CN107041881B - Application of calycosin in preparing anti-rotavirus medicine - Google Patents
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Abstract
The invention relates to the technical field of medicines, in particular to application of calycosin in preparation of anti-rotavirus medicines. The application of calycosin in preparing anti-rotavirus medicaments reveals that calycosin can be applied to preparing anti-rotavirus medicaments, and the application can provide significant value for developing safe and effective medicaments for preventing and treating rotavirus infection. In addition, the application of the calycosin in preparing the anti-rotavirus medicine reveals that the molar concentration of the calycosin in the anti-rotavirus medicine is 10-80 umol/L, namely, the calycosin can well prevent and treat rotavirus infection under certain medicine concentration, and reveals that the molar concentration of the calycosin in the anti-rotavirus medicine, which can generate toxicity to cells, is more than 80 umol/L, thereby providing good guiding value for utilizing the calycosin in the anti-rotavirus medicine.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of calycosin in preparation of anti-rotavirus medicines.
Background
Rotavirus (RV) is a main pathogen causing acute diarrhea of infants, about 60 thousands of infants die of RV infection every year, and no specific medicine for preventing and treating the RV infection exists at present. 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, the research on the rotavirus infection mechanism is strengthened, and the development of safe and effective medicaments for preventing and treating rotavirus infection is very important.
Disclosure of Invention
The invention aims to provide the application of calycosin 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 calycosin in preparing anti-rotavirus medicaments.
In the anti-rotavirus medicine, the molar concentration of the calycosin against rotavirus is 10 to 80 mu mol/L.
In the rotavirus resisting medicine, the molar concentration of calycosin to produce toxicity to cells is more than 80 mu mol/L.
Compared with the prior art, the invention has the beneficial effects that:
(1) the application of calycosin in preparing the rotavirus resisting medicine disclosed by the invention discloses that calycosin can be applied to preparing the rotavirus resisting medicine, and the application can provide significant value for developing safe and effective medicines to prevent and treat rotavirus infection.
(2) The application of calycosin in preparing the anti-rotavirus medicament provided by the invention discloses that the anti-rotavirus molar concentration of the calycosin in the anti-rotavirus medicament is 10-80 mu mol/L, namely, the calycosin can well prevent and treat rotavirus infection under certain medicament concentration, and discloses that the molar concentration of the calycosin which generates toxicity to cells in the anti-rotavirus medicament is more than 80 mu mol/L, thereby providing good guiding value for utilizing the calycosin to resist rotavirus medicament.
Drawings
FIG. 1 is a graph showing the results of experiments on the cytotoxic effect of calycosin on MA 104.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following 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.
Among them, DMEM referred to in the present invention is a medium containing various amino acids and glucose.
Example 1.
Application of calycosin in preparing medicine for resisting rotavirus is provided.
The application of calycosin in preparing anti-rotavirus medicaments reveals that calycosin can be applied to preparing anti-rotavirus medicaments, and the application can provide significant value for developing safe and effective medicaments for preventing and treating rotavirus infection.
Moreover, the application of calycosin in the embodiment in the preparation of the anti-rotavirus medicament reveals that the anti-rotavirus molar concentration of calycosin in the anti-rotavirus medicament is 10 to 80 mu mol/L, namely, the calycosin can well prevent and treat rotavirus infection under certain medicament concentration, and reveals that the molar concentration of calycosin which generates toxicity to cells in the anti-rotavirus medicament is more than 80 mu mol/L, thereby providing good guiding value for utilizing the calycosin to resist rotavirus medicament.
Application experiment of calycosin in preparing anti-rotavirus medicine:
first, experimental material
Experimental drugs:
calycosin (Dowmansite, Inc., 99.08% purity, lot number: MUST-16031110).
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;
olymps inverted optical microscope CKX 41;
enzyme linked immunosorbent assay (ELISA) DG 3022A;
carbon dioxide incubator, available from Thermo corporation, usa;
cells and viruses;
MA104 cells (rhesus monkey embryonic kidney cells), Wa strain rotavirus.
Second, Experimental methods
Cell and virus:
MA104 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 calycosin solution: weighing 5mg of calycosin sample, dissolving with DMSO (dimethyl sulfoxide), adding appropriate amount of DMEM, dissolving with DMSO final 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 1.25 mu mol/L-320 mu mol/L.
Cytotoxicity experiments: MA104 cells at 10X 104Cell density of one/ml, added to 200. 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, and firstly, mixing the growth liquid according to the ratio of 1:1 and 1: 10. 1: 100. 1: 1000 was diluted with serum-free DMEM medium to obtain a series of concentrations of drug solution, and then 100. mu.l/well was added to a 96-well plate, and 6 wells were repeated 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 three other drug concentrations were normal within 72 hours except for the change in cytotoxicity between the first and second drug concentrations. This is achieved byIndicating an effective concentration between 1:1 and 1: 100. According to the ratio of 1:1, 1: 2. 1: 4. 1: 8. 1: 16. 1: 32. 1: 64 to obtain a series of concentrated solutions, and repeating the above steps, wherein the concentration is higher than 1: 4, a change in cytotoxicity occurred.
37℃ 5％CO2Incubating, continuously observing for 3d with optical microscope, sucking out the medicinal liquid, keeping out of the sun, adding 5mg/ml MTT 25 μ l/well (prepared with serum-free DMEM medium, and standing in 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 570nm 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 plate of MA104 cells grown as a monolayer, and each liquid medicine was repeated 4 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 group 50100 μ l/well of virus (virus reacted with 10 μ g/ml pancreatin at 37 ℃ for 30min), 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 is read by an enzyme linked immunosorbent assay at 570 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.
They were added to 96-well culture plates grown to a monolayer of MA104 cells, and the cells were washed 2 times with PBS before. 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 200. 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 solution (virus was allowed to act with trypsin at a concentration of 10. mu.g/ml for 30min) was added to 100. mu.l/well in a 96-well culture plate of MA104 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 200 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
Results of Calycosin cytotoxicity test:
the results of experiments on the cytotoxic effect (n-3) of calycosin on MA104 are shown in figure 1, and show that the calycosin has no cytotoxicity at the concentration of 1.25-80 mu mol/L, and has obvious cytotoxicity when the concentration is more than 160 mu mol/L.
(II) the results of experiments on the prevention of rotavirus invading cells by calycosin:
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 preventing rotavirus invading cells.
(III) results of experiments for direct inactivation of rotavirus by calycosin:
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 Calycosin on Virus biosynthesis:
calycosin biosynthesis on RV (n ═ 3)
The results show that the higher the inhibition rate of the anti-RV biosynthesis of calycosin along with the increase of the concentration of the calycosin, the higher the anti-RV biosynthesis of calycosin is, the higher the concentration of the calycosin is, but the effects of preventing rotavirus invading cells and directly killing rotavirus are not achieved. The research provides experimental basis for developing calycosin as a new drug for resisting RV.
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)
1. Application of calycosin in preparing anti-rotavirus medicine is provided; in the anti-rotavirus medicine, the molar concentration of the calycosin against rotavirus is 10 to 80 mu mol/L; in the rotavirus resisting medicine, the molar concentration of calycosin to produce toxicity to cells is more than 80 mu mol/L.
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| CN101073592A (en) * | 2006-05-18 | 2007-11-21 | 天津天士力制药股份有限公司 | Method for separating and extracting Milkvetch Root |
| CN101653438A (en) * | 2009-07-14 | 2010-02-24 | 澳门大学 | Application of calycosin in radix astragali in preparing medicament for vascular protection and angiogenesis promotion |
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