Kyllinga brevifolia formula for preventing and treating EV71 type infection

Abstract

The invention discloses a formula of kyllinga breviscapus for preventing and treating EV71 type infection, and relates to the technical field of medicines, and the formula comprises the following steps: S1, taking kyllinga breviscapus, and adding 6 times of volume of water into the kyllinga breviscapus; s2, decocting for three times, and decocting for 1 hour each time; s3, filtering the decoction, and merging the filtrate; and S4, carrying out reduced pressure concentration at-80 DEG C (the vacuum degree is-0.090 to-0.1 MPa) to obtain freeze-dried powder. Compared with the prior art, the application has the advantages that the enterovirus 71 is a microRNA viridae enterovirus group A member EV71, the kyllinga breviscapus has good inhibitory activity on the enterovirus 71, and the kyllinga breviscapus has important clinical application value on treatment of diseases infected with the enterovirus 71.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a water centipede formula for preventing and treating EV71 infection. Background Art

[0002] Enterovirus 71 (EV71) is a single-stranded positive-strand RNA virus belonging to the family Parvoviridae and the genus Enterovirus. It is highly infectious and has a high morbidity rate. EV71 virus particles are 20-sided symmetrical spherical structures with a diameter of approximately 24 to 30 nm. They have no envelope or protrusions, so they are highly resistant to the surrounding environment and are tolerant to detergents, gastric acid, and ethanol. Moreover, as an RNA virus, EV71 is prone to mutation during replication. There is a lack of effective measures to block EV71 infection and effective therapies. So far, there is no specific treatment or globally available vaccine.

[0003] EV71 is a virus that can cause hand, foot and mouth disease and central nervous system infection. The main transmission route is fecal-oral transmission. Infection can cause low fever, mouth pain, vomiting, diarrhea, etc. At the same time, because EV71 is highly infectious to the central nervous system, clinical symptoms also include aseptic meningitis, myocarditis, muscle stiffness, etc. Although patients with no symptoms or mild symptoms will recover naturally and produce antibodies, patients infected with EV71 and developing severe symptoms still have a high chance of dying from cardiopulmonary failure and extensive brainstem damage.

[0004] Water centipede is a perennial herbaceous plant of the genus Cyperus of the family Cyperaceae, also known as golden button grass, three-pod grass, golden bull grass, etc. The whole plant is used as medicine and can be harvested throughout the year, washed and dried for use. Water centipede is named because its rhizome is nearly cylindrical, slender and jointed, and it crawls flat on the ground. Water centipede tastes spicy, slightly bitter, sweet, and is flat in nature. It enters the lung and liver meridians. It has the effects of dispelling wind and detoxifying, clearing heat and removing dampness, and promoting blood circulation and detoxifying. Water centipede has the effects of dispelling wind and relieving exterior pathogens, clearing heat and removing dampness, relieving cough and reducing phlegm, and removing blood stasis and swelling. It is used for colds, bronchitis, whooping cough, malaria, dysentery, hepatitis, chyluria, traumatic injury, rheumatoid arthritis; external use for snake bites, skin itching, and furunculosis. However, there are no reports on water centipede inhibiting enteroviruses including EV71. Summary of the invention

[0005] In order to solve the above technical problems, the present invention first provides the use of water centipede in preparing medicine for preventing, alleviating or treating diseases caused by enterovirus EV71.

[0006] Specifically, the technical solution provided by the present invention is: a water centipede formula for preventing and treating EV71 infection, comprising the following steps:

[0007] S1. Take the water centipede and add 6 times the volume of water;

[0008] S2, decoct three times, each time for 1 hour;

[0009] S3, the decoction is filtered and the filtrate is combined;

[0010] S4. Concentrate under reduced pressure at -80°C (vacuum degree: -0.090 to -0.1 MPa) to obtain a freeze-dried powder.

[0011] Furthermore, the vacuum degree of S4 is -0.090 to -0.1 MPa.

[0012] Furthermore, the water extract of water centipede can inhibit enterovirus 71, can treat diseases caused by enterovirus 71 infection, and improve diseases caused by enterovirus 71 infection; inhibiting enterovirus 71 means inhibiting enterovirus 71 at the body level or the cellular level.

[0013] Furthermore, diseases caused by enterovirus 71 infection may include hand, foot and mouth disease, herpangina, encephalitis, aseptic meningitis, acute weak limb paralysis, acute hemorrhagic conjunctivitis, etc.; symptoms caused by enterovirus 71 infection may include headache, fever, vomiting, muscle stiffness, etc.

[0014] Furthermore, the inhibition of enterovirus 71 occurs when the water centipede enters the host or host cells, i.e., the endocytosis stage. The water centipede is suitable for preparing products for treating diseases caused by enterovirus 71 infection.

[0015] Furthermore, enterovirus 71 is divided into three subtypes, namely group A, group B and group C; enterovirus 71 group B and group C each contain five subgroups, namely viruses of subgroups B1-B5 and C1-C5.

[0016] Compared with the prior art, the advantages of the present invention are: enterovirus 71 is a member of group A of the enterovirus genus of the Picornaviridae family, EV71; water centipedes have good inhibitory activity against enterovirus 71; and water centipedes have important clinical application value for the treatment of diseases caused by infection with enterovirus 71. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a structural comparison diagram of embodiment 1.

[0018] Figure 2 It is a schematic diagram of the cytotoxicity and inhibition rate of the water centipede in Example 1.

[0019] Figure 3 It is a schematic diagram of the relative expression level of EV71 mRNA in cells and the concentration of water centipede in Example 2. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0021] Embodiment 1

[0022] This embodiment provides a specific implementation method, which includes three parts:

[0023] 1. Cell virus culture

[0024] The human rhabdomyosarcoma cell line RD was obtained from the American Type Culture Collection (ATCC, CRL-3216TM) and cultured in a 37°C, 5% CO2 incubator using DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen).

[0025] EV71, a member of the Enterovirus family A, was propagated in RD cells and the virus titer was determined by TCID50. All infection experiments were performed in a biosafety level 2 (BLS2) laboratory.

[0026] 2. EC50 and CC50 determination of water centipedes inhibiting EV71, a member of the Enterovirus group A of the Picornaviridae family

[0027] EC50 assay: 1×10 4 RD cells were inoculated into 96-well plates and cultured in an incubator at 37°C and 5% CO2 for 24 h. The freeze-dried powder of the water extract of water centipede was diluted to 1 mg / mL with DMEM medium containing 2% FBS (i.e., maintenance medium), and the drug solution was filtered with a 0.45 μm filter membrane to remove the residue. Subsequently, the drug was diluted twice with cell maintenance medium to obtain 10 concentrations of water centipede solution, and each concentration was inoculated into 2 wells (50 μL / well). At the same time, the virus dilution was added to the cell well (50 μl / well) so that the virus MOI after entering the well was 0.1, and the drug concentrations after entering the well were 1.00 mg / mL, 0.50 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.0312 mg / mL, 0.0156 mg / mL, 0.008 mg / mL, 0.004 mg / mL, 0.002 mg / mL, and 0.001 mg / mL. Continue to culture for about 24 hours until the negative control group (i.e., the group with EV71 virus added but no reagent added) can see obvious lesions under the microscope and take pictures. At the same time, a blank group was set up. The expression of intracellular viral RNA and the intracellular reference gene GAPDH was quantitatively detected by qRT-PCR. GraphPad-Prism 9 software was used for data analysis and EC50 calculation.

[0028] CC50 detection: CC50 was detected using the CCK-8 method. RD cells were inoculated into 96-well cell culture plates. The test was performed when the cell density reached 60%-80%. After the RD cells were replaced with diluted drugs, the final concentrations were 1.00 mg / mL, 0.50 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.0312 mg / mL, 0.0156 mg / mL, 0.008 mg / mL, 0.004 mg / mL, 0.002 mg / mL, and 0.001 mg / mL, and 2 wells were inoculated for each concentration. The cells were cultured at 37°C and 5% CO2 for 24 hours. The luminescence intensity at 450 nm was detected using a multifunctional microplate reader (Bio Tek). GraphPad-Prism 9 software was used for data analysis and calculation of CC50.

[0029] EC50 refers to the drug concentration that can effectively inhibit 50% of cells from being infected with the virus. The smaller the value, the better the inhibitory effect on the virus.

[0030] CC50 is the drug concentration that causes 50% of cells to become diseased. The higher the value, the lower the toxicity to cells.

[0031] SI: Selectivity index, which is the ratio of CC50 to EC50. The larger the value, the higher the possibility of drug development.

[0032] 3. Virus Sample Processing and Detection

[0033] RNA was extracted using the Super Fast Pure Cell RNA Isolation Kit (Vazyme, Catalog No. RC102) according to the manufacturer's instructions. Reverse transcription used the Hifair II 1st strand cDNA synthesis kit (Shanghai Yisheng Biotechnology, Catalog No.: 11121ES60) that can digest gDNA, and qRT-PCR used Quantstudio real-time PCR detection reagent (Applied biosystems, Foster City, CA, USA). The sequence information of the primers used is shown in Table 1. SYBR-Green method qPCR amplification: 95°C 5min, 40 cycles, 95°C 10s, 55°C 20s, 72°C 31s. Normalization was achieved by detecting the GAPDH gene.

[0034]

[0035] Table 1 Primer sequences used in the study

[0036] Results: Figure 1As shown, compared with the negative well (A) (i.e., the negative control group) in which only the virus was added but no water centipede, the water centipede with a concentration of 1 mg / ml (C) and 0.5 mg / ml (D) in the experimental group showed a good antiviral effect. Figure 1 Figure B is a picture of untreated cells (blank group), i.e., the test results without adding centipede water or virus (EV71). Figure 1 As can be seen from (C) and 1 (D), 1 mg / ml (C) and 0.5 mg / ml (D) solutions of water centipede can significantly inhibit cytopathic effects.

[0037] According to the relative expression of viral mRNA in cells, Figure 2 As shown, the EC50 and CC50 of water centipede were calculated using the log(inhibitor) vs.response formula in the nonlinear analysis of GraphpadPrism 9 software. The experimental results showed that on RD cells, the EC50 of water centipede against EV71 was 0.01798 mg / mL, CC50>1 mg / mL, SI>55.617, and it had an anti-EV71 effect of more than 90% (94% to 99%) in the four concentration ranges of 0.125 mg / ml to 1 mg / ml.

[0038] Embodiment 2

[0039] In this example, the dosing time method was used to preliminarily determine at which stage of the virus life cycle the antiviral active ingredient (water centipede) exerted its inhibitory effect. The specific experimental steps were as follows: 3×10 4 RD cells were infected with EV71 strain with MOI=0.1 24 hours later, and the solution was changed 2 hours after virus infection. 0.25 mg / mL water centipede solution was added to the three groups of samples during the whole infection cycle (when adding virus and 2 hours after adding virus), before entering the cell (when adding virus), and after entering the cell (2 hours after adding virus incubation). RNA in the cells was extracted 16 hours after adding drugs and poisons, and the viral replication in the cells and the expression of the cellular reference gene GAPDH were determined by qRT-PCR. The specific steps were the same as those in Example 1. In addition, a negative control was set, that is, a group in which only virus was added without water centipede solution.

[0040] The results are as follows Figure 3 As shown, when water centipede (0.25 mg / mL, 0.025 mg / ml, 0.0025 mg / ml) was added only at the virus infection cell stage, the relative expression level of EV71 mRNA in the cells with 0.25 mg / mL water centipede was reduced by 98% (the relative expression level of EV71 mRNA = 2^(GAPDH-EV71)), indicating that water centipede can inhibit the virus entry process and the effect is significant.

[0041] The above shows and describes the basic principles and main features of the present invention and the advantages of the invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A water centipede formula for preventing and treating EV71 infection, characterized in that The following steps are involved: S1. Take the water centipede and add 6 times the volume of water; S2, decoct three times, each time for 1 hour; S3, the decoction is filtered and the filtrate is combined; S4. Concentrate under reduced pressure at -80°C (vacuum degree: -0.090 to -0.1 MPa) to obtain a freeze-dried powder.

2. A water centipede formula for preventing and treating EV71 infection according to claim 1, characterized in that: The vacuum degree of S4 is -0.090 to -0.1 MPa.

3. A water centipede formula for preventing and treating EV71 infection according to claim 1, characterized in that: The water extract of the water centipede can inhibit enterovirus 71, treat diseases caused by enterovirus 71 infection, and improve diseases caused by enterovirus 71 infection; inhibiting enterovirus 71 means inhibiting enterovirus 71 at the body level or the cell level.

4. A water centipede formula for preventing and treating EV71 infection according to claim 3, characterized in that: The diseases caused by enterovirus 71 infection may include hand, foot and mouth disease, herpetic pharyngitis, encephalitis, aseptic meningitis, acute weak limb paralysis, acute hemorrhagic conjunctivitis, etc.; the symptoms caused by enterovirus 71 infection may include headache, fever, vomiting, muscle stiffness, etc.

5. A water centipede formula for preventing and treating EV71 infection according to claim 1, characterized in that: The inhibition of enterovirus 71 occurs when the water centipede enters the host or host cells, i.e., the cell entry stage. The water centipede is suitable for preparing products for treating diseases caused by enterovirus 71 infection.

6. A water centipede formula for preventing and treating EV71 infection according to claim 3, characterized in that: The enterovirus 71 type is divided into three subtypes, namely group A, group B and group C; the enterovirus 71 type group B and group C each contain five subgroups, namely viruses of the B1-B5 and C1-C5 subgroups.

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