Daphniphyllum calycinum alkaloid compound with anti-EV71 virus activity and preparation and application thereof

Abstract

The invention belongs to the technical field of medical chemistry, and particularly relates to an alkaloid compound with enterovirus 71 (EV71) resisting activity as well as preparation and application of the alkaloid compound. Four novel alkaloid compounds are separated from branches and leaves of daphniphyllum calycinum, the structures of the compounds are novel, skeletons of three compounds (compounds 1-3) are rare cyano-substituted polycyclic skeletons, and one compound (compound 4) is rare Daphmanidin type alkaloid. The four alkaloid compounds have significant anti-EV71 virus activity, and have good application prospects in the aspect of developing new drugs for preventing and treating infant hand-foot-and-mouth diseases induced by EV71 viruses.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a nephrolepis alkaloid compound having anti-EV71 virus activity and a preparation method and application thereof. Background Art

[0002] Enterovirus 71 (EV71) is one of the main pathogens that cause hand, foot and mouth disease (HFMD) in infants and young children. It can spread rapidly through excrement, water, air and other channels. It is highly infectious and has a high morbidity rate, especially complications of the nervous system. Children of all age groups can be infected and develop the disease, especially those under 3 years old. A small number of seriously ill children may develop complications such as aseptic meningitis, brainstem encephalitis, and neurogenic pulmonary edema. Very few are in critical condition and can cause death. Survivors may have sequelae, which seriously endangers the health and life safety of children. It is known as the "poliomyelitis" of the 21st century. Hand, foot and mouth disease caused by EV71 has a cyclical epidemic trend every 2-3 years in many places (such as Sarawak, Malaysia). EV71 infection has become an important public health issue in the world, especially in the Asia-Pacific region. EV71 is a single-stranded positive-strand RNA virus belonging to the Picornaviridae family, Enterovirus genus. The genome is about 7,500 bases long, with only one open reading frame encoding a polyprotein. This polyprotein can produce four structural proteins (VP1-VP4) and seven non-structural proteins (2A-2C and 3A-3D) of the virus after further hydrolysis and shearing, flanked by 5′ and 3′ uncoding regions (UTRs). When the virus begins to infect, the appearance and structure of the four structural proteins change, making it easier to bind to host cells. After binding, the virus begins to replicate. At this time, the structural protein undergoes structural changes again, making it easier for the single-stranded RNA in the protein to enter the infected cell body. After a series of gene replication and amino acid coding, the precursor protein of the virus is initially formed, and then modified and cut by the virus's own functional protein, so that the precursor protein is quickly converted into EV71 virus protein, and then infects other cells.

[0003] At present, the main anti-EV71 drugs in clinical practice are ribavirin and acyclovir, but their therapeutic effects are limited, and long-term use can easily cause liver damage. Therefore, the development of anti-EV71 drugs with specificity, high efficiency and low toxicity is of great significance to the health of infants and young children and social development. Among them, natural products, as one of the important sources of drug development, have the advantages of rich and diverse structures and wide biological activities. Therefore, it is of great scientific significance and development prospects to explore natural products with anti-EV71 activity in plants in nature. Summary of the invention

[0004] In order to overcome the deficiencies of the above-mentioned prior art, the present invention isolated four new alkaloid compounds from the branches and leaves of Daphniphyllum calycinum. These compounds have good anti-EV71 virus activity and can be used to prepare anti-EV71 lead compounds and antiviral drugs, providing candidate compounds for the research and development of new anti-EV71 drugs.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a genus Acer alkaloid compound, wherein the genus Acer alkaloid compound comprises at least one of compounds 1-4, and the structural formulas of compounds 1-4 are as follows:

[0007]

[0008] Preferably, the Achyranthes bidentata alkaloid compound comprises at least one of compounds 3 and 4, and the structural formulas of compounds 3 and 4 are as follows:

[0009]

[0010] After EV71 (MOI=0.1) infection for 48h, the EC 50 Under the same infection conditions, the EC values ​​of compounds 3 and 4 were 2.826 μg / mL and 3.783 μg / mL, respectively. 50 are smaller than ribavirin (Ribavirin's EC 50 It was 65.770 μg / mL), indicating that it had good anti-EV71 virus activity and could be used to prepare anti-EV71 lead compounds and antiviral drugs, providing candidate compounds for the research and development of new anti-EV71 drugs.

[0011] The second aspect of the present invention provides the use of the Achyranthes bidentata alkaloid compounds described in the first aspect in the preparation of anti-EV71 virus drugs or drugs for preventing and treating hand, foot and mouth disease in infants and young children induced by EV71 virus.

[0012] The third aspect of the present invention provides an anti-EV71 drug, wherein the anti-EV71 drug uses at least one of compounds 1-4 as an active ingredient, and the structural formulas of compounds 1-4 are as follows:

[0013]

[0014] Preferably, the anti-EV71 drug uses at least one of compounds 3 and 4 as an active ingredient, and the structural formulas of compounds 3 and 4 are as follows:

[0015]

[0016] Preferably, the anti-EV71 drug also includes a pharmaceutically acceptable excipient.

[0017] More preferably, the excipients are diluents, adhesives, lubricants, disintegrants, solubilizers, stabilizers, etc. that can be used in the pharmaceutical field, as well as some pharmaceutical matrices; they can also be functional pharmaceutical excipients available in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0018] Preferably, the dosage forms of the anti-EV71 drug include tablets, granules, capsules, pills, sustained-release preparations, oral liquid preparations, powder for injection, and injection.

[0019] Preferably, the anti-EV71 drug is administered orally or by injection.

[0020] The pharmaceutical preparation of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically), i.e., flexibly by oral, subcutaneous, intramuscular and intravenous administration. If certain drugs are unstable under gastric conditions, they can be prepared into enteric-coated tablets.

[0021] The fourth aspect of the present invention also provides a method for preparing four Achyranthes bidentata alkaloid compounds, comprising the following steps:

[0022] (1) soaking and extracting dried Acer truncatum branches and leaves with an ethanol solution, and recovering the solvent by rotating and reducing pressure to obtain an extract;

[0023] (2) dissolving the extract with water and adjusting the pH of the aqueous solution;

[0024] (3) extracting the aqueous solution of step (2) with ethyl acetate, adjusting the pH of the aqueous layer after extraction, and extracting again with dichloromethane and concentrating;

[0025] (4) extracting the aqueous layer from step (3) with dichloromethane and concentrating;

[0026] (5) The dichloromethane layer extract is separated and purified to obtain the alkaloid compounds of the Herba Lycopodii.

[0027] Preferably, in step (1), the ethanol is an ethanol aqueous solution with a volume fraction of 90% to 95%.

[0028] Preferably, in step (2), the pH of the aqueous solution is adjusted to pH = 2-3 using 3% tartaric acid and hydrochloric acid.

[0029] Preferably, in step (3), the pH of the water layer is adjusted to pH = 9-10 using a saturated sodium carbonate solution.

[0030] Preferably, in step (5), the separation is first eluted by silica gel column chromatography and then separated by Sephadex LH-20 column.

[0031] More preferably, the elution system for elution by silica gel column chromatography is dichloromethane and methanol in a volume ratio of (80:1)-(1:1).

[0032] Preferably, in step (5), the purification is performed by semi-preparative high performance liquid chromatography using a reverse phase C18 column, and the mobile phase is methanol and water.

[0033] Preferably, the volume ratio of methanol to water is (100-10):(0-90), and the flow rate of the mobile phase is 1-3 mL / min.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] In order to screen out compounds with anti-EV71 activity from natural products, the present invention isolated four new Daphniphyllum calycinum alkaloid compounds from the branches and leaves of Daphniphyllum calycinum. The compounds have novel structures, among which the skeletons of compounds 1-3 are rare polycyclic skeletons substituted with cyano groups, and compound 4 is a rare Daphmanidin-type alkaloid. The compounds have enterovirus 71 inhibitory activity, especially compounds 3 and 4. After 48 hours of EV71 (MOI = 0.1) infection, the EC 50 Under the same infection conditions, the EC values ​​of compounds 3 and 4 were 2.826 μg / mL and 3.783 μg / mL, respectively. 50 are smaller than ribavirin (Ribavirin's EC 50 The results show that the four new alkaloid compounds of Achyranthes bidentata provided by the present invention have good application prospects in the development of new drugs for the prevention and treatment of hand, foot and mouth disease in infants and young children induced by EV71 virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The H NMR spectrum of the alkaloid compound 1 from Niuerfeng ( 1 HNMR spectrum);

[0037] Figure 2 The C NMR spectrum of the alkaloid compound 1 from Niuerfeng ( 13 C NMR spectroscopy) and distortionless polarization transfer technique spectroscopy (DEPT spectroscopy);

[0038] Figure 3 It is the high-resolution electrospray ionization mass spectrum (HRESIMS spectrum) of the alkaloid compound 1 of Niuerfeng;

[0039] Figure 4 This is the infrared spectrum (IR spectrum) of the Niuerfeng alkaloid compound 1;

[0040] Figure 5 This is the ultraviolet spectrum (UV spectrum) of the alkaloid compound 1 of the Herba Cynanchifoliae;

[0041] Figure 6 It is a compound of the alkaloids of Acer truncatum 1 HNMR spectrum;

[0042] Figure 7 It is a compound of the alkaloids of Acer truncatum 13 C NMR and DEPT spectra;

[0043] Figure 8 This is the HRESIMS spectrum of the alkaloid compound 2 from Niuerfeng;

[0044] Fig. 9 This is the IR spectrum of the alkaloid compound 2 from the herbaceous pine;

[0045] Fig.10 This is the UV spectrum of the alkaloid compound 2 from the genus Niuerfeng;

[0046] Fig.11 The alkaloids of Acer truncatum L. 1 HNMR spectrum;

[0047] Fig.12 The alkaloids of Acer truncatum L. 13 C NMR and DEPT spectra;

[0048] Fig.13 This is the HRESIMS spectrum of the alkaloid compound 3 from Niuerfeng;

[0049] Fig.14 This is the IR spectrum of the alkaloid compound 3 from the herbaceous pine;

[0050] Fig.15 This is the UV spectrum of the alkaloid compound 3 from the genus Niuerfeng;

[0051] Fig.16 The alkaloid compound 4 of Achyranthes bidentata 1 HNMR spectrum;

[0052] Fig.17 The alkaloid compound 4 of Achyranthes bidentata 13 C NMR and DEPT spectra;

[0053] Fig.18 This is the HRESIMS spectrum of the alkaloid compound 4 from Niuerfeng;

[0054] Fig.19 This is the IR spectrum of the alkaloid compound 4 from the herbaceous pine;

[0055] Fig. 20 This is the UV spectrum of the alkaloid compound 4 from the genus Niuerfeng;

[0056] Fig.21 This is the result of the initial screening of the antiviral activity of the alkaloid compounds 1–4 from Achyranthes bidentata using the CCK8 method;

[0057] Fig. 22 The EC of alkaloids 3 from Niuerfeng was determined by CCK8 method 50 Result graph;

[0058] Fig.23 Determination of EC of alkaloids 4 from Niuerfeng by CCK8 method 50 Result graph;

[0059] Fig.24 The EC of positive control drug ribavirin was determined by CCK8 method 50 Result graph. DETAILED DESCRIPTION

[0060] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0061] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0062] Example: Isolation and identification of alkaloid compounds from Rhizoma Cyperi

[0063] 1.1 Reagents and instruments used in the experiment

[0064] (1) Analytical ethanol, ethyl acetate, dichloromethane, and methanol were purchased from Guangzhou Bell Biotechnology Co., Ltd.; chromatographic grade methanol was purchased from TEDIA; the water used for HPLC was Wahaha purified water (Hangzhou Wahaha Company);

[0065] (2) Silica gel for column chromatography (80-120 mesh, 200-300 mesh, 300-400 mesh) and thin layer chromatography silica gel plates (GF254) were purchased from Shanxi Nuotai Biotechnology Co., Ltd.;

[0066] (3) Analytical column XB-C185μM (250mm×4.6mm), semi-preparative column XB-C 18 5μM (250mm×10mm), Sephadex TM LH-20 was purchased from Merck, Germany.

[0067] (4) The color developer used for thin layer chromatography (TLC) was 10% sulfuric acid-ethanol solution.

[0068] (5) Others: (WHF-203B) UV spectrophotometer; (EYEKAN-1300) rotary evaporator; (XD-5210A) rotary evaporator; Agilent 1120 high performance liquid chromatograph (HPLC); Waters e2695 (HPLC); Bruker AM-500, Bruker AM-600 nuclear magnetic resonance spectrometer (Bruker, Karlsruhe, Germany).

[0069] 1.2. Acquisition of alkaloid compounds from Rhizoma Atractylodis Macrophyllae

[0070] The sample of Glehnia littoralis used in the experiment was collected in Liannan Yao Autonomous County, Guangdong Province in August 2022, and was identified by Professor Zheng Xilong of Guangdong Pharmaceutical University. The sample is stored in the Department of Natural Medicine, School of Pharmacy, Sun Yat-sen University (Shenzhen). The specimen number is SYSUSZ-2022-Galk.

[0071] The specific preparation method comprises the following steps:

[0072] (1) 15 kg of dried branches and leaves of Acer truncatum were soaked and extracted three times in 20 L of 95% ethanol aqueous solution at room temperature, each time for 3 h. The obtained extracts were filtered, combined and concentrated into an extract.

[0073] (2) Add water to stir the extract into a suspension (the mass ratio of extract to water is 1:2), and adjust the aqueous solution to pH 2-3 with 3% tartaric acid and hydrochloric acid, and then use ethyl acetate for extraction, the volume ratio of the water layer to the ethyl acetate layer is 1:1, extract 3-5 times, and separate the water layer and the ethyl acetate layer for later use.

[0074] (3) The aqueous layer in step (2) is adjusted to a pH of 9-10 with saturated sodium carbonate, extracted three times with dichloromethane and water in a volume ratio of 1:1, and the dichloromethane layer is evaporated and concentrated to prepare an extract for later use.

[0075] (4) The dichloromethane layer extract was taken and subjected to silica gel column chromatography using a dichloromethane / methanol (80:1–3:1, V / V, with 1‰ diethylamine) elution system to obtain five fractions (Fr.1 to Fr.5).

[0076] (5) Fr.2 was purified by silica gel column chromatography (mobile phase: petroleum ether / ethyl acetate 20:1–0:1, with 1‰ diethylamine) to obtain six components (Fr.2.A–Fr.2.F). Component Fr.2.D was further separated by Sephadex LH-20 (MeOH) column to obtain six subcomponents Fr.2.4.1–Fr.2.4.6. Component Fr.2.4.2 was purified by semi-preparative RP-HPLC (chromatographic column RP-C 18 , 5μmol / L, 250×10mm, Welch Materials, Inc.) (the mobile phase was methanol / water with a volume ratio of 65:35, and the flow rate was 3mL / min) to prepare compound 2 (1.4 mg, retention time t R 32.0min); component Fr.2.4.3 was subjected to semi-preparative RP-HPLC (chromatographic column RP-C 18 , 5μmol / L, 250×10mm, Welch Materials, Inc.) (the mobile phase was methanol / water with a volume ratio of 80:20, and the flow rate was 3mL / min) to prepare compound 3 (1.7 mg, retention time t R 30.3min) and compound 4 (1.5mg, retention time t R 26.0min);

[0077] (6) Fr.4 was purified by silica gel column chromatography (mobile phase: petroleum ether / ethyl acetate 15:1–0:1, with 1‰ diethylamine) to obtain four components (Fr.4.1–Fr.4.4). Component Fr.4.3 was further separated by Sephadex LH-20 (MeOH) column to obtain six subcomponents Fr.4.3.1–Fr.4.3.6. Component Fr.4.3.3 was purified by semi-preparative RP-HPLC (chromatographic column RP-C 18 , 5μmol / L, 250×10mm, Welch Materials, Inc.) (the mobile phase was methanol / water with a volume ratio of 40:60, and the flow rate was 3mL / min) to prepare compound 1 (3.4 mg, retention time t R is 28.0min).

[0078] The four alkaloid compounds 1–4 have the following molecular structures:

[0079]

[0080] The above compounds 1-4 were subjected to structural analysis, and the obtained carbon-hydrogen data are shown in Table 1.

[0081] Table 1 Assignment of carbon and hydrogen data in the NMR spectra of compounds 1–4

[0082]

[0083]

[0084] a Deuterated chloroform was used as solvent. b Deuterated methanol is used as solvent. Continuing from the above table:

[0085]

[0086] a Deuterated chloroform was used as solvent. b Deuterated methanol was used as solvent.

[0087] Combined with the carbon and hydrogen data of compounds 1-4, the structural analysis of the above compounds 1-4 was performed, and the physical and chemical property data obtained are as follows:

[0088] Compound 1: colorless crystals; 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150MHz), such as Figure 1 –2; HRESIMS [M+H] + m / z 379.2386 (calcd.for C 24 H 31 N2O,2379.2386);IRν max 3434,2927,1701,1632cm –1 ; UV(MeOH)λ max (logε)204.0nm,298.5nm,such as Figure 3 –5.

[0089] Compound 2: colorless crystals; 1 H NMR (CD3OD, 600 MHz) and 13 C NMR (CD3OD, 150 MHz), such as Figure 6 –7; HRESIMS [M+Na] + m / z 417.2154 (calcd.for C 24 H 30 N2O3Na,417.2156);IRν max 3439,2950,2926,2140,1699,1653,1632,1507,1438,1384,1352,1326,1187,1069,1043cm –1 ; UV(MeOH)λmax (logε)203.0nm,298.0nm,such as Figure 8 –10.

[0090] Compound 3: colorless crystals; 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150MHz), such as Fig.11 –12; HRESIMS [M+Na] + m / z 417.2154 (calcd.for C 24 H 30 N2O3Na,417.2153);IRν max 3433,2958,2924,2854,2140,1703,1637,1463,1383,1263,1189,1081,1026,968,804,559cm –1 ; UV(MeOH)λ max (logε)233.5nm,291.5nm,such as Fig.13 –15.

[0091] Compound 4: white solid; 1 H NMR (CD3OD, 600 MHz) and 13 C NMR (CD3OD, 150 MHz), such as Fig.16 –17; HRESIMS [M+H] + m / z 368.2226 (calcd.for C 23 H 30 NO3,368.2226);IRν max 3427,2928,2872,1702,1648,1630,1438,1384,1351,1254,1115,1066,1030,763,584,567cm –1 ; UV(MeOH)λ max (logε)204.0nm,299.5nm,such as Fig.18 –20.

[0092] Experimental example: Anti-EV71 activity experiment of Acer truncatum alkaloid compounds 1–4

[0093] 1. Experimental Materials

[0094] (1) Main reagents: Ribavirin (Rb) was purchased from Selleck; DMEM (high glucose) medium was purchased from Sangon Biotechnology Co., Ltd.; penicillin-streptomycin was purchased from Nanjing Shenghang Biotechnology Co., Ltd.; fetal bovine serum (FBS) was purchased from ZETAlife; 0.25% trypsin (containing EDTA) and CCK-8 were purchased from Suzhou New Saimei Biotechnology Co., Ltd.

[0095] (2) Test cells and virus strains: RD cells and EV71 strain were from Shanghai Institute of Major Infectious Diseases and Biosafety.

[0096] 2. Test methods and results

[0097] First, all compounds 1–4 were preliminarily screened for activity, and the compounds with initial screening activity better than the positive control drug were rescreened and their EC 50 curve, obtain EC 50 value.

[0098] 2.1. Preliminary screening

[0099] RD cells were plated in a 96-well cell culture plate overnight. When the cells proliferated to about 90% in DMEM (high glucose) medium, the test compounds 1–4 (25 μg / mL) were fully mixed with EV71 virus dilution (MOI = 0.1), added to the cell wells and cultured for 2 hours, then the cell supernatant was aspirated and the cells were washed. Fresh medium containing the corresponding concentration of the compound was replaced and cultured for another 48 hours. CCK-8 was used to detect cell activity, that is, 10 μL of CCK-8 reagent was added to each well, incubated at 37°C for 1.5 hours, and then the OD value at a wavelength of 450 nm was measured using an enzyme reader. Three replicate wells were set for each sample, and a cell control well without drug and a virus infection control well were also set. Cell survival rate (%) of the drug-treated group = (OD 450nm – OD of virus control wells 450nm / cell control well OD 450nm – OD of virus control wells 450nm )×100%. Graph PadPrism9.5.1 software was used for drawing.

[0100] like Fig.21 As shown in the figure, when the concentration of compounds 3 and 4 was 25 μg / mL, the cell survival rate was greater than 50% after EV71 (MOI = 0.1) infection for 48 hours, and the antiviral effect was better than that of the positive control drug Ribavirin, indicating that they have certain anti-EV71 virus activity. The subsequent screening will be carried out and the EC 50 Although compounds 1 and 2 also have certain anti-EV71 activity, their activity is less than that of the positive control drug Ribavirin, so they are not rescreened.

[0101] 2.2. Rescreening

[0102] RD cells were plated in a 96-well cell culture plate overnight. When the cells proliferated to about 90%, the test compounds 3 and 4 (25 μg / mL) were fully mixed with the virus dilution solution (MOI=0.1), added to the cell wells and cultured for 2 hours, then the cell supernatant was discarded, the cells were washed, and the fresh culture medium containing the corresponding concentration of the compound was replaced. The culture was continued for 48 hours, and the cell activity was detected using CCK-8, that is, 10 μL of CCK-8 reagent was added to each well, and the OD value at a wavelength of 450 nm was measured using an enzyme marker after incubation at 37°C for 1.5 hours. Three replicate wells were set for each sample, and a cell control well without drug and a virus infection control well were set at the same time. Cell survival rate (%) of the drug-treated group = (OD 450nm – OD of virus control wells 450nm / cell control well OD 450nm – OD of virus control wells 450nm ) × 100%. Graph PadPrism 9.5.1 software was used for drawing.

[0103] like Fig. 22 As shown in Figure 24, after EV71 infection (MOI = 0.1) for 48 h, the EC 50 The EC values ​​of compounds 3 and 4 were 2.826 μg / mL, 3.783 μg / mL, and 65.770 μg / mL, respectively. 50 They are smaller than ribavirin, indicating that they have better anti-EV71 virus activity.

[0104] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A compound of alkaloids from Rhizoma Cyperi, characterized in that: The oxalicum alkaloid compound comprises at least one of compounds 1-4, and the structural formulas of compounds 1-4 are shown below:

2. The Herba Lysimachiae alkaloids compound according to claim 1, characterized in that: The oxalicum alkaloid compound comprises at least one of compounds 3 and 4, and the structural formulas of compounds 3 and 4 are as follows:

3. Use of the Achyranthes bidentata alkaloid compound according to claim 1 or 2 in the preparation of anti-EV71 virus drugs or drugs for preventing and treating hand, foot and mouth disease in infants and young children induced by EV71 virus.

4. An anti-EV71 drug, characterized in that: The anti-EV71 drug uses at least one of compounds 1-4 as an active ingredient, and the structural formulas of compounds 1-4 are shown below:

5. An anti-EV71 drug according to claim 4, characterized in that: The anti-EV71 drug uses at least one of compounds 3 and 4 as an active ingredient, and the structural formulas of compounds 3 and 4 are as follows:

6. An anti-EV71 drug according to claim 4 or 5, characterized in that: The anti-EV71 drug also includes pharmaceutically acceptable excipients.

7. An anti-EV71 drug according to claim 4 or 5, characterized in that: The dosage forms of the anti-EV71 drug include tablets, granules, capsules, pills, sustained-release preparations, oral liquid preparations, powder for injection, and injections.

8. An anti-EV71 drug according to claim 4 or 5, characterized in that: The administration methods of the anti-EV71 drug include oral administration and injection.

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