CN110898100A

CN110898100A - Application of selfheal aqueous extract in preparation of medicine for preventing and treating Zika virus infection

Info

Publication number: CN110898100A
Application number: CN201911377007.4A
Authority: CN
Inventors: 李耿; 龙海珊; 喻良文; 吕东勇; 刘小虹
Original assignee: Guangzhou University of Traditional Chinese Medicine
Current assignee: Guangzhou University of Traditional Chinese Medicine; Guangzhou University of Chinese Medicine
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-03-24

Abstract

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.

Description

Application of selfheal aqueous extract in preparation of medicine for preventing and treating Zika virus infection

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% CO 2. The drug was diluted with cell growth media (containing 10% serum) at final concentrations of 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 4500 μ g/mL. The medium was aspirated off and 100. mu.l of the corresponding drug solution was added to each well. Blank groups (containing only PBS without cells) and normal cells were also set as controls. After 48 hours, 20. mu.L/well of 5mg/mL MTT solution was added to each well and placed in a 37 ℃ incubator containing 5% CO2 for 4 hours. (while discarding the surrounding PBSSolution, MTT solution added as zero adjustment group). After 4h, the 96-well plate was removed. The MTT solution was gently spun off, left to stand for 1min, and then spun off again. 150. mu.L/well of DMSO solutions were added, respectively, and the OD was measured at 490mm with shaking for 10 min.

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 2X 10⁵Each cell/ml/well was inoculated into a 12-well plate and cultured in a 30 ℃ incubator containing 5% CO2 for 18 hours. The medium was aspirated off, 100. mu.l/well of diluted virus dilutions of different concentrations were added, then 400. mu.l/well of medium was supplemented to 500. mu.l, and 500. mu.l/well of serum-free DMEM medium was used as a blank. Mixing, and adsorbing in a constant temperature incubator containing 5% CO2 at 30 deg.C for 2 hr. Removing virus solution, adding 1 ml/well 0.8% CM-2% FBS-DMEM culture solution, and culturing in constant temperature incubator containing 5% CO2 at 30 deg.C for 7 d. 1.5 ml/well of 4% paraformaldehyde fixative was added and fixed overnight at room temperature. The supernatant was discarded, 250. mu.l/well of a 1% crystal violet solution was added, and the mixture was incubated at room temperature for 30min, and the number of plaques was counted, Pfu/ml being the reciprocal of the number of plaques x (1000/100) × dilution factor.

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-antibody 4 ℃ low-speed shaking table incubation overnight, second-antibody room temperature low-speed incubation for 50min), exposing by using a Bio-Rad ChemiDocXRS + ultra-high sensitivity chemiluminescence imager, analyzing a strip, and obtaining a result shown in figure 6.

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 rest 5 mice have good states, and the phenomena such as arch back, hind limb paralysis and the like do not appear.

On day 3, the copy number of serum virus RNA of mice in the high-dose group of the selfheal aqueous extract is remarkably reduced (P is less than 0.001) compared with that of mice in a virus control group and a low-dose group, and the copy number of serum virus RNA of mice in the low-dose group of the selfheal aqueous extract is remarkably reduced (P is less than 0.05) compared with that of mice in the virus control group. At day 5, the serum viral RNA copy number of mice in the high dose group of selfheal aqueous extract was significantly reduced (P < 0.001) compared with those in the virus control group and the low dose group.

The experiments all prove that the selfheal aqueous extract has the effect of resisting Zika virus.

Drawings

FIG. 1 is a graph of the relationship between the survival rate of Vero cells and the concentration of aqueous extract of Prunellae Spica.

FIG. 2 is a graph showing the relationship between the survival rate of HeLa cells and the concentration of aqueous extract of Prunellae Spica.

FIG. 3 is a photomicrograph of the effect of aqueous extracts of Prunella vulgaris on Zika virus-induced cytopathic effects.

FIG. 4 is a bar graph of the apoptotic effect of aqueous extracts of Prunella vulgaris on infection of Vero cells by Zika virus under different modes of administration.

FIG. 5 is a graph of the effect of aqueous extracts of Prunella vulgaris on the inhibition rate of Zika virus in Vero cells.

FIG. 6 is an electrophoretic band showing the effect of aqueous extracts of Prunella vulgaris on the expression of the Zika virus protein in Vero cells, wherein the electrophoretic band is shown from left to right: lane 1 is a blank control group with only normal cells, lane 2 is a control group containing Zika virus, and lanes 3-9 are aqueous Prunella vulgaris extracts at concentrations of 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 150. mu.g/ml, 200. mu.g/ml, 250. mu.g/ml and 300. mu.g/ml, respectively.

FIG. 7 is a graph showing the body weight of each group as a function of the time of viral infection, after infection of INFAR1-/-C57BL/6 mice with aqueous extracts of Prunella vulgaris against Zika virus.

FIG. 8 is a graph in which the number of survivors of mice in each group was counted as the time elapsed since the aqueous extract of Prunella vulgaris was infected with INFAR1-/-C57BL/6 mouse against Zika virus.

FIG. 9 is a graph showing the variation of the copy number of viruses detected at the RNA level on

days

3 and 5, respectively, of an aqueous extract of Prunella vulgaris in comparison with the sera of INFAR1-/-C57BL/6 mice; in the figure, the difference between the two groups is significant, and P is less than 0.05; indicates that the difference between the two groups is significant, P < 0.001.

Detailed description of the preferred embodiments

Example 1: (preparation of aqueous extract of Prunella vulgaris)

Taking 10kg of selfheal, adding 100L of distilled water, soaking for 30 minutes, decocting for 30 minutes, filtering while hot, concentrating to obtain a thick extract with the specific gravity of 1g/ml, and drying to obtain a selfheal water extract.

Example 2: (granules)

Taking 9g of the aqueous extract obtained in the example 1, adding auxiliary materials such as cane sugar, dextrin and the like, placing the mixture into a boiling granulator, mixing, granulating, drying, uniformly mixing with starch, cross-linked PVP and sodium carboxymethyl starch, using 75% ethanol solution of 5% PVP as a binding agent, preparing a soft material, granulating by using a 18-mesh sieve, drying at 60 ℃ for 1h, subpackaging, and preparing granules with the specification of 30mg per bag for oral administration. Drying and packaging. The product is grayish brown, bitter and pungent in flavor. For oral administration. 2-3 times a day, and one bag at a time. Other items should meet the related regulations under the item of granules in pharmacopoeia 2015 edition of the people's republic of China.

Example 3: (capsules)

Taking 15g of the aqueous extract in example 1, adding auxiliary materials such as starch and the like, placing in a boiling granulator, mixing, granulating and drying. The obtained granules are filled into No. 1 hard gelatin capsules, and each capsule is subpackaged with 300 mg. For oral administration. The product is in the form of capsule, and the content is in the form of ash brown granule or powder, bitter and pungent in taste, and can be orally administered. 2-3 times a day, one granule at a time. Other items should meet the relevant regulations under the capsule item of pharmacopoeia 2015 edition of the people's republic of China.

Example 4: (tablet)

Taking 15g of the aqueous extract of the example 1, adding auxiliary materials such as starch and the like, placing in a boiling granulator, mixing, granulating, drying, adding a proper amount of magnesium stearate, tabletting, and subpackaging 300mg of each tablet. For oral administration. The product is a gray brown tablet, bitter and pungent in taste, and is administered orally. 2-3 times a day, one tablet at a time. Other items should meet the relevant regulations under the 2015 release tablets of the people's republic of China.

Example 5: (oral liquid)

Taking 15g of the aqueous extract of the example 1, adding a proper amount of water for dissolving, filtering, adding a proper amount of simple syrup and sodium benzoate, adding water to 1000ml, uniformly stirring, standing, filtering, encapsulating and sterilizing. The product is a brown clear liquid, bitter and pungent in flavor, and is administered orally. One for 2-3 times a day. Other items should meet the relevant regulations under the oral liquid item in pharmacopoeia 2015 of the people's republic of China.

Claims

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.