CN110974867B

CN110974867B - Application of cassia twig aqueous extract in preparation of medicine for preventing and treating Zika virus infection

Info

Publication number: CN110974867B
Application number: CN201911376943.3A
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
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2021-10-15
Anticipated expiration: 2039-12-27
Also published as: CN110974867A

Abstract

The invention relates to application of a cassia twig aqueous extract in a medicament for preventing and treating Zika virus infection. The cassia twig aqueous extract in the application of 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 a cassia twig aqueous extract and a conventional amount of medically acceptable auxiliary materials.

Description

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

Technical Field

The invention relates to a pharmaceutical preparation containing unknown structures from plants, in particular to a new application of woody plants in the Lauraceae family in pharmacy.

Background

Zika virus belongs to the family of Flaviviridae, the genus Flaviviridae. Zika virus is a single-stranded positive-strand RNA virus, mature Zika virus is spherical and comprises a ssRNA genome with 3 'and 5' untranslated regions and a single open reading frame encoded by multiple proteins, and the genome is 10.8kb in length.

Zika virus (Zika virus, Zikv) is an arbovirus, first discovered in 1947 from rhesus monkeys of the Wugan Dazhai forest and named Zika virus. The first separation from Aedes Africana after one year. In the middle of the 20 th century, cases of Zika virus infection were rare and mainly concentrated in sub-Saharan Africa and southeast Asia. The first outbreak of Zika virus outside Africa occurred in Yapu island 2007, and approximately three quarters of people aged three years old were suspected of infecting Zika virus. In 2013, Zika virus had an even larger outbreak in French Pornia, about 29000 cases, and some people developed complications when infected with Zika virus. From 2015 to 2016, Zika virus began to become prevalent in the America, so far Zika virus spread worldwide.

The transmission pathway of Zika virus is mainly through Aedes mosquito, and later researches show that Zika virus can also transmit sexually. The global prevalence of Zika virus has prompted the world health organization to declare it a sudden public health event of international concern in 2016. Overwhelming experience over the past few years has shown that Zika virus infection and related neurological complications are long-standing challenges for public health, and despite the declining number of cases of Zika virus infection in the western hemisphere since 2016, the need for basic research and anti-ZIKV drug development remains urgent. Viruses such as ZIKV, which reappear and propagate at an extremely fast rate, are an ever-present threat in the 21 st century.

Several FDA-approved drugs have been generated to date for resistance to Zika virus infection that are effective against viruses in infected adult mouse models and, most importantly, prevent maternal-fetal transmission and severe microcephaly in neonates in pregnant mouse models.

Ramulus Cinnamomi belongs to dried twig of Cinnamomum cassia Presl of Lauraceae, and is also called cortex Cinnamomi. Has special fragrance, sweet taste, slight pungent and warm property, and enters heart, lung and bladder meridians. Has the effects of inducing sweat, expelling pathogenic factors from muscles, warming and activating channels, supporting yang, regulating qi, and lowering blood pressure. It is used to treat common cold due to wind-cold, abdominal psychroalgia, amenorrhea due to cold, arthralgia, phlegm and fluid retention, edema, palpitation, and galloping. At present, pharmacological studies on cassia twig indicate that cassia twig has certain efficacies in the aspects of virus resistance, oxidation resistance, tumor resistance and the like. In the aspect of antivirus, the study mainly on influenza virus is performed, and a small amount of studies on Human Respiratory Syncytial Virus (HRSV) and Herpes Simplex Virus (HSV) are performed, but no study on Zika virus resistance exists in the current literature reports.

Disclosure of Invention

The invention aims to solve the technical problem of providing a new application of cassia twig in pharmacy.

The new application is specifically the application of the cassia twig 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 cassia twig aqueous extract and a conventional amount of medically acceptable auxiliary materials.

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 above application, the cassia twig aqueous extract can be extracted by a method commonly used in the field, and the method recommended by the inventor is as follows:

taking cassia twig, adding 6 times of distilled water, carrying out reflux extraction for 30 minutes at 60 ℃, carrying out suction filtration while the cassia twig is hot, adding distilled water with the same volume into dregs, carrying out heating reflux extraction for 30 minutes at 60 ℃, and carrying out suction filtration while the cassia twig is hot; mixing the filtrates, cooling, centrifuging, concentrating, and freeze drying to obtain ramulus Cinnamomi water extract.

The preparation method of the solid oral preparation is a conventional preparation method specified in pharmacopoeia, namely the solid oral preparation is prepared by mixing the cassia twig aqueous extract and common auxiliary materials of the solid oral preparation according to a clinically acceptable ratio.

The preparation method of the liquid oral preparation is a conventional preparation method specified in pharmacopoeia, namely, the cassia twig aqueous extract and the auxiliary materials commonly used by the liquid oral preparation are mixed according to a clinically acceptable proportion, stirred evenly, filtered, filled and sterilized to obtain the liquid oral preparation.

The solid oral preparation or the liquid oral preparation has the effect of resisting Zika virus and can be used for preventing Zika virus infection.

The safety and beneficial effects of the drug of the present invention are further illustrated by experiments below.

The invention uses a virus plaque titer method to determine the inhibition effect of the cassia twig aqueous extract on Zika virus, and observes the toxicity of the cassia twig aqueous extract on Vero cells and Hela cells and the inhibition effect on Zika virus. The invention utilizes MTT method to measure the half Toxic Concentration (TC) of tested medicine₅₀)>1000. mu.g/ml, a maximum effect concentration on the semi-complete Zika virus of 500. mu.g/ml, the selection index SI>2, high efficiency and low toxicity. The specific experimental method is as follows.

First, cell experiments

1. Experimental Material

1.1 cell lines

A green simian kidney epithelial cell line (Vero-E6) was given by the stressed laboratory of the university of Wuhan virology country. And (4) storing in a laboratory.

1.2 Experimental groups and corresponding tested drugs

And (3) testing a sample: the aqueous extract of Cassia twig obtained in example 1 below was prepared in DMEM medium to give a drug stock solution at a concentration of 10 mg/ml.

1.3 Virus strains

Asian Zika virus strains (SZ01, 2016), gifted by the Guangdong province disease prevention and control center (CDC). ZV strain was expanded in C6/36 cells, and the supernatant was filtered through a 0.45 millipore filter and stored in liquid nitrogen. Viral titers were determined by plaque assay in Vero cells.

1.4 reagents and instruments

Ilex fetal calf serum (Hangzhou ilex); DMEM medium (Thermo Fisher Scientific-Life); DMSO (tianjin fuyu); trypsin (Thermo Fisher Scientific-Life); inverted microscope (nikon) type S1 OOF.

1.5 animals

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. Feeding the animal in ABSL-2 laboratory of Guangzhou Chinese medicinal university laboratory animal center (Guangdong animal preparation GZL 0004), controlling the temperature at 22-25 ℃ and the relative humidity at 40-70%, performing illumination/darkness for 12h day and night circulation, feeding sterile granular mouse material, drinking water as ultrapure water, padding as sterile corncob, and carrying out animal experiments by approval of animal ethics committee of Guangzhou Chinese medicinal university laboratory animal

2. Method of producing a composite material

2.1 determination of toxicity concentration of aqueous Cassia twig extract on Vero cells (HeLa cells)

Vero cells (HeLa cells) grown in pieces in cell flasks were counted by trypsinization at 2X 10⁴Per 100. mu.l/well were plated in 96-well plates. Placing in a container containing 5% CO₂And culturing for 18h in a constant temperature incubator at 30 ℃. The aqueous extract of Cassia twig obtained in the following example 1 was cultured in DMEM medium containing 10% of SIJIQING serum at a final concentration of 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500. mu.g/mlAnd (3) a medicinal solution. The medium was aspirated off, 100 μ l of drug solution was added to each well, normal cell controls were set, and 100ul of DMEM medium containing 10% tetrastigme serum was added to each well. Placing in a container containing 5% CO₂And culturing in a constant temperature incubator at 30 ℃ for 48 hours. The drug-containing medium was aspirated, 20. mu.l/well of 5mg/ml MTT solution was added, and the mixture was placed in a container containing 5% CO₂And culturing for 4 hours in a constant temperature incubator at 30 ℃. The MTT solution was aspirated, 150. mu.l/well DMSO solution was added, the mixture was shaken for 10min, and the OD was measured at 490 nm. The cell survival rate calculation formula is as follows:

percent cell survival rate ═ 100% (OD experimental group-OD zero control group)/(OD control group-OD zero control group) ×

Calculating 50% toxic concentration as half Toxic Concentration (TC)₅₀)。

The drug toxicity results are shown in tables 1, 2 below and in figures 1, 2:

TABLE 1 toxic Effect of Cassia twig on Vero cells

TABLE 2 toxic Effect of Cassia twig on Hela cells

2.2 determination of the antiviral Effect in vitro

2.2.1CPE method for observing anti-Zika virus effect of cassia twig aqueous extract in Vero cells

Taking 10mg/ml of drug mother liquor, and preparing 50, 100, 200, 300, 400, 500, 600, 700, 800 and 900 mu g/ml of drug solution (A) by using DMEM medium containing 2% fetal bovine serum. Drug solutions (B) were prepared at 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800. mu.g/ml in DMEM basal medium.

Pancreas used for Vero cells grown in slices in cell bottlesProtease digestion count at 2X 10⁵Per 100. mu.l were inoculated in 12-well plates. Placing in a container containing 5% CO₂And culturing for 18h in a constant temperature incubator at 30 ℃. The medium was aspirated off, and the drug solutions (A) were added to 12-well plates according to a concentration gradient at 500. mu.l/well, respectively, and a blank was set. At the same time, 250. mu.l of the drug solution (B) was mixed with 250. mu.l of Zika virus with MOI of 0.2 to prepare a drug-containing solution (C), and 250. mu.l of DMEM basal medium was mixed with 250. mu.l of the virus to prepare a drug blank. Placing in a container containing 5% CO₂And culturing for 2 hours in a constant temperature incubator at 30 ℃. Removing the culture medium by suction, adding 500 μ l/well of drug-containing virus solution (C), and placing in a container containing 5% CO₂And culturing for 2 hours in a constant temperature incubator at 30 ℃. Removing culture medium by suction, adding 1000 μ l/well of Chinese medicinal solution (A), and placing in a container containing 5% CO₂And culturing in a constant temperature incubator at 30 ℃ for 48 hours. Cytopathic effect CPE was observed microscopically. The results of observing the effect of the drug against Zika virus in Vero cells by the CPE method are shown in FIG. 3.

2.2.2RT-qPCR method for determining Zika virus resistance of cassia twig aqueous extract in Vero cells

RNA extraction: in a 12-well plate, cassia twig aqueous extracts with different concentration gradients are taken and tested for anti-Zika virus effect in Vero cells. Discarding the supernatant, adding 500. mu.l/well Trizol solution, lysing for 30min at room temperature, blowing to remove cells, and collecting. Adding 100 μ l chloroform solution, shaking vigorously, standing for 2min, and centrifuging at 4 deg.C 12000r/min for 10 min. Adding 200 μ l of the supernatant into a new 1.5ml EP tube, adding 200 μ l of 70% ethanol solution, mixing, transferring to an adsorption column, and centrifuging at 12000r/min for 30 s. Discarding the collected solution, adding 700. mu.l of BufferRW1, centrifuging at 12000r/min for 30s, discarding the collected solution, adding 500. mu.l of BufferRW2, centrifuging at 12000r/min for 30s, discarding the collected solution, adding again 500. mu.l of BufferRW2, centrifuging at 12000r/min for 30s, and air-drying the column at 12000r/min for 2 min. Discarding the collection tube, opening the cover of the adsorption column, and air-drying at room temperature for 5 min. The adsorption column was set in another fresh RNase-Free1.5mLEP tube, 35. mu.l of RNase-Free water was added to the center of the adsorption column, and the mixture was allowed to stand for 1min, centrifuged at 12000rpm for 60s, and the RNA solution was collected.

Reverse transcription: mu.g of lDT primer and 0.5. mu.l of random primer were put into a PCR tube, 17. mu.l of the RNA solution obtained by extraction was added as a template, and the mixture was briefly centrifuged and annealed at 70 ℃ for 5 min. The PCR tube was removed and cooled for 5 min. Preparing a reaction system, mixing, adding into a PCR tube, centrifuging for a short time, and performing reverse transcription (reverse transcription reaction temperature is 37 ℃ for 90 min; reaction termination temperature is 70 ℃ for 10 min; and temperature reduction is 16 ℃ for 5 min).

Real-time fluorescent quantitative PCR: designing a real-time fluorescent 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 virus inhibition ratio%

The effect of aqueous cinnamon twig extracts on zika virus nucleic acid replication in Vero cells was plotted by MTT method using graphpadprism5.0 software, and the results are shown in table 3 below.

TABLE 3 Effect of aqueous Cassia twig extracts on replication of Zika Virus nucleic acids in Vero cells

2.2.3 Using Western blotting to examine the Effect of aqueous extract of Cassia twig on the protein expression of Zika Virus

Vero cells grown in pieces in cell flasks were counted by trypsinization, seeded into 6-well plates at 2 ml/well, and placed in a flask containing 5% CO₂Culturing in a constant-temperature incubator at 30 ℃ for 48h, discarding the supernatant, adding 1 ml/hole PBS solution, scraping all Vero cells by using a cell scraper, transferring the Vero cells into a 1.5ml EP tube, centrifuging at 4000r/min at 4 ℃ for 5min, discarding the supernatant, adding Cocktail1 containing protease inhibitor: 50 μ l of RIPA cell lysate (i.e., Cocktai L1.6 μ l, RIPA78.4 μ l) was repeatedly aspirated and lysed in a refrigerator at 4 ℃ for 60min, followed by centrifugation at 12000r/min at 4 ℃ for 10 min.

Taking a small amount of samples to prepare a protein standard curve: preparing a BCA working solution (the proportion of the BCA reagent A to the BCA reagent B is 50: 1, mixing uniformly), taking a certain amount of supernatant, completely lysing the supernatant by using RIPA lysate, taking 10 mu l of supernatant, diluting the supernatant by 10 times to 100 mu l of supernatant, and enabling the final concentration to be 0.5 mg/ml. And adding 0, 1, 2, 4, 8, 12, 16 and 20 mul of protein standard solution into an eight-way tube respectively, and adding RIPA lysate to make up to 20 mul respectively so that the concentrations of the standard solutions are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/ml respectively. Add 200. mu.l/well BCA working solution, set at 37 ℃ for 30min, set 3 duplicate wells for each concentration. And (3) measuring the absorbance at 562nm, drawing a protein standard curve by taking the protein concentration as an abscissa and the absorbance as an ordinate, and solving a protein standard curve equation.

Taking a small amount of supernatant for protein quantification: preparing BCA working solution (the proportion of the BCA reagent A to the BCA reagent B is 50: 1, mixing uniformly), taking 2 mul/tube of supernatant, adding into an octal tube, respectively adding RIPA lysate to make up to 20 mul, adding 200 mul/hole BCA working solution, and standing at 37 ℃ for 30 min. And (3) measuring the absorbance at 562nm, obtaining the protein concentration according to a standard curve equation, and setting 3 multiple holes for each concentration.

Preparing polyacrylamide gel for electrophoresis, adding 8 mul/hole of each protein sample into a loading hole, adding 4 mul of protein maker, setting the voltage of concentrated gel to 80V, setting the voltage of separation gel to 120V after 30min, and ending the electrophoresis after about 60 min. Preparing a film transfer liquid, taking out gel, installing film transfer interlayers in the sequence of black negative pole → sponge layer → filter paper layer → polyacrylamide gel → NC film (activated by methanol) → filter paper layer → sponge layer → white positive pole from bottom to top, placing the film transfer interlayers in a film transfer groove, and paying attention to remove bubbles in each layer. The transmembrane current is set to be 180mA for 2 h. After the membrane transfer is finished, preparing a sealing solution (5g of skimmed milk, 100ml of PBST solution, uniformly mixing by vortex), putting the NC membrane into a box, adding the sealing solution, oscillating by a shaking table (10r/min), and sealing for 50 min. The blocking solution was discarded and PBST solution was added and rinsed 3 times for 5min each time on a shaker. 4ml of each protein primary antibody solution (GAPDH: PBST diluted 1: 5000, other viral proteins diluted 1: 1000, 4ml each) was added to the incubation chamber, the NC membrane was removed, the respective target protein band was cut, and the membrane was placed in the corresponding primary antibody and incubated overnight at 4 ℃ with shaking at a low speed on a shaker. Primary antibody was recovered and washed 3 times 5min each time in a shaker with PBST solution. Preparing a second antibody diluent (3g of skimmed milk powder, 100ml of PBST solution, uniformly mixing by vortex), preparing a solution (except that GAPDH is diluted by a mouse antibody: second antibody diluent according to a ratio of 1: 5000, other viral proteins are diluted by a rabbit antibody: second antibody diluent according to a ratio of 1: 5000, preparing four milliliters of each part), discarding a membrane washing solution, respectively adding 4ml of each protein second antibody solution, and placing on a shaking table for low-speed incubation at room temperature for 50 minutes.

Exposure: turn on the Bio-RadChemiDocXRS + ultra high sensitivity chemiluminescent imager. Taking HRP luminescent substrate solution A and solution B, and carrying out reaction according to the weight ratio of 1: 1, and reacting with each protein band for 5 min. The developing solution was gently blotted dry with filter paper, and the protein band was transferred to the developing zone. The exposure time was set to 15s and the exposure was 15 times. And after the exposure is finished, storing the data.

The results of the cassia twig aqueous extract on zika virus protein expression in Vero cells are shown in figure 4.

2.3 determination of viral titre

Vero cells grown in pieces in cell flasks were counted by trypsinization at 2X 10⁵The individual cells/ml/well were seeded in 12-well plates and placed in a 5% CO₂And culturing for 18h in a constant temperature incubator at 30 ℃. 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 adding into a container containing 5% CO₂And adsorbing for 2 hours in a constant temperature incubator at 30 ℃. Removing virus solution, adding 1 ml/well 0.8% CM-2% FBS-DMEM culture solution, and placing in a medium containing 5% CO₂And culturing in a constant temperature incubator at 30 ℃ 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.

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, high dose group of cassia twig water extract, and low dose group of cassia twig water extract. The virus control group, the cassia twig 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. 5, 6 and 7.

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 5 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 cassia twig aqueous extract in the high-dose and low-dose groups starts to gradually decrease after 5 days, the low-dose group starts to appear the phenomena of arch back and the like after 7 days, the typical nervous system symptoms such as hind limb paralysis, paralysis and the like appear in 9 days, all mice die in 11 days, the weight of the mice in the high-dose group stops decreasing after 12 days, the mice die in 11 days, 2 mice die in 12 days, and the rest 4 mice have good states and do not appear the phenomena of arch back, hind limb paralysis and the like.

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

The above experiments all prove that the cassia twig aqueous extract has the effect of resisting Zika virus.

Drawings

FIG. 1 is a graph of Vero cell viability versus concentration of aqueous extract of Cassia twig.

FIG. 2 is a graph of the relationship between the survival rate of HeLa cells and the concentration of aqueous extract of cinnamon twig.

FIG. 3 photomicrograph of the effect of aqueous extract of Cassia twig on Zika virus-induced cytopathic effect.

FIG. 4 electrophoretic bands of effect of aqueous extract of Cassia twig on the expression of Zika virus protein in Vero cells, from left to right in the figure are: lane 1 is a blank control with only normal cells, lane 2 is a control containing Zika virus, and lanes 3-9 are aqueous extracts of Cassia twig at concentrations of 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, 300. mu.g/ml, 400. mu.g/ml and 500. mu.g/ml, respectively.

FIG. 5 is a graph of body weight of each group as a function of time of viral infection following infection of mice with INFAR1-/-C57BL/6 from aqueous extracts of Cassia twig to Zika virus.

FIG. 6 is a graph showing the survival of mice in each group as a function of infection time after infection of INFAR1-/-C57BL/6 mice with an aqueous extract of Cassia twig against Zika virus.

FIG. 7 is a graph showing the variation of copy number of viruses detected at RNA level on

days

3 and 5, respectively, of an aqueous extract of Cassia twig in the sera of mice with INFAR1-/-C57 BL/6; in the figure, indicates that 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

Example 1: (preparation of aqueous extract of Cassia twig)

Extracting 50g of dried ramulus Cinnamomi with 300ml of distilled water at 60 deg.C under reflux for 30min, hot filtering, extracting the residue with 300ml of distilled water at 60 deg.C under reflux for 30min, hot filtering, and mixing the filtrates. Cooling, centrifuging, removing precipitate, concentrating the supernatant at 60 deg.C under reduced pressure to 1g/ml crude drug (about 50ml), freezing, and drying to obtain ramulus Cinnamomi water extract.

Example 2: (granules)

Taking 10g 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 10mg of granules per bag for oral administration. The product is gray brown, sweet and slightly pungent. 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 3g 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 were filled into a No. 1 hard gelatin capsule and made into capsules of 10 mg/granule for oral administration. The product is capsule, and the content is gray brown granule or powder, and has sweet and slightly pungent taste. Other items should meet the relevant regulations under the capsule item of pharmacopoeia 2015 edition of the people's republic of China. 1 granule for 2-3 times a day.

Example 4: (tablet)

Taking 10g of the aqueous extract of 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, and tabletting. After finishing the granules, adding a proper amount of talcum powder, mixing uniformly, tabletting and preparing tablets with the specification of 10 mg/tablet for oral administration. The preparation is taken 1 tablet at a time 2-3 times a day. The product is a gray brown tablet, sweet and slightly pungent in taste, and can be orally administered. Other items should meet the relevant regulations under the 2015 release tablets of the people's republic of China.

Example 5: (oral liquid)

Taking 10g of the aqueous extract of the example 1, adding a proper amount of water to dissolve, filtering, adding a proper amount of simple syrup and sodium benzoate, adding water to 1000ml, stirring uniformly, standing, filtering, encapsulating, and sterilizing, wherein each 10 ml. The product is brown clear liquid, sweet and slightly pungent in taste, and can be orally administered. The medicine is taken 1 dose 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 cassia twig aqueous extract in preparing medicine for preventing and treating Zika virus infection.

2. Use according to claim 1, characterized in that: the medicine is a solid or liquid oral preparation prepared from cassia twig aqueous extract and medically acceptable auxiliary materials.

3. Use according to claim 2, characterized in that: the solid or liquid oral preparation is granules, capsules, tablets or oral liquid.

4. Use according to claim 1, 2 or 3, characterized in that: the cassia twig aqueous extract is prepared by the following method:

taking cassia twig, adding 6 times of distilled water, heating and refluxing for extraction for 30 minutes at 60 ℃, carrying out suction filtration while the cassia twig is hot, adding the distilled water with the same volume as the cassia twig into the decoction dregs, heating and refluxing for extraction for 30 minutes at 60 ℃, and carrying out suction filtration while the cassia twig is hot; and combining the two filtrates, cooling, centrifuging, concentrating, and freeze-drying to obtain the cassia twig aqueous extract.