CN117982563A - Application of traditional Chinese medicine composition in preparation of dengue virus resistant medicines - Google Patents
Application of traditional Chinese medicine composition in preparation of dengue virus resistant medicines Download PDFInfo
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Abstract
The invention discloses an application of a traditional Chinese medicine composition in preparing an anti-dengue virus medicine, wherein the traditional Chinese medicine composition comprises safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica. The research of the invention shows that the traditional Chinese medicine composition has remarkable inhibition effect on dengue II virus, the half effective concentration (EC 50) on dengue II virus is 1.238 mu L/mL, the cytotoxicity is low, the half toxic concentration (CC 50) on cells is 225.6 mu L/mL, and the Selection Index (SI) is 182.2. Therefore, the traditional Chinese medicine composition can be used for preparing an anti-dengue virus medicine with safety, effectiveness and small toxic and side effects, and has important clinical significance for inhibiting dengue virus and radically preventing dengue virus infection.
Description
Technical Field
The invention belongs to the technical field of medicines. More particularly relates to an application of a traditional Chinese medicine composition in preparing anti-dengue virus medicines.
Background
Dengue virus belongs to the genus flaviviridae in the flaviviridae family, comprising four serotypes: type I, II, III, IV, especially type II, is the most serious hazard and the most widely spread. Most patients are infected with dengue virus and then have a series of clinical symptoms after infection, wherein the clinical symptoms mainly include hyperpyrexia, headache, muscle and joint pain, can be accompanied with rash and lymphadenitis, and can cause large-scale epidemic. Dengue virus has stronger infectivity, and the main transmission way is that the virus is transmitted by mosquito bites, enters the skin after being bitten by the mosquito, gradually grows and grows in capillary endothelial cells and mononuclear-phagocyte systems, and finally is spread by blood flow to cause diseases. Dengue virus transmission has obvious seasonality, cases can occur all year round in regions with high temperature and high humidity all year round, but mainly occur in summer and autumn rainy seasons, and the incidence rate of the dengue virus in local epidemic areas is increased every other year, and the dengue virus has the tendency of epidemic once every 4-7 years.
At present, no specific anti-dengue virus drug exists for dengue viruses, and supporting and symptomatic treatment measures are mainly adopted after virus infection. However, the treatment after infection can only be aimed at symptom improvement, and the problem of the source of infection cannot be solved. Therefore, research on inhibition of dengue virus, prevention of dengue virus infection from root, search for effective and specific drugs for inhibition and prevention of dengue virus, and have important clinical significance.
Disclosure of Invention
The invention aims to overcome the defects of the existing dengue virus resistant medicaments and provide the application of the traditional Chinese medicine composition in preparing the dengue virus resistant medicaments.
The first object of the invention is to provide the application of the traditional Chinese medicine composition in preparing the anti-dengue virus medicine.
The second object of the invention is to provide the application of the traditional Chinese medicine composition in preparing the medicine for preventing dengue virus infection.
A third object of the present invention is to provide an anti-dengue virus drug.
The above object of the present invention is achieved by the following technical scheme:
according to a large number of researches, the traditional Chinese medicine composition prepared from safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica has remarkable inhibition effect on dengue virus, has a half effective concentration (EC 50) of 1.238 mu L/mL on dengue II virus which is the most widely transmitted and most pathogenic, has low cytotoxicity, has a half toxic concentration (CC 50) of 225.6 mu L/mL on cells, and has a Selection Index (SI) of 182.2. Therefore, the traditional Chinese medicine composition can be used for resisting dengue virus and radically preventing dengue virus infection. The present invention therefore protects the following scheme:
the application of the traditional Chinese medicine composition in preparing the anti-dengue virus medicine comprises safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica.
The application of a traditional Chinese medicine composition in preparing a medicine for preventing dengue virus infection is provided, wherein the traditional Chinese medicine composition comprises safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica.
The invention also discloses an anti-dengue virus drug which contains a traditional Chinese medicine composition and a pharmaceutically acceptable carrier.
The traditional Chinese medicine composition accounts for 1-99% of the total mass of the medicine.
The Chinese medicinal composition comprises Carthami flos, radix Paeoniae Rubra, rhizoma Ligustici Chuanxiong, saviae Miltiorrhizae radix, and radix Angelicae sinensis. In one specific scheme, the traditional Chinese medicine composition consists of safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica.
More specifically, the traditional Chinese medicine composition is a composition of safflower extract, red paeony root extract, szechuan lovage rhizome extract, red sage root extract and Chinese angelica extract.
Preferably, the safflower extract is an alcohol extract, and the red peony root extract, the ligusticum wallichii extract, the red sage root extract and the angelica sinensis extract are water extracts.
More specifically, the preparation process of the safflower extract comprises the following steps:
1. Adding 30% ethanol into flos Carthami decoction pieces, and leaching;
2. filtering, adding ethanol into the leaching solution to make the ethanol content reach 70%, and cooling;
3. filtering, concentrating the filtrate, adding ethanol into the concentrated solution to reach alcohol content of 80%, and cooling;
4. Filtering, removing ethanol from the filtrate, and drying to obtain Carthami flos extract.
Specifically, the dosage ratio of the safflower decoction pieces to 30% ethanol in the step 1 is 1: (1-8) (w/v), said leaching being for 1-12 hours.
Preferably, the dosage ratio of the safflower decoction pieces to 30% ethanol in the step 1 is 1:8 (w/v), the leaching being for 8 hours.
Specifically, the cold storage in the steps 2 and 3 is cold storage for 1-72 hours.
Preferably, the cold storage in steps 2 and 3 is cold storage for 48 hours.
As an alternative, the drying in step 4 may be vacuum drying.
The preparation process of the red paeony root extract, the szechuan lovage rhizome extract, the red sage root extract and the Chinese angelica extract comprises the following steps of:
(1) Adding process water into the medicinal decoction pieces, boiling, keeping micro boiling for 0.5-3 hours, filtering, and collecting filtrate I and medicinal residues;
(2) Adding process water into the residue, boiling, keeping micro boiling for 0.5-3 hours, filtering, and collecting filtrate II;
(3) Mixing the filtrates I and II, concentrating, adding gelatin solution into the concentrated solution during stirring, adding ethanol to ethanol content of 70%, and cold preserving;
(4) Filtering, concentrating the filtrate, adding water saturated n-butanol into the concentrated solution, and extracting;
(5) Taking the extract, removing n-butanol until no alcohol smell exists, and drying to obtain radix Paeoniae Rubra extract, rhizoma Ligustici Chuanxiong extract, saviae Miltiorrhizae radix extract or radix Angelicae sinensis extract;
The medicinal material decoction pieces are radix paeoniae rubra decoction pieces, rhizoma ligustici wallichii decoction pieces, radix salviae miltiorrhizae decoction pieces or Chinese angelica decoction pieces.
As an alternative embodiment, the process water may be purified water, water for injection, sterilized water for injection.
Specifically, the dosage ratio of the traditional Chinese medicinal decoction pieces to the process water in the step (1) is 1: (6-12) (w/v).
Preferably, the dosage ratio of the traditional Chinese medicinal decoction pieces to the process water in the step (1) is 1:10 (w/v).
Preferably, the keeping of the micro-boiling in the step (1) is keeping the micro-boiling for 2 hours.
Specifically, the dosage ratio of the traditional Chinese medicinal decoction pieces to the process water in the step (2) is 1: (6-8) (w/v).
Preferably, the dosage ratio of the traditional Chinese medicinal decoction pieces to the process water in the step (2) is 1:8 (w/v).
Preferably, the keeping of the micro-boiling in the step (2) is keeping the micro-boiling for 1 hour.
Specifically, the concentration of the gelatin solution in the step (3) is 0.1-20%, and the cold storage is cold storage for 1-72 hours.
Preferably, the concentration of the gelatin solution in the step (3) is 2-5%, and the cold storage is cold storage for 24 hours.
More preferably, the concentration of the gelatin solution in step (3) is 2%.
Specifically, the volume ratio of the water-saturated n-butanol to the concentrated solution in the step (4) is 1: (1-4).
Preferably, the volume ratio of the water-saturated n-butanol to the concentrated solution in the step (4) is 1:2.
Specifically, the extraction in the step (4) is carried out for 1 to 6 times.
Preferably, the extraction in step (4) is 4 times.
As an alternative, the drying in step (5) may be vacuum drying.
In addition, based on antiviral synergistic effect, the traditional Chinese medicine composition can be combined with other dengue virus resistant components in practice to enhance the dengue virus resistant effect, so that the dengue virus resistant medicine can also contain other dengue virus resistant components.
In addition, different pharmaceutical dosage forms can be selected according to the specific condition of patients infected by dengue virus, so the dosage form of the anti-dengue virus drug is any pharmaceutically acceptable pharmaceutical dosage form.
As one of the possible embodiments, the anti-dengue virus drug may be formulated as a liquid dosage form.
As an embodiment, the method for preparing the liquid preparation with dengue virus resisting effect from the traditional Chinese medicine composition comprises the following steps:
s1, dissolving safflower extract, red paeony root extract, szechuan lovage rhizome extract, red sage root extract and Chinese angelica extract with water for injection, and cooling;
S2, adding anhydrous glucose or sodium chloride into the cold storage liquid, regulating the pH value to 5.5-7.0, and cold storage;
S3, taking cold storage liquid, and performing ultrafiltration to obtain ultrafiltrate;
and S4, encapsulating and sterilizing the ultrafiltrate to obtain the traditional Chinese medicine liquid preparation.
Specifically, in the step S1, the mass ratio of any two extracts of the safflower extract, the red peony root extract, the ligusticum wallichii extract, the red sage root extract and the angelica sinensis extract is as follows: 1 to 10.
Preferably, in the step S1, the mass ratio of any two extracts of the safflower extract, the red peony root extract, the ligusticum wallichii extract, the red sage root extract and the angelica sinensis extract is as follows: 1:1.
Specifically, the content of the anhydrous glucose or sodium chloride in the step S2 is 0.5-10% (w/v) of the traditional Chinese medicine liquid preparation.
Preferably, the anhydrous glucose or sodium chloride content in the step S2 is 4.5% (w/v) of the traditional Chinese medicine liquid preparation.
The invention has the following beneficial effects:
The invention researches the inhibition effect of a drug prepared from a traditional Chinese medicine composition consisting of safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica on dengue II virus and the toxic effect on cells, and research data show that the traditional Chinese medicine composition has a half toxic concentration (CC 50) of 225.6 mu L/mL on cells, a half effective concentration (EC 50) of 1.238 mu L/mL on dengue II virus and a Selection Index (SI) of 182.2, is a low-toxicity and high-efficiency anti-dengue virus drug prescription, can be used for preparing a safe and effective anti-dengue virus drug with small toxic and side effects, and fills the defects of the existing anti-dengue virus drug.
Drawings
FIG. 1 shows the cytotoxicity of a liquid preparation of Chinese medicinal materials in vitro;
FIG. 2 shows the in vitro anti-dengue virus effect of the liquid traditional Chinese medicine preparation.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Unless otherwise indicated, reagents and materials used in the following examples were those available commercially.
African green monkey kidney cells (Vero cells) used in the following examples were purchased from Shanghai China society of sciences stem cell bank and stored in liquid nitrogen using a freezing tube.
Dengue II strains used in the examples below were from the institute of Marhan virus, academy of sciences of China.
The reagents and instrumentation used in the following examples were as follows:
(1) New calf serum (Calf serum) and DMEM medium are purchased from Gibco company;
(2) DMSO was purchased from guangzhou city, kangyang chemical engineering limited;
(3) Trypsin was purchased from DIFCO corporation of the united states (Shanghai bioengineering company agency);
(4) Olympus PM-6 inverted microscope was purchased from OLYMPUS corporation, japan;
(5) E-52AA rotary evaporator was purchased from Shanghai Asia Biochemical instruments works;
(6) BP221S electronic analytical balance was purchased from Shanghai precision instruments list limited.
The following virus experiments were performed in the university of chinese medicine laboratory animal center biosafety secondary (ABSL-2) laboratory.
Safflower decoction pieces, red peony decoction pieces, ligusticum wallichii decoction pieces, red sage root decoction pieces, chinese angelica decoction pieces used in the following examples are all purchased from Guangzhou to Xin pharmaceutical industry Co., ltd.
EXAMPLE 1 preparation of the Chinese medicinal composition into liquid preparation
The Chinese medicinal composition (Carthami flos, radix Paeoniae Rubra, rhizoma Ligustici Chuanxiong, saviae Miltiorrhizae radix, and radix Angelicae sinensis) is made into liquid preparation.
The formula of the traditional Chinese medicine composition is as follows: 100g of safflower, 100g of red paeony root, 100g of szechuan lovage rhizome, 100g of red sage root, 100g of Chinese angelica.
The preparation method of the traditional Chinese medicine liquid preparation comprises the following steps:
1. Preparation of safflower dry paste: taking 100g of safflower decoction pieces, adding 30% ethanol with the volume of 8 times, and leaching for 8 hours; filtering, taking leaching liquor with the volume of 4-6 times, adding 95% ethanol to ensure that the ethanol content is 70%, and cooling for 48 hours; filtering, and concentrating the filtrate to 100mL under reduced pressure; adding 95% ethanol into the concentrated solution to make the ethanol content reach 80%, and cooling for 48 hr; filtering, concentrating under reduced pressure to remove ethanol, and vacuum drying to obtain Carthami flos dry extract;
2. Preparation of radix paeoniae rubra dry paste: taking 100g of red paeony root decoction pieces, adding 10 times of purified water, heating and boiling, keeping micro boiling for two hours, filtering, and collecting filtrate I; continuously heating and boiling the residues with 8 times of process water, keeping micro-boiling for one hour, discarding the residues, and collecting filtrate II; combining the filtrates I and II, and concentrating to 100mL (namely, first concentrated solution); adding 2% gelatin solution into the first concentrated solution under stirring, adding 95% ethanol to make ethanol content reach 70%, and cooling for 24 hr; filtering, concentrating the filtrate to 100mL (namely, second concentrated solution); adding water saturated n-butanol into the second concentrated solution, extracting four times, adding 50mL of water saturated n-butanol each time, mixing the extractive solutions, concentrating under reduced pressure to remove n-butanol until no alcohol smell exists, and vacuum drying to obtain radix Paeoniae Rubra dry extract;
3. preparation of rhizoma Ligustici Chuanxiong dry extract, saviae Miltiorrhizae radix dry extract and radix Angelicae sinensis dry extract: taking 100g of decoction pieces of ligusticum wallichii, radix salviae miltiorrhizae and angelica sinensis, treating the decoction pieces with a preparation process of red paeony root dry paste, wherein each concentration solution is 300mL, and 150mL of water-saturated n-butanol is used for each extraction to prepare ligusticum wallichii dry paste, radix salviae miltiorrhizae dry paste and angelica sinensis dry paste respectively;
4. Taking 5 dry pastes prepared in the steps 1-3 according to the following weight ratio of 1:1:1:1:1, dissolving with water for injection, diluting to 200mL, and cooling for 24 hours; taking cold stock solution, adding anhydrous glucose according to the mass volume ratio of 4.5% of the total amount of the traditional Chinese medicine liquid preparation, and continuously adding water for injection to 1000mL; regulating the pH value to 5.5-7.0 by using 10% sodium hydroxide, and cooling and storing for 24 hours; taking cold storage liquid, and ultrafiltering to obtain ultrafiltrate; encapsulating and sterilizing to obtain the traditional Chinese medicine liquid preparation.
EXAMPLE 2 in vitro cytotoxicity of liquid formulations of traditional Chinese medicine
1. Experimental method
(1) Vero cells with the density of 0.5 multiplied by 10 5 cells/mL are inoculated into a 96-well cell culture plate, 100 mu L of vero cells are used for each well when the cells are attached and the density on the culture plate reaches 80% -90%;
(2) Experimental grouping:
The liquid preparation of traditional Chinese medicine prepared in example 1 was diluted with 2% new born calf serum-containing DMEM medium in gradient, each gradient differing by 2 times, and prepared into liquid preparation of traditional Chinese medicine with concentration of 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 μL/mL as pharmaceutical group;
simultaneously setting a cell control group (DMEM medium containing 2% of new calf serum and cells, and no traditional Chinese medicine liquid preparation) and a blank control group (DMEM medium containing 2% of new calf serum, and no cells and traditional Chinese medicine liquid preparation);
(3) Adding the medicine:
Adding 100 mu L of the traditional Chinese medicine liquid preparation with each concentration into each hole of a medicine group, adding 100 mu L of a DMEM culture medium containing 2% of new calf serum into each hole of a cell control group, adding 100 mu L of a DMEM culture medium containing 2% of new calf serum into each hole of a cell-free culture hole of a blank control group, repeating 3 times of each group, and culturing in a 5% CO 2 incubator at 37 ℃;
(4) After 48h of medicated culture, 100. Mu.L of DMEM medium (2% fresh calf serum) containing 10% CCK-8 reagent was added to each group, and after incubation at 37℃for 1h, the cell culture plates were placed in a multifunctional microplate detector and absorbance at 450nm was measured for each group.
Toxicity of the liquid traditional Chinese medicine preparation to vero cells is expressed by cell survival rate, and the calculation formula is as follows:
cell survival (%) = (drug group-blank control group)/(cell control group-blank control group) ×100%.
2. Experimental results
As shown in FIG. 1, the half toxic concentration (CC 50) of the traditional Chinese medicine liquid preparation measured by the CCK-8 method is 225.6 mu L/mL, and cells can still maintain higher cell viability under higher drug concentration, which indicates that the traditional Chinese medicine liquid preparation has low toxicity and can be used for preparing anti-dengue virus drugs with high safety.
EXAMPLE 3 in vitro anti-dengue Virus Effect of liquid preparation of traditional Chinese medicine
1. Experimental method
1. Experiment grouping design: blank (mock group), virus group (virus group), drug group (0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100. Mu.L/mL of the liquid preparation of the Chinese medicine prepared in example 1)
2. Anti-dengue virus effect test:
(1) Cell pre-incubation:
When Vero cells in the 12-well plate cling to the wall and the density on the culture plate reaches 80% -90%, taking out the well plate from the cell culture box, sucking old culture medium, and washing the cells once by using 1 XPBS buffer solution; and then adding the medicine:
Each hole of the medicine group is added with 500 mu L of traditional Chinese medicine liquid preparations with different concentrations which are diluted by serum-free DMEM culture medium in advance;
500. Mu.L of DMSO-containing serum-free DMEM medium (DMSO is added at a 1:1000 ratio, no drug) is added to each well of mock and virus groups;
the cell plates were then placed in a cell incubator (37 ℃,5% co 2) and incubated for 2 hours;
(2) Pre-incubation of virus:
Meanwhile, dengue virus (moi=0.1) diluted with serum-free DMEM medium was aliquoted into 1.5mL sterile EP tubes, 500 μl each; drug groups were added with the corresponding concentrations of drug in the EP tube, and virus groups were added with DMSO-containing serum-free DMEM medium (DMSO was added at a ratio of 1:1000, no drug contained);
Another dengue virus-free EP tube was taken and 500 μl of serum-free DMEM medium was added as mock group;
Each set of EP tubes was placed in a cell incubator (37 ℃,5% CO 2) and incubated for 2 hours;
(3) After the incubation in the steps (1) and (2) is finished, taking out the 12-hole cell plates and the EP tubes, sucking the culture medium in the 12-hole cell plates, washing the cells once by using 1 XPBS buffer solution, adding the premixed mixture solution of the medicines and viruses in each group of the EP tubes into the 12-hole cell plates in the corresponding group, adding 500 mu L of the premixed mixture solution into each hole, and finally placing the cell plates into a cell incubator (37 ℃ C., 5% CO 2) for incubation for 2 hours;
(4) After the incubation in step (3) was completed, the 12-well cell plates were removed, the medium in the 12-well cell plates was aspirated, the cells were rinsed once with 1×pbs buffer, 1 mL% dmem medium (containing 10% fbs and 1% diabody) containing the corresponding concentration of the drug was added to each well of the drug group, 2% dmem medium (containing 10% fbs and 1% diabody) containing DMSO was added to each well of the mock group and the virus group, DMSO was added in a ratio of 1:1000, and then the cell plates were placed in a cell incubator (37 ℃,5% co 2) and incubated for 48 hours.
3. Extracting and detecting viral RNA in dengue virus infected cell samples:
(1) Extracting total RNA:
The cell plates after the incubation of step (4) of method 2 of this example were removed, the medium was aspirated, the cells were rinsed once with 1 XPBS buffer, then 500. Mu. L TRlzol Reagent cell lysates were added to each well, and after 5 minutes of action, the samples from each well were transferred to a new individual EP tube and total RNA from the samples from each well were extracted according to the extraction procedure described in Ultrapure RNAkit kit.
(2) CDNA synthesis by reverse transcription
The total RNA was reverse transcribed into cDNA using HiScript II Q RT SuperMix for qPCR kit, for specific procedures in accordance with the instructions of the kit. Wherein the cDNA reverse transcription system is configured as shown in Table 1 below:
TABLE 1
The reverse transcription reaction gene amplification procedure was set as shown in table 2 below:
TABLE 2
(3) Detection of
And (3) adding SYBR Green fluorescent markers into the cell sample cDNA obtained after the reverse transcription in the step (2), and performing real-time fluorescent quantitative PCR detection. The change of fluorescent signals in the reaction system, namely the copy number change of the target gene dengue II virus DENV II gene, is directly detected by a fluorescent signal detector, so that the relative quantitative detection is carried out, and the dengue II virus inhibition rate is calculated according to the relative quantitative detection.
Inhibition ratio = (1-genome virus gene expression amount/genome virus gene expression amount) ×100%.
Wherein, the expression quantity of the virus gene is calculated as 2-delta ct.
Deltact = mean of drug or virus deltact values-mean of blank deltact values,
Delta ct value = target gene (DENV ii) ct value-reference Gene (GAPDH) ct value.
The ct value is generated by the program of the qPCR instrument.
The dengue II virus PCR primer sequences are shown in Table 3 below:
TABLE 3 Table 3
The real-time fluorescent quantitative PCR detection system configuration is shown in Table 4 below:
TABLE 4 Table 4
The real-time fluorescent quantitative PCR detection procedure set forth in table 5 below:
TABLE 5
2. Experimental results and analysis
The embodiment explores the inhibition effect of the traditional Chinese medicine liquid preparation on dengue II virus after the dengue II virus infects Vero cells, and the half-effective concentration (EC 50) of the traditional Chinese medicine liquid preparation on the dengue II virus infects Vero cells is calculated by a qPCR method. As shown in FIG. 2, the half effective concentration of the traditional Chinese medicine liquid preparation on the Vero cells infected by the dengue II virus is 1.238 mu L/mL, which indicates that the traditional Chinese medicine liquid preparation has excellent ability of inhibiting the dengue II virus and can still effectively inhibit the dengue II virus under low concentration.
For antiviral drugs, the most important index characterizing their antiviral activity in vitro is the Selection Index (SI), which refers to the ratio of the cytotoxic effect of the drug to the inhibitory effect on viral replication, namely: si=cc 50/EC50, reflecting a balance of drug effectiveness and safety. The results of the comprehensive analysis of the results of the example 1 and the example 2 show that the selection index SI of the traditional Chinese medicine liquid preparation is 182.2, and the traditional Chinese medicine liquid preparation is low in toxicity and high in efficiency.
EXAMPLE 4 in vivo anti-dengue Virus Effect of liquid preparation of traditional Chinese medicine
1. Experimental method
1. Experimental materials:
Experimental animals: type I interferon receptor deficient mice IFNAR-/-C57BL/6SPF grade female mice 60, 6-7 weeks old, weight 13-17g, given by the university of Guangzhou medical science Zhao Jincun teaching, raised in the university of Guangzhou traditional Chinese medicine animal laboratory building 3 SPF grade laboratory (license number [ SYXK (Guangdong) 2018-0001 ]). In the experimental process, the 3R principle is followed, the humanization care is given to mice, and the mice are fed into an independent ventilation and ventilation cage box (IVC) according to 6 mice/cage, wherein the temperature (24+/-2) DEG C and the humidity (40% -60%). The small mice are given a quasi-feed and sterilized water, and are free to eat.
Virus strain: the DENV II TSV strain DENV II TSV was given away by the university of nuance Wu Jianguo and stored and amplified in the biosafety secondary (ABSL-2) laboratory.
Animal virus experiments were performed in a biosafety secondary (ABSL-2) laboratory (record number: guangdong animal Equipment GZL 0004).
Medicament: the liquid preparation of traditional Chinese medicine prepared in example 1.
2. Experimental grouping:
3. in vivo viral inhibition assay
Animal experiments on days 2, 5 and 8, 50 mu L of cheek blood of each experimental group of mice is taken and rapidly placed in a 1.5mL EP tube containing 500 mu L TRIzon Reagent, covered with a cover, shaken and shaken well, and stored in a refrigerator at-80 ℃. The amounts of copies of viral RNA at different times in the blood samples of mice of each of the above groups were measured according to the method described in "extraction and detection of viral RNA in dengue virus-infected cell samples" in example 3.
2. Experimental results
The test results of the inhibition effect of the traditional Chinese medicine liquid preparation on dengue virus in mice are shown in the following table 6:
TABLE 6
( And (3) injection: the data in Table 6 are obtained by log of the original viral RNA copy amount data, and represent significant differences between the traditional Chinese medicinal liquid preparation group and dengue virus group data, wherein P < 0.05 and P < 0.01 )
The results in table 6 show that dengue virus group mice can detect dengue virus infection with high copy number on days 2, 5 and 8, and compared with dengue virus groups, the virus copy number of the traditional Chinese medicine liquid preparation in the high-dose group and the low-dose group is obviously reduced along with the attack time, so that the traditional Chinese medicine liquid preparation can obviously inhibit replication of dengue virus in the mice. Meanwhile, the in vivo virus copy quantity of mice in a positive control group (temozolomide mesylate group) is reduced on the 5 th day of virus attack, but is rapidly increased on the 8 th day, which shows that the effective antiviral action duration of temozolomide mesylate is shorter, and compared with the traditional Chinese medicine liquid preparation, the virus copy quantity of the high-dose group and the low-dose group can still maintain lower virus copy quantity after being obviously reduced, thus indicating that the traditional Chinese medicine liquid preparation has the capability of stably and long-term inhibiting dengue virus.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The application of the traditional Chinese medicine composition in preparing the anti-dengue virus medicine is characterized in that the traditional Chinese medicine composition comprises safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica.
2. The application of the traditional Chinese medicine composition in preparing the medicine for preventing dengue virus infection is characterized in that the traditional Chinese medicine composition comprises safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica.
3. The use according to claim 1 or 2, wherein the Chinese medicinal composition is composed of safflower, red peony root, ligusticum wallichii, red sage root, chinese angelica.
4. The use according to any one of claims 1 to 3, wherein the Chinese medicinal composition is a composition of safflower extract, red peony root extract, ligusticum wallichii extract, red sage root extract, angelica sinensis extract.
5. The use according to claim 4, wherein the safflower extract is an alcohol extract, and the red peony root extract, the ligusticum chuanxiong rhizome extract, the red sage root extract and the angelica sinensis extract are water extracts.
6. An anti-dengue virus medicine is characterized by comprising a traditional Chinese medicine composition comprising safflower, red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica and a pharmaceutically acceptable carrier.
7. The medicine according to claim 6, wherein the Chinese medicinal composition accounts for 1-99% of the total mass of the medicine.
8. The medicament according to claim 6 or 7, characterized in that it also contains other anti-dengue virus components.
9. The medicament according to any one of claims 6 to 8, wherein the medicament is a liquid preparation of a traditional Chinese medicine.
10. The medicine according to claim 9, wherein the preparation method of the liquid preparation of the traditional Chinese medicine is as follows:
s1, extracting safflower with alcohol, extracting red paeony root, szechuan lovage rhizome, red sage root and Chinese angelica with water, combining the extracts, dissolving the extracts with water for injection, and cooling and storing;
S2, adding anhydrous glucose or sodium chloride into the cold storage liquid obtained in the step S1, regulating the pH value to 5.5-7.0, and cold storage;
S3, taking the cold storage liquid obtained in the step S2, and performing ultrafiltration to obtain ultrafiltrate;
and S4, encapsulating and sterilizing the ultrafiltrate to obtain the traditional Chinese medicine liquid preparation.
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