CN111773234B - Application of icariside II in preparation of anti-enterovirus 71 medicament - Google Patents
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- CN111773234B CN111773234B CN202010814738.7A CN202010814738A CN111773234B CN 111773234 B CN111773234 B CN 111773234B CN 202010814738 A CN202010814738 A CN 202010814738A CN 111773234 B CN111773234 B CN 111773234B
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Abstract
The application discloses application of a specific monomer icariside II in Chinese herbal medicine epimedium herb in the technical field of traditional Chinese medicine pharmacy in preparing a pharmaceutical composition, in particular application in preparing a pharmaceutical composition for preventing and treating Enterovirus 71 (Enterovirus 71, EV 71). The results of studies carried out by in vitro EV71 infected cell model experiments show that icariside II can inhibit the replication of EV71 at the gene and protein level. Proves that the icariside II has obvious effect of resisting EV71 and can be used for preparing medicines for resisting EV 71.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine pharmacy, in particular to application of a specific monomer icariside II in Chinese herbal medicine epimedium herb in preparation of an antiviral medicament, and specifically application in preparation of an anti-enterovirus 71 medicament.
Background
Hand-Foot-Mouth disease (HFMD) is an acute intestinal infectious disease caused by various enteroviruses, is better developed in preschool age, particularly in children under 3 years old, and has the main transmission routes of digestive tracts, respiratory tracts and close contact. Enterovirus type 71 (Enterovirus 71, EV71) is the major pathogen of HFMD, a highly phagocytic neurovirus, and can cause severe nervous system damage. Therefore, the treatment and prevention are very slow from the root. At present, the HFDM is treated by combined administration of interferon, ribavirin, methylprednisolone and the like in clinic, but the drugs have strong effect on severe HFMD caused by EV71, and the search for new and specific anti-EV 71 drugs for preventing and treating the HFMD is particularly important.
The Chinese medicine Epimedium herb (Epimedium brevicornum Maxim) is also called Epimedium, climbing fern herb, common loosestrife herb and the like, is a plant of the genus Epimedium in the family berberidaceae, and is generally distributed in Guizhou, Sichuan, Liaoning, Jiangxi, Shaanxi, Hunan, Hubei and other places in China. Herba epimedii is recorded in Shen nong Ben Cao Jing at the earliest, has extremely high medicinal value and long application history, and is recorded to have the main effects of tonifying kidney and strengthening yang, dispelling wind and removing dampness and the like. Modern researches show that epimedium can be clinically used for treating diseases such as breast lumps, hypertension and the like, and also has the effects of enhancing the human body immunity, delaying senescence, preventing and treating neurological diseases and the like.
Prior art CN201580055424.0 discloses an antiviral composition containing epimedium koreanum extract as an active ingredient, which contains epimedium koreanum (epimedium) extract as an active ingredient. Disclosed is a Korean epimedium extract which can be used for the preparation of a medicament for the prevention or treatment of viral diseases such as hand-foot-and-mouth disease. However, there is no evidence that the virus composition can be used for preventing and treating hand-foot-and-mouth disease in the whole specification of the above documents, and there is no specific indication which kind of virus causes hand-foot-and-mouth disease, and the active ingredients in epimedium extract are unknown, so that the prior art lacks a specific component extracted from epimedium for treating EV 71-caused diseases.
In addition, icariside II is a polyhydroxy flavonoid monomer component extracted from epimedium herb, is one of effective components of epimedium herb, and has the effects of treating erectile dysfunction, resisting ischemic brain injury, resisting tumors and the like in literature reports, but the effect of icariside II on resisting EV71 is not reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of icariside II in preparing the anti-enterovirus 71 medicament.
One of the purposes of the invention is to provide the application of icariside II or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers or excipients as active ingredients in the preparation of anti-enterovirus drugs.
Wherein the enterovirus is enterovirus 71.
The diseases caused by the enterovirus 71 infection comprise hand-foot-and-mouth disease, viral angina, aseptic meningitis, brainstem encephalitis, neurogenic pulmonary edema, poliomyelitis and central nervous system infection.
The second purpose of the invention is to provide a medicament, which is prepared by taking icariside II as an active ingredient and is used for preventing and treating enterovirus infection.
In particular to a medicament for resisting enterovirus 71 type infection, which is prepared by taking icariside II as an active ingredient.
The molecular formula of icariside II is as follows: c27H30O10(commercially available).
The invention is also directed to the above formula C27H30O10Icariside II was subjected to the following experiment:
detecting the influence of icariside II on Cell cytopathic effect (CPE) after EV71 infection by using EV71 infected RD (human malignant embryonal rhabdomyoma) cells as a research model; effect on virus titer in supernatant; effects on EV71VP1 mRNA and VP1 protein. The detection result shows that icariside II can inhibit CPE caused by EV71, reduce virus titer, inhibit the transcription level of EV71VP1 mRNA, and inhibit the expression of VP1 protein.
Experimental results prove that icariside II has an obvious EV71 resistance effect and can be used for preparing medicines for resisting EV71 infection.
Drawings
FIG. 1 is a schematic diagram of the effect of icariside II on the growth of RD cells detected by the CCK8 method;
FIG. 2 is a schematic diagram showing the effect of icariside II on CPE caused by EV71 observed under a light microscope;
FIG. 3 is a graph showing the change of virus titer after icariside II is applied to EV 71-infected RD cells calculated by the Reed-Muench formula;
FIG. 4 is a graph showing the effect of icariside II on EV71VP1 mRNA transcription levels after detecting its effect on EV 71-infected RD cells by fluorescent quantitative PCR;
FIG. 5 is a schematic diagram showing the effect of icariside II on EV71VP1 protein after detecting the effect of icariside II on EV 71-infected RD cells by Western Blot method.
Detailed Description
The following is further detailed by way of specific embodiments:
1. CCK8 screening maximum nontoxic concentration of icariside II on RD cells
RD cells in logarithmic growth phase were taken, digested with 0.25% trypsin, and seeded in 96-well plates at 1X 104And (3) removing the culture solution after the cells grow into a monolayer, adding icariside II (12.5 mu M, 25 mu M, 50 mu M, 100 mu M and 200 mu M) with different concentrations into a 96-well plate, inoculating 5 wells into each dilution to serve as an experimental group, adding only culture medium into 5 wells to serve as an experimental control group, adding only culture medium into 5 wells to serve as a blank control, and adding 0.1mL into each well. Standing at 37 deg.C for 5% CO2Culturing in an incubator, measuring OD value by adopting a CCK8 method after culturing for 24h and 48h respectively, and calculating the cell survival rate.
As shown in figure 1, the invention uses a CCK8 analysis method to detect the influence of the drug on the cell growth, adopts a cell model RD accepted by the field experiment, the selected icariside II concentrations are respectively 12.5 mu M, 25 mu M, 50 mu M, 100 mu M and 200 mu M, and the experimental data are subjected to one-factor variance analysis, and the result shows that the icariside II with the concentration of more than 50 mu M has obvious influence (p is less than 0.05) on the growth of RD cells.
2. Observation of influence of icariside II on CPE (CPE) caused by EV71 under light microscope
RD cells in logarithmic growth phase were taken, digested with 0.25% trypsin, and seeded in 6-well plates at 5X 105And (3) removing the culture solution after the cells grow into a monolayer, washing with PBS for 2-3 times, infecting RD cells with EV71 with the MOI of 0.1, sucking out the virus solution after 2 hours, replacing the virus solution with a culture medium which is prepared from a complete culture medium and contains icariside II with different concentrations, and setting an icariside II single administration group, an EV71 infection group and a negative culture medium control group. CPE was observed under an optical microscope after 24 h. As shown in fig. 2, CPE is a series of changes that occur in the cell after the virus replicates in the host cell, such as: initially, the nuclei change, chromatin borders, nuclei condense, and subsequently the cell membrane changes, losing adhesion, cells become rounded, fall off the flask wall, or cells fuse with each other, etc. These changes can be examined microscopically. The results show that: compared with the EV71 infected group, the icariside II concentration of 12.5. mu.M, 25. mu.M and 50. mu.M can effectively inhibit CPE caused by EV 71.
3. Effect of icariside II on EV71 Virus Titer
Plating, inoculating poison and adding medicine in the same way as 2, adding medicine for 24h, collecting cell supernatant, centrifuging at 4 ℃ and 12000rpm for 10min, and collecting cell supernatant again for later use.
Vero cells in logarithmic growth phase were taken, digested with 0.25% trypsin, at 1X 104Inoculating into 96-well plate at a concentration of 100 μ L per well, removing culture medium when the cells are full of monolayer, washing with PBS twice, and sequentially diluting the collected supernatant virus solution 10 times (10 times) with DMEM/F-12 medium-1~10-10) 8 wells were inoculated at each dilution, 8 additional wells were used as blanks, and serum-free DMEM/F-12 medium was added at 0.1mL per well. Standing at 37 deg.C for 5% CO2Culturing in incubator, observing and recording CPE every day for 72h, and calculating 50% cell infection dose (TCID) of virus solution by Reed-Muench formula50)。
As shown in FIG. 3, icariside II was present at 25. mu.M and 50. mu.M and was able to reduce the titer in the cell supernatant caused by EV71 infection, compared to the virus-infected group. The results show that icariside II 25. mu.M and 50. mu.M decreased the titer in the cell supernatant caused by EV71 infection compared to the virus-infected group.
4. Influence of icariside II on VP1 Gene transcript level
Inoculating a plate, inoculating and adding drugs in the same mode as 2, adding drugs for 24h, collecting cells and extracting total RNA, wherein the process comprises the following steps: taking out a 6-hole plate, placing the 6-hole plate on ice, washing the 6-hole plate for 2 times by PBS (phosphate buffer solution), adding 1mL of Trizol to perform lysis for 5-10min, and transferring a lysate to an EP (EP) tube; ② adding 0.2mL chloroform, shaking for 30s by a vortex shaking instrument, and standing for 15min at room temperature. Centrifuging at 12000rpm and 4 deg.C for 15 min; thirdly, placing the upper colorless aqueous phase into a new EP tube, adding isopropanol with the same volume, turning upside down and mixing uniformly, standing at room temperature for 10min,12000rpm, and centrifuging at 4 ℃ for 10 min; fourthly, abandoning the supernatant, washing the RNA sediment by 1mL of 75 percent ethanol, reversing the solution to wash the RNA sediment upside down, and centrifuging the RNA sediment for 8min at the temperature of 4 ℃ at 12000 rpm; fifthly, abandoning the supernatant, taking 30 microliter RNase-free water to dissolve RNA precipitate after ethanol remained in the precipitated RNA is volatilized, and freezing the RNA precipitate in a refrigerator at the temperature of minus 80 ℃.
From the obtained RNA concentration value, the quantitative amount was 1. mu.g, and the amount of the desired RNA sample was calculated. The extracted RNA is reversely transcribed into cDNA in a 20-mu L system, and the reaction procedure is as follows: 15min at 37 ℃; the reaction system is stored at 85 ℃, 5sec and 4 ℃ as follows:
TABLE 1 real-time fluorescent quantitative PCR reverse transcription system
TABLE 2 real-time fluorescent quantitative PCR primer sequences
Taking the obtained cDNA as a template, repeating the cDNA in three times for each sample, taking beta-actin as an internal reference, and carrying out amplification in a fluorescent quantitative PCR instrument, wherein the reaction program is as follows: at 95 ℃ for 30 s; (95 ℃, 3s → 60 ℃, 30s) × 40 cycles, dissolution curve apparatus default, reaction system as in table:
TABLE 3 real-time fluorescent quantitative PCR reaction System
The experiment is repeated three times, the Graphpad prism5 software is used for carrying out data analysis and mapping, and beta-actin is used as an internal reference and 2-△△CtThe results were calculated, and the relative levels between the groups were calculated.
As shown in FIG. 4, icariside II 50. mu.M was effective in reducing the level of transcription of EV71VP1 mRNA.
5. Influence of icariside II on VP1 protein expression
Inoculating the plate, inoculating the virus and adding the medicine in the same way as 2, adding the medicine for 24 hours, collecting cells and extracting total protein, and adopting the following process: cell proteins were extracted using RIPA lysate and protein concentration was determined by the bicinchoninic acid (BCA) method. Carrying out electrophoretic separation on 10% SDS-PAGE, wherein the sample loading amount is 40 mu g, and then transferring the sample to a PVDF membrane; 5% BSA was blocked at room temperature for 2h, and primary antibody VP1, beta-actin were incubated overnight at 4 ℃ for 6min × 5 times in TBST; incubating corresponding secondary antibody at room temperature for 2h, and TBST 6min × 5 times; using ELC hypersensitive luminescent liquid A, B according to the proportion of 1: 1 proportion of mixed luminescence by ChemiDocTMThe Touch Imaging System instruments perform exposures. As shown in FIG. 5, icariside II 50. mu.M was effective in reducing the level of transcription of EV71VP1 mRNA. Meanwhile, the expression of the EV71VP1 protein is detected by Western blot, and the result shows that icariside II can effectively inhibit the expression of the EV71VP1 protein and has concentration dependence.
The experiment results are combined to obtain that icariside II has obvious EV71 effect, and can be used for preparing medicines for preventing and treating hand-foot-and-mouth disease, viral angina, aseptic meningitis, brainstem encephalitis, neurogenic pulmonary edema, poliomyelitis and central nervous system infection caused by EV 71.
Claims (2)
1. Application of icariside II or pharmaceutically acceptable salts thereof as active ingredients in preparation of anti-enterovirus 71 medicines.
2. Application of icariside II or pharmaceutically acceptable salt thereof as an active ingredient in preparing a medicament for preventing and treating hand-foot-and-mouth disease, viral angina, aseptic meningitis, brain stem encephalitis, neurogenic pulmonary edema and central nervous system infection caused by enterovirus 71.
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CN106377537A (en) * | 2016-08-19 | 2017-02-08 | 江苏康缘药业股份有限公司 | Application of acetytastragaloside |
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Antiviral Efficacy of Flavonoids against Enterovirus 71 Infection in Vitro and in Newborn Mice;Wenwen Dai等;《Viruses》;20190707;第11卷(第625期);第1-14页 * |
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复旦大学***/卫健委/医科院医学分子病毒学重点实验室2019年度开放课题评审结果通知;复旦大学***/卫健委/医科院医学分子病毒学重点实验室;《http://www.klmmv.fudan.edu.cn/CN/Show.aspx?info_lb=30&infoid=327&flag=4》;20190605;第1页 * |
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