml are stirred and dissolved, filtered by a 0.22 mu m filter membrane for sterilization, and are stored at minus 20 ℃ in dark for standby after being subpackaged.

Experimental equipment:

a 96-hole cell culture plate, an adjustable microsyringe, a cell culture bottle, a filter, a suction pipe, a thermostat and a disinfection pot;

clean bench SIK-202, available from Anhui Union clean plants;

OLYMP μm S inverted light microscope CKX 41;

enzyme linked immunosorbent assay (ELISA) DG 3022A;

carbon dioxide incubator, available from Thermo corporation, usa;

cells and viruses;

caco-2 cells (colon cancer cells), MA104 (rhesus monkey kidney cells), Wa strain rotavirus.

Second, Experimental methods

Cell and virus:

caco-2 cells were grown and passaged in DMEM medium containing 10% fetal bovine serum, 100. mu.g/ml penicillin, 100. mu.g/ml streptomycin. The infectious virus is human Wa strain rotavirus suitable for cell culture proliferation.

(II) cytotoxicity test of drugs:

preparing a baicalin solution: weighing 2mg of baicalin sample, dissolving with DMSO (dimethyl sulfoxide), adding appropriate amount of DMEM, dissolving with DMSO concentration not more than 0.5% as standard, and setting solvent control group. The aseptic operation in the clean bench is used for filtering and sterilizing the disposable filter head. The initial concentration was calculated. The final concentration of each drug is 25-200 mu mol/L.

Cytotoxicity experiments: caco-2 cells were cultured at 6X 10³Cell density of one/ml, 100. mu.l per well in 96 well cell culture plates. 5% CO at 37 ℃₂Incubate until the cells grow into a monolayer. Sucking out growth liquid, diluting with serum-free DMEM culture solution according to the proportion of 1:1, 1:10, 1:100 and 1:1000 to obtain liquid medicines with a series of concentrations, adding 100 mu l/hole into a 96-hole plate, and repeating 6 holes for each concentration. Normal controls were supplemented with an equal volume of DMEM maintenance solution without serum only. 5% CO at 37 ℃₂After incubation and continuous observation by light microscope, the cells at the other three drug concentrations were normal within 48 hours except for the change in cytotoxicity between the first and second drug concentrations. This indicates an effective concentration between 1:1 and 1: 100. A series of concentrations of medicinal liquid are obtained according to the ratio of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64, and the operations are repeated, and the cytotoxicity change of the medicinal liquid with the concentration higher than 1:4 within 72 hours is found under an optical microscope.

37℃5％CO₂Incubating, continuously observing for 2d with optical microscope, sucking out the liquid medicine, keeping out of the sun, adding 5mg/ml MTT25 μ l/well (prepared with serum-free DMEM medium, keeping out of the sun), and keeping at 37 deg.C with 5% CO₂After incubation for 4h, centrifuging at 800r/ml, removing supernatant, adding DMSO (DMSO) 50 μ l/well, oscillating at room temperature for about 10min, dissolving crystals, mixing, and detecting Optical Density (OD) value of each well at 490nm wavelength on a microplate reader. The cell viability was found by the following formula:

cell viability ═ mean absorbance value of drug group/mean absorbance value of cell control group × 100%.

(III) virus proliferation and titer determination:

proliferation and titer determination of Wa strain RV was performed on MA 104. RV was inoculated when cells were grown to monolayer in culture flasks. Prior to virus inoculation, the cell monolayer was washed once with phosphate buffered saline (PBS, pH7) and twice with serum-free DMEM medium. Before inoculation, the original virus solution was removed from a-80 ℃ freezer, dissolved at 4 ℃, exposed to 10. mu.g/ml pancreatin at 37 ℃ for 30min, and the MA104 cells grown as a monolayer were washed twice with PBS. Then adding virus liquid after incubation with pancreatin, wherein the cell maintenance liquid after virus inoculation is a DMEM culture liquid without fetal bovine serum and containing 1 mu g/ml pancreatin. Incubating the cells inoculated with the virus in a carbon dioxide incubator (containing 5% of carbon dioxide) at 37 ℃ until the virus infected MA104 cytopathic effect (CPE cytopathic effect) reaches + + + and then putting the culture bottle into a culture flask for freezing and storing at-20 ℃, thawing at 4 ℃, repeatedly freezing and thawing for three times, centrifuging at a low temperature and a high speed of 12000r/min, taking the supernatant to obtain virus liquid, repeating the virus passage experiment in the cells by the same method, and harvesting the virus. Subpackaging and storing at-80 deg.C.

Viral infectivity titer determination was performed in 96-well plates using MA104 cells (microtiter method). Diluting the obtained product to 10 according to the ratio of 1:10 in a serum-free DMEM culture solution^-110^-210^-3…10^-6At equal serial concentrations, 96-well plates cultured as monolayers of MA104 cells were washed 2 times with PBS, and different dilutions of virus were added to 96-well cell culture plates, repeating for 8 wells at each concentration at 25 μ l/well. Setting normal control cells, 5% CO at 37 ℃₂After 2h incubation, virus fluid was aspirated and cell culture medium without serum was added at 200. mu.l/well. 5% CO at 37 ℃₂Incubation and continuous observation. When no CPE is continuously appeared in the virus wells with the lowest dilution capable of showing cytopathic CPE, counting the number of the virus wells with CPE appearing in each dilution, and calculating the TCID of the virus according to the Reed-M [ mu ] nch method₅₀(Tissue culture infective dose)。

(IV) the anti-RV effect of the medicine:

in the experiment, the concentration of the TC90-95 drug is firstly selected for carrying out the following anti-RV experiment, and then the selected drug with the anti-RV effect is diluted by a DMEM culture solution (without serum) in a multiple ratio to examine the anti-RV effect at different concentrations.

(V) the effect of the medicine on virus-adsorbed cells:

the liquid medicine was added to a 96-well culture plate of Caco-2 cells grown as a monolayer, and each liquid medicine was repeated 6 wells at 100. mu.l/well. Normal cell control groups were set, and virus control groups were each supplemented with an equal volume of DMEM medium (no serum). 5% CO at 37 ℃₂And (5) incubating for 2 h.

Sucking out the medicinal liquid, adding 100TCID except for normal control group₅₀Of (1)Toxin (virus reacts with pancreatin with concentration of 10 mug/ml at 37 ℃ for 30min)100 mug/well, 5% CO at 37 ℃₂Incubating for 2h, aspirating the virus, adding 200. mu.l/well of cell maintenance medium, 5% CO at 37 ℃₂Incubation was followed by continuous observation.

After the cells had CPE at ++++ time, the maintenance solution was aspirated, 5mg/ml MTT50 μ l/well was added, incubated for 2h and the supernatant discarded. Add DMSO 25. mu.l/well and mix well with shaking at room temperature for about 10 min. Subsequently, the absorbance value was read by an enzyme linked immunosorbent assay at 490 nm.

The virus inhibition rate and therapeutic index of the drug were calculated according to the following formulas:

the inhibition ratio of the drug to the virus (average absorbance value of drug group-average absorbance value of virus control group)/(average absorbance value of normal cell control group-average absorbance value of virus control group) × 100%, and the experiment was repeated three times.

The Therapeutic Index (TI) is used as an evaluation index to measure the RV inhibition efficacy of the drug, and the TI is CC50/IC 50.

Note: observed under an optical microscope, without CPE, recorded as: a; CPE appeared in 25% of the cells and was recorded as: +; between 25% and 50% of the cells develop CPE, which is recorded as: + +; between 50% and 75% of the cells develop CPE, which is recorded as: + + + +; between 75% and 100% of the cells develop CPE, which is recorded as: ++++.

(VI) direct inactivation of the virus by the drug:

the drug was mixed with 100TCID50 in a virus solution (virus was exposed to pancreatin at a concentration of 10. mu.g/ml for 30min) in equal volume for 2 h.

This was added to 96-well culture plates grown as monolayers of Caco-2 cells, which were previously washed 2 times with PBS. Normal cell control groups were set, and virus control groups were each supplemented with an equal volume of DMEM only. 5% CO at 37 ℃₂Incubating for 2h, then aspirating the mixture, adding 100. mu.l/well of cell maintenance medium, 5% CO at 37 ℃₂And (4) continuously observing incubation, carrying out MTT detection by the method when the cells have CPE within the range of ++ - +++ and calculating the inhibition rate and the therapeutic index of the drug to the virus.

(VII) Effect of drugs on Virus biosynthesis:

(1) will 100TCID₅₀The virus liquid of (1)(the virus was allowed to react with trypsin at a concentration of 10. mu.g/ml for 30min) was added to 100. mu.l/well in 96-well culture plates of Caco-2 cells grown as a monolayer, and the cells were washed 2 times with PBS before. And setting a normal cell control, and adding an equivalent volume of DMEM culture solution. 5% CO at 37 ℃₂And (3) incubating for 2h, then sucking out the virus liquid, adding 100 mu l/hole of the virus liquid, setting virus control, and only adding an equal volume of DMEM culture solution.

(2)37℃5％CO₂Incubating for 2 hr, sucking out the medicinal liquid, adding cell maintenance liquid 100 μ/well, and adding 5% CO at 37 deg.C₂Incubation was followed by continuous observation.

(3) Namely, MTT detection is carried out when cell CPE is between ++ - +++ according to the method, and the inhibition rate and the therapeutic index of the drug to the virus are calculated.

(eight) statistical analysis

SPSS13.0 software is adopted, and One-Way ANOVA method is selected, and the OD value of the medicine group is compared with the OD value of the model group by mean. And (4) selecting a PROBIT regression method, and performing regression analysis on the drug concentration, the cell survival rate, the drug concentration and the inhibition rate of the drug on the virus. Obtaining the median cell survival concentration CC of the drug₅₀Inhibitory concentration of drug on half of the virus-infected cells IC₅₀And calculating the therapeutic index according to the therapeutic index TI which is the concentration of the cell median survival drug/the concentration of the median virus inhibiting drug.

Third, experimental results

(I) results of baicalin cytotoxicity test:

the experimental result of the cytotoxic effect (n is 3) of baicalin on Caco-2 is shown in figure 1, and the result shows that no cytotoxicity occurs in the baicalin at the concentration of 3.125-400 mu mol/L, and obvious cytotoxicity occurs when the concentration is more than 800 mu mol/L.

(II) the experimental result of preventing rotavirus invading cells by baicalin is as follows:

TABLE 1 baicalin action for preventing rotavirus invading cell (n ═ 3)

The results in Table 1 show that the inhibition rate of baicalin for preventing rotavirus invading cells is maximum at 200 mu mol/L, and that the inhibition rate of baicalin for resisting RV invading cells is between 100 mu mol/L and 250 mu mol/L, and the inhibition rate is weakened after being strengthened with the concentration increase.

(III) the experiment result of the baicalin direct inactivation rotavirus:

MTT shows that the OD value of each drug concentration group is not significant compared with that of a virus control group, and P is more than 0.05(n is 3). The results show that the medicine has no effect of directly killing rotavirus.

(IV) Effect of baicalin on Virus biosynthesis:

TABLE 2 Effect of baicalin on Virus biosynthesis (n ═ 3)

The results in Table 2 show that the inhibition rate of the anti-RV biosynthesis effect is maximum at 175 mu mol/L with the increase of the concentration of the baicalin, and show that the inhibition rate of the anti-RV biosynthesis effect of the baicalin is between 100 mu mol/L and 200 mu mol/L, and the inhibition rate is weakened after being enhanced with the increase of the concentration, but the inhibition rate has no effect of directly killing rotavirus.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of baicalin in preparing anti-rotavirus medicine; in the rotavirus resisting medicine, the molar concentration of the baicalin is 3.125-400 mu mol/L; in the rotavirus resisting medicine, the molar concentration of the baicalin generating toxicity on cells is more than 800 mu mol/L.

2. The use of baicalin in the preparation of an anti-rotavirus medicament according to claim 1, which is characterized in that: the rotavirus resisting medicine is a tablet, a granule, powder, a capsule, a pellet, a liquid preparation, a semi-solid preparation or a liposome preparation.

3. The use of baicalin in the preparation of an anti-rotavirus medicament according to claim 1, which is characterized in that: the rotavirus resisting medicine includes composition or compound preparation with baicalin as component.