Application of selfheal aqueous extract in preparation of medicine for preventing and treating Zika virus infection
阅读说明:本技术 夏枯草水提物在制备防治寨卡病毒感染的药物中的应用 (Application of selfheal aqueous extract in preparation of medicine for preventing and treating Zika virus infection ) 是由 李耿 龙海珊 喻良文 吕东勇 刘小虹 于 2019-12-27 设计创作,主要内容包括:本发明涉及夏枯草水提物在制备防治寨卡病毒感染的药物中的应用。本发明应用中的夏枯草水提物对寨卡病毒有明显的抑制作用,可用于制备防治寨卡病毒感染的药物。本发明所述的防治寨卡病毒感染的药物是由夏枯草水提物医学上可接受的辅料制成临床上可接受的固体或液体口服制剂。(The invention relates to an application of a selfheal aqueous extract in preparing a medicament for preventing and treating Zika virus infection. The selfheal aqueous extract applied in the invention has obvious inhibition effect on Zika virus and can be used for preparing medicaments for preventing Zika virus infection. The medicine for preventing and treating Zika virus infection is a clinically acceptable solid or liquid oral preparation prepared from medically acceptable auxiliary materials of a selfheal aqueous extract.)
1. Application of Prunellae Spica water extract in preparing medicine for preventing and treating Zika virus infection is provided.
2. Use according to claim 1, characterized in that: the medicine is a solid or liquid oral preparation which is composed of a common dose of auxiliary materials acceptable in medicine and a common water extract of selfheal.
3. Use according to claim 2, characterized in that: the pharmaceutical preparation is granules, capsules, tablets or oral liquid.
4. The use of claim 1, 2 or 3, wherein the aqueous extract of Prunella vulgaris is prepared by:
taking selfheal, adding water with the dose of 10 times of that of selfheal, reflux-extracting for 30 minutes, cooling, filtering, taking filtrate, concentrating and drying to obtain the selfheal water extract.
Technical Field
The invention relates to a medicinal preparation containing an undetermined structure from selfheal plants, in particular to a new application of the selfheal plants in the Labiatae in pharmacy.
Background
Zika virus is divided into two major lineages, the african and asian lineages. Zika virus (Zika virus) is classified as a Flaviviridae family, a flavivirus genus, belonging to a single-stranded positive-strand RNA virus, 10,794kb in length, having an open reading frame encoding the viral polyprotein, flanked by 5 'and 3' noncoding regions. The ZIKV-encoded polyprotein can be cleaved into 3 structural proteins (glycoprotein E, envelope glycoprotein prM/M, nucleocapsid protein C) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) by host cell protease and viral NS2B-NS3 protease, with the structural proteins located at the amino terminus and the non-structural proteins located at the carboxy segment and having serine protease, RNA helicase, and RNA-dependent RNA polymerase (RdRP) functions. In recent years, ZIKV has been prevalent worldwide, and studies have shown that ZIKV can trigger Congenital microcephaly (Congenital microcephaly), Guillain-barre syndrome (Guillain-barre syndrome), and other neurological symptoms. ZIKV was also transmitted by mother and infant, which is a major cause of microcephaly, in addition to mosquito transmission and sexual transmission. At present, many research institutions in the world are developing Zika virus vaccines, but no specific medicine or vaccine exists for treating Zika virus, and the virus mainly adopts preventive modes such as mosquito prevention and killing. Further research on Zika virus is necessary and urgent to find a drug for effectively treating Zika virus.
Prunellae Spica belongs to dried ear of Prunellae Spica of Labiatae, is pungent, bitter and cold in taste, and enters liver and gallbladder channels, has effects of clearing liver-fire, improving eyesight, decreasing swelling, and resolving hard mass, and can be used for treating conjunctival congestion, swelling and pain, eyeball nyctalgia, headache, giddiness, goiter, scrofula, mammary abscess, nodules, etc. Modern clinical treatment is mainly used for treating diseases such as thyroid diseases, hyperplasia of mammary glands, hypertension, skin diseases, lymphoma and the like, and is matched with other medicines for treating insomnia, prostatic hyperplasia, tuberculosis, cyst, acute and chronic pharyngitis, rhagadia manus et pedis and desquamation, hemoptysis, hysteromyoma, pelvic inflammation, epilepsy, irregular menstruation and the like. At present, antiviral researches on selfheal are mainly focused on resisting HSV, RSV, HIV, Ebola virus and the like, and researches on selfheal resisting Zika virus are not found in literatures.
Disclosure of Invention
The invention aims to solve the technical problem of providing a new application of selfheal in pharmacy.
The new application is specifically the application of the selfheal aqueous extract in preparing the medicine for preventing and treating Zika virus infection.
In the application, the medicament is a clinically acceptable solid or liquid oral preparation prepared from the selfheal aqueous extract and a conventional amount of medically acceptable auxiliary material preparation.
In the application, the solid oral preparation is specifically clinically acceptable granules, capsules or tablets.
In the application, the liquid oral preparation is a clinically acceptable oral liquid.
In the application, the selfheal aqueous extract can be obtained by adopting a method commonly used in the field, and the method recommended by the inventor is as follows:
taking a selfheal medicinal material, adding water with the dose of 10 times, carrying out reflux extraction for 30 minutes, cooling, filtering, taking filtrate, concentrating and drying to obtain a selfheal water extract.
The preparation method of the solid oral preparation is a conventional preparation method specified in pharmacopoeia, namely the selfheal aqueous extract and the auxiliary materials commonly used by the solid oral preparation are mixed according to a clinically acceptable proportion to prepare the solid oral preparation.
The preparation method of the liquid oral preparation is a conventional preparation method specified in pharmacopoeia, namely, the prunella vulgaris aqueous extract and auxiliary materials commonly used by the liquid oral preparation are mixed according to a clinically acceptable ratio to prepare the liquid oral preparation, and the liquid oral preparation is obtained by uniformly stirring, filtering, canning and sterilizing.
The solid oral preparation or the liquid oral preparation has the effect of resisting the Zika virus and can be used for preventing and treating the infection of the Zika virus.
The safety and beneficial effects of the drug of the present invention are further illustrated by experiments below.
The invention adopts a cytopathic effect inhibition experiment (CPE phenomenon) to research the inhibition effect of the selfheal on Zika virus, observes the toxic effect of the selfheal aqueous extract on Vero cells and the inhibition effect on the Zika virus, and utilizes an MTT method to determine the half toxic concentration and the half effective concentration of the selfheal aqueous extract. RNA extraction is carried out, and then the effect of selfheal on resisting Zika virus is detected by an RT-PCR method; and detecting the influence of the selfheal aqueous extract on the protein expression of the Zika virus in Vero cells by a Western blot method.
First, cell experiments
1. Experimental Material
1.1 cell lines
C6/36 cell line: aedes albopictus cell line (C6/36) was purchased from Shanghai academy of sciences;
Vero-E6 cell line: the green ape kidney epithelial cell line (Vero-E6) was given by the stressed laboratory of the virology nation of Wuhan university;
zika virus strain: SZ01/2016, Asian, gifted by the Guangdong province disease prevention control center CDC.
1.2 test drugs
And (3) testing a sample: an aqueous extract of Prunella vulgaris was prepared as described in example 1 below.
1.3 Virus strains
Zika Vrius (GenBank accession KU963796) was obtained from the Guangdong provincial disease prevention and control center, amplified in C6/36 cells, and the supernatant was filtered through a 0.45 μm microporous membrane and stored at-80 ℃. Viral titers were determined by plaque assay on Vero cells.
1.4 reagents and instruments
Neonatal calf serum and DMEM medium (Gibco); DMSO (Sigma company); trypsin (DIFCO, usa). Olympus PM-6 inverted microscope (OLYMPUS, Japan).
2. Method of producing a composite material
2.1 determination of toxicity concentration of Prunella vulgaris on Vero and HeLa cells
Vero and Hela cells with proper growth state in a cell culture flask are mixed at 2 x 104Per 100. mu.l/well in 96-well plates (white flat bottom) and incubated overnight at 37 ℃ with 5%
The cell survival rate calculation formula is as follows:
percent cell viability ═ drug group-blank group)/(normal group-blank group) × 100% the 50% toxic concentration was calculated as the drug median Toxic Concentration (TC)50)。
The curve of the relationship between the survival rate of Vero cells and the concentration of the selfheal aqueous extract obtained by the method is shown in figure 1; the relationship between the survival rate of HeLa cells and the concentration of aqueous extract of Prunellae Spica obtained by the above method is shown in FIG. 2.
2.2 determination of the titre of Zika Virus
Vero cells grown in pieces in cell flasks were counted by trypsinization at
2.3 Effect of aqueous Prunellae Spica extract on Zika Virus-induced cytopathic Effect in Vero cells
Sterile PBS is used for preparing mother liquor of the selfheal medicine. Vero and Hela cells are evenly inoculated into a 96-well cell culture plate until the cells grow to 4 multiplied by 104Changing into serum-free DMEM medium at each hole, adding the drug to be tested with the final concentration of 100, 75, 50, 25, 10, 2, 1, 0.1 and 0.01 mu M DMEM medium for gradient dilution, and incubating for 1h in a cell incubator at 37 ℃. After incubation, multiplicity of infection (mul) was added per welltiplicity of infection) zika virus with an MOI of 0.5 was adsorbed in a cell culture chamber at 37 ℃ for 1 hour. Discarding the virus solution, rinsing with PBS once, adding the culture medium containing the medicine, and culturing in a cell culture box at 37 ℃ for 48 h. Cell supernatants were collected to determine virus titers. The 50% virus inhibitory concentration was calculated as the drug median Effective Concentration (EC)50) Selection index SI ═ CC50/EC50。
The observation under a microscope by using a CPE (CPE) observation method is shown in figure 3; statistical analysis is carried out on the data of the apoptosis influence of Zika virus infected Vero cells by the selfheal aqueous extract in different administration modes, and the result is shown in figure 4; the data for the effect of aqueous Prunella vulgaris extract on the inhibition rate of Zika virus in Vero cells are plotted and the results are shown in FIG. 5.
2.4RT-qPCR method for determining Zizania Spica aqueous extract anti-Zika virus effect in Vero cells
Firstly, extracting RNA, then carrying out reverse transcription to obtain the required RNA, finally carrying out real-time fluorescence quantitative PCR, designing a real-time fluorescence quantitative PCR primer pair, taking the product cDNA obtained by the reverse transcription, and preparing a reaction system. Each sample was used for Zika virus genome and GAPDH group at the same time, each group was set with 3 duplicate wells, after the sample addition was completed, the PCR plate was closed with cellophane, and centrifuged at 2000r/min for 2 min. And (5) detecting by using a qPCR instrument, and processing data. The virus inhibition rate calculation formula is as follows:
the percent virus inhibition ratio (control gene expression level-experimental group gene expression level)/control gene expression level × 100%.
The calculation results are shown in table 1 below:
TABLE 1 influence of aqueous Prunella vulgaris extracts on replication of Zika virus nucleic acids in Vero cells (X + -SEM, n ═ 3)
2.5 measurement of inhibition Effect on Zika Virus protein in vitro (Western blotting)
The previous steps are the same as the drug effect test method 2.3, the virus is added for stimulation, selfheal aqueous extracts with different concentrations are added, Vero cell samples are collected after 48 hours of action, centrifugation is carried out for 5 minutes at 4000r/min, the supernatant is discarded, and Cocktail1 containing protease inhibitor is added: and repeatedly blowing and beating 80 mu l of 50 RIPA cell lysate, performing assisted lysis in a refrigerator at 4 ℃ for 60min, centrifuging at 12000r/min at 4 ℃ for 10min, taking a small amount of supernatant, performing protein quantification, and taking a small amount of samples to prepare a protein standard curve. Preparing polyacrylamide gel, performing electrophoresis (80V electrophoresis for 30min, 120V electrophoresis for 60min), transferring a membrane (180mA, 2h), sealing with 5% milk solution (0-10r/min, sealing for 60min), performing antibody hybridization (first-
Second, animal experiment
3. Animal(s) production
INFAR1-/-C57BL/6 mice, SPF grade, 3-4 weeks old, 13-15 g weight, half female and half male, 24 in total, provided by professor Zhao Yong, Guangzhou university of medical science. The animal experiment is bred in ABSL-2 laboratory of Guangzhou Chinese medicinal university laboratory animal center (Guangdong Guangsheng animal preparation GZL 0004), the temperature is 22-25 ℃, the relative humidity is 40-70%, the illumination/darkness is carried out for 12h day and night circulation respectively, sterile granular mouse materials are fed, drinking water is ultrapure water, padding materials are sterile corncobs, and the animal experiment development is approved by the Guangzhou Chinese medicinal university laboratory animal ethical committee.
4. Method of producing a composite material
4.1 animal grouping and model building
Mice were randomly divided into 4 groups of 6 mice each, half of each, normal control group, virus control group, selfheal aqueous extract high dose group and selfheal aqueous extract low dose group. The virus control group, the selfheal aqueous extract high-dose group and the low-dose group are inoculated to 10 percent of mice in each mouse by the abdominal cavity route5PFU Zika virus. The mice in the high and low dose groups are administrated by a gastric lavage way, the administration dose is 900mg/kg and 450mg/kg respectively, and the virus control group and the normal control group are subjected to gastric lavage by sterile PBS with equal volume; each group was gavaged 1 time per day for 12 consecutive days.
4.2 clinical observations
After inoculation of zika virus from mice, mice were continuously observed for clinical symptoms, such as: activity, mental state, arch and back, quadriplegia, dying or death, etc. for 12 days, and recording survival and weight change of each group. The results are shown in fig. 7, 8 and 9.
5. Conclusion
The body weight of the normal control group mice shows a continuous increasing trend, and the mice have no diseases or death. The body weight of the mice in the virus control group is obviously and gradually reduced on the 4 th day, the phenomena of arch, back and the like begin to appear on the 6 th day, the typical nervous system symptoms such as hind limb paralysis, paralysis and the like appear on the 7 th day, and the mice all die on the 9 th day. The weight of the selfheal aqueous extract in the high-dose and low-dose groups starts to gradually decrease after 4 days, the low-dose group starts to appear phenomena such as arch back and the like after 7 days, typical nervous system symptoms such as hind limb paralysis, paralysis and the like appear in 8 days, all mice die in 10 days, the weight of the mice in the high-dose group stops decreasing trend after 8 days, the mice show a slow increasing trend, 1 mouse dies in 12 days, the
On
The experiments all prove that the selfheal aqueous extract has the effect of resisting Zika virus.
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