WO2017133468A1

WO2017133468A1 - Pulchinenoside and application as inhibitor of ev71 virus

Info

Publication number: WO2017133468A1
Application number: PCT/CN2017/071656
Authority: WO
Inventors: 刘琦
Original assignee: 刘琦
Priority date: 2016-02-02
Filing date: 2017-01-19
Publication date: 2017-08-10
Also published as: CN105535004A; CN105535004B

Abstract

A pulchinenoside and application thereof for preparing a pharmaceutical product for treating an EV71 virus infection or a disease caused by the EV71 virus infection (such as hand-foot-and-mouth disease, an issue of central nervous system activity or neurogenic pulmonary edema).

Description

Use of Pulsatilla saponins as EV71 virus inhibitors

Technical field

The invention belongs to the field of medical technology. In particular, the present invention relates to the use of a Pulsatilla saponin compound for the treatment of an EV71 virus infection or a disease caused by an EV71 virus infection (for example, hand, foot and mouth disease, central nervous system damage or neurogenic pulmonary edema). The present application also relates to the use of a Pulsatilla saponin for the preparation of a medicament for the treatment of an EV71 virus infection or a disease caused by an EV71 virus infection.

Background technique

EV71 virus (Enterovirus 71, enterovirus type 71) is a highly pathogenic enterovirus belonging to the family Picornaviridae and Enterovirus. EV71 virus is one of the main pathogens of hand-foot-mouth disease (HFMD), which is mainly transmitted by fecal-oral route, airborne transmission and contact sexual transmission. Hand, foot and mouth disease is an acute infectious disease caused by enterovirus that is harmful to infants and young children under 5 years of age. Since it was included in the national epidemic reporting system in 2008, there have been more than 1 million cases of hand, foot and mouth disease reported annually. By the end of 2015, the country had reported nearly 14 million cases of hand, foot and mouth disease, and more than 3,300 infant and child deaths. The incidence rate ranked first among the legal infectious diseases, and the case fatality rate ranked the top 5. Therefore, hand, foot and mouth disease has had a major impact on social stability and economic development, and has also caused widespread concern. The main manifestations of hand, foot and mouth disease are fever, oral mucosal ulcerative herpes and blister-like rash at the extremities. Individual patients have fatal complications such as myocarditis and pulmonary edema caused by the virus invading the heart and brain. In addition, EV71 virus infection also has obvious neurotropic, easy to infect the host nervous system, causing severe central nervous system damage such as aseptic cerebrospinal meningitis, polio-like paralysis, brainstem encephalitis, and severe cases. Has a high mortality rate. In recent years, the outbreak of EV71 virus in Asia is on the rise, and there is no specific drug at present. Therefore, the development of effective anti-EV71 virus drugs for the treatment of hand, foot and mouth disease is imminent.

Compared with traditional western medicines, which are expensive and have low antiviral activity, traditional Chinese medicine has the advantages of low price, wide drug source and remarkable activity. Some traditional Chinese herbal medicines have the functions of inhibiting viral replication, preventing viral cytopathic diseases, regulating immune function, improving pulmonary circulation, analgesic and anti-inflammatory effects. At present, with the improvement of screening and separation technology, searching for ingredients with antiviral activity from traditional Chinese medicine has become one of the important ways to develop antiviral drugs.

A large number of in vitro antiviral screening and clinical studies have found that some compound and single-flavor Chinese medicines have shown certain anti-EV71 virus activity. Studies have shown that Qingkailing, Shuanghuanglian and many other traditional Chinese medicine compounds and single-flavor drugs can alleviate the disease and shorten the course of EV71 hand-foot-mouth disease (Song Chunmei, Inner Mongolia Traditional Chinese Medicine, 2010.29(4); Guo Xiaoyuan, Chinese Medicine Emergency, 2009. 18(9); Chen Yonghong, Chinese Journal of Integrated Traditional Chinese and Western Medicine, 2006.13(1)). The Shuanghuanglian powder for injection is mainly made from forsythia, silver flower and astragalus, and has the effect of cooling, detoxifying and clearing heat. Yi Shihong et al.'s in vitro antiviral activity against Shuanghuanglian powder injection showed that it has the effect of inhibiting EV71 virus and has certain curative effect in the clinical application of Hand, Foot and Mouth Disease (Yi Shihong et al., Journal of Bethune Medical University) , 2001.27(5)). Zhang Wen et al. randomly divided 79 children with hand, foot and mouth disease diagnosed as pediatric hand, foot and mouth disease. The control group was given ribavirin granules. The observation group was given Qingkailing granules. The efficacy and side effects of the two groups were observed. There was no significant difference in the efficacy between the two groups, but the incidence of adverse reactions in the control group was significantly higher than that in the treatment group, indicating that Qingkailing Granules are better than ribavirin in the treatment of hand, foot and mouth disease in children (Zhang Wen et al., Chinese Medicine) Herald, 2008.5(7)). One of the main components of the geranium plant, the old Chinese herb, is the ancient Chinese medicine "Zihuazi Benjing", "Compendium of Materia Medica" and "Minnan Materia Medica" all have its effects of clearing away heat and detoxification. Modern pharmacology shows its antiviral, antibacterial and liver protection effects. Experiments show that geranium has better anti-EV71 effect in vivo and in vitro (Zhang He, SME Management and Technology, 2008 (13)).

Pulsatilla is a dry root of the genus Pulsatilla chinensis (Bunge Regel) of the Ranunculaceae family. It has the effects of clearing away heat and detoxifying, cooling blood and stopping phlegm. The current study found that the main pharmacological effects of Pulsatilla chinensis include antibacterial action, anti-amebic effect, anti-cancer effect, spermicidal action and sedative analgesia. The main active ingredients of Pulsatilla chinensis include Pulsatilla and Pulsatilla saponins, among which Pulsatilla saponins can be obtained by water extraction or alcohol extraction. At present, it has been found that Pulsatilla saponins have the functions of enhancing immune function, anti-inflammatory, anti-tumor and anti-pathogenic microorganisms (Mimaki, Y. et al., J Nat Prod, 1999. 62(9): p.1279-83; Liu, T Et al., Int J Biol Macromol, 2013.54: p. 225-9), has received extensive research and attention, but there have been no reports of antiviral activity of Pulsatilla saponins.

Summary of the invention

In the present invention, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise stated. Moreover, the steps of cell culture, biochemistry, cell biology and the like used herein are conventional steps widely used in the corresponding fields. Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the terms "EV71" or "EV71 virus" are used interchangeably herein to refer to a small RNA enterovirus capable of causing diseases such as hand, foot and mouth disease, central nervous system damage, and the like. Those skilled in the art understand that the EV71 virus includes multiple genotypes, and thus in the present invention, the term "EV71" or "EV71 virus" includes the A, B1-B5, C1-C5 subtypes.

As used herein, the term "sugar residue" refers to a residue resulting from the removal of a group, such as hydrogen (-H) or hydroxyl (-OH), from a monosaccharide, oligosaccharide or polysaccharide. In the present invention, the term "sugar residue composed of n monosaccharide units" means a residue derived from a sugar containing n monosaccharide units (n is an integer of 1 or more). When the sugar residue contains at least 2 monosaccharide units (i.e., n is an integer greater than or equal to 2), the n monosaccharide units may be linked together by glycosidic linkages. Further, the sugar residue is not limited by the order in which the monosaccharide units are arranged and the type of glycosidic bond formed between the respective monosaccharide units.

As used herein, the term "pharmaceutically acceptable salt" means that an acidic functional group (eg, -COOH, -OH, etc.) present in a compound provided by the present invention is formed with a suitable inorganic or organic cation (base). Salts, and include, but are not limited to, alkali metal salts such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, etc.; other metal salts such as aluminum salts, iron salts, zinc salts, Copper salt, nickel salt, cobalt salt, etc.; inorganic base salt, such as ammonium salt; organic base salt, such as t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester Salt, ethylenediamine salt, N-methylglucamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloropu Rucaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine salt, tris(hydroxymethyl)aminomethane salt.

As used herein, the term "pharmaceutically acceptable ester" refers to an ester of -OH present in a compound provided herein with an appropriate acid (eg, a carboxylic acid or an oxy-containing mineral acid), or An ester of -COOH and a suitable alcohol present in the compounds provided herein. Suitable ester groups include, but are not limited to, formate, acetate, propionate, butyrate, acrylate, ethyl succinate, hard fatty acid ester or palmitate.

As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, an effective amount for treating a disease (eg, EV71 infection) means that the treatment process is sufficient to cure or at least partially arrest the disease and complications of the patient already suffering from the disease, improve one or more clinical symptoms, or achieve the desired biological consequences. The dose or amount. Determination of such an effective amount is well within the capabilities of those skilled in the art. For example, the particular dosage for any particular patient depends on a number of factors, including the condition being treated and the severity of the condition; the age, weight, general health, sex, and daily diet of the patient; time of administration, route of administration And the rate of excretion of the particular compound used; the duration of treatment; the drug used in conjunction with or concurrent with the particular compound employed; and the subjective judgment of the treating physician within the scope of sound medical judgment. In certain embodiments, an effective amount is also one in which the therapeutically beneficial effect is greater than any toxic or detrimental effect.

The inventors of the present application have found for the first time that Pulsatilla saponins can inhibit infection caused by EV71 virus. Apoptosis, with significant anti-EV71 virus activity, can be used to treat EV71 infection or diseases caused by EV71 infection. Prior to this application, anti-EV71 viral activity of such compounds has not been reported.

Accordingly, in one aspect, the present invention provides the use of a compound of the formula (I) or a pharmaceutically acceptable salt or ester thereof for the treatment of a disease caused by EV71 infection or infection by EV71, or for the preparation of a treatment Use of a drug for EV71 infection or a disease caused by EV71 infection;

among them,

R ₁ is hydrogen or a sugar residue consisting of n monosaccharide units;

R ₂ is hydrogen or a sugar residue composed of m monosaccharide units;

n is an integer, and 1 ≤ n ≤ 7, for example, n is 1, 2, 3, 4, 5, 6, or 7;

m is an integer, and 1≤n≤7, for example, m is 1, 2, 3, 4, 5, 6, or 7;

n+m≤7.

In certain preferred embodiments, the monosaccharide units are each independently selected from the group consisting of glucose, rhamnose or arabinose.

In certain preferred embodiments, R ₁ is hydrogen or a saccharide residue consisting of 1, 2 or 3 monosaccharide units; for example, R ₁ is hydrogen, or an arabinose residue, or 1 Arab A sugar residue composed of a sugar and one rhamnose monosaccharide unit, or a sugar residue composed of one arabinose, one rhamnose, and one glucose monosaccharide unit.

In certain preferred embodiments, R _{1 is} selected from the group consisting of H-, α-L-ara-, α-L-rha-(1→2)-α-L-ara- or β-D-glc-(1 →4)-[α-L-rha-(1→2)]-α-L-ara-.

In certain preferred embodiments, R ₂ is hydrogen or a saccharide residue consisting of 1 or 3 monosaccharide units; for example, R ₂ is hydrogen, or R ₂ is a glucose residue, or R ₂ is 1 A sugar residue composed of rhamnose and two glucose monosaccharide units.

In certain preferred embodiments, R _{2 is} selected from H-, β-D-glc- or

α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-.

In certain preferred embodiments, R ₁ and R ₂ are selected from the group consisting of a combination of:

1) R ₁ is H-;

R ₂ is H- or β-D-glc-;

2) R ₁ is α-L-ara-;

R ₂ is H- or β-D-glc-;

3) R ₁ is α-L-rha-(1→2)-α-L-ara-;

R ₂ is H- or α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-;

4) R ₁ is β-D-glc-(1→4)-[α-L-rha-(1→2)]-α-L-ara-;

R ₂ is H-.

In certain preferred embodiments, the compound is selected from the compounds in Table 1.

Table 1.

In certain preferred embodiments, the disease caused by EV71 infection is hand, foot and mouth disease, central nervous system damage (eg, aseptic cerebrospinal meningitis, polio-like paralysis, or brainstem encephalitis) Or neurogenic pulmonary edema.

In another aspect, the present invention provides a method of treating an EV71 infection or a disease caused by EV71 infection, comprising administering to a subject in need thereof an effective amount of a compound of the formula (I) or a pharmaceutically acceptable salt or ester;

among them,

R ₁ is hydrogen or a sugar residue consisting of n monosaccharide units;

R ₂ is hydrogen or a sugar residue composed of m monosaccharide units;

n is an integer, and 1 ≤ n ≤ 7, for example, n is 1, 2, 3, 4, 5, 6, or 7;

m is an integer, and 1≤n≤7, for example, m is 1, 2, 3, 4, 5, 6, or 7;

n+m≤7.

In certain preferred embodiments, R ₂ is hydrogen or a sugar residue of a monosaccharide or 3 units; for example, R ₂ is hydrogen, or a glucose residue, or consists of a rhamnose and 2 A sugar residue consisting of glucose monosaccharide units.

In certain preferred embodiments, R _{2 is} selected from H-, β-D-glc- or

α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-.

In certain preferred embodiments, R ₁ and R ₂ are a combination selected from the group consisting of:

1) R ₁ is H-;

R ₂ is H- or β-D-glc-;

2) R ₁ is α-L-ara-;

R ₂ is H- or β-D-glc-;

3) R ₁ is α-L-rha-(1→2)-α-L-ara-;

R ₂ is H- or α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-;

4) R ₁ is β-D-glc-(1→4)-[α-L-rha-(1→2)]-α-L-ara-;

R ₂ is H-.

Optionally, the method further comprises administering to the subject an effective amount of a drug having antiviral activity (eg, ribavirin, cleavage or shuanghuanglian).

In certain preferred embodiments, the subject is a mammal, such as a human.

In certain preferred embodiments, the compounds can be administered by a variety of methods known in the art, such as by oral, parenteral, rectal, pulmonary or topical administration. An exemplary route of administration is oral administration. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing And emulsifiers, such as ethanol, isopropanol, ethyl acetate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil) , peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof. Liquid dosage forms for oral administration in addition to inert diluents may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. The solid dosage form may contain, in addition to the active compound, a pharmaceutically acceptable inert excipient or carrier such as a filler such as lactose, sucrose, glucose, mannitol, starch, microcrystalline cellulose, galactose, crospovidone And calcium sulphate); binders (such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia); wetting agents (such as cetyl alcohol and glyceryl monostearate); disintegrants (such as agar, calcium carbonate, starch, alginic acid, sodium carboxymethylcellulose, sodium carboxymethyl starch); lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, laurel Sodium sulphate); and mixtures thereof. The compounds of the invention may also be administered by non-oral routes, such as parenteral administration. The dosage form for parenteral administration may be an injection preparation, including an injection solution, a sterile powder for injection or a concentrated solution for injection. In addition to the active compound, the injectable preparation may contain a pharmaceutically acceptable carrier such as sterile water, Ringer's solution, and isotonic sodium chloride solution, and may also contain suitable additives such as antioxidants, buffers, and the like depending on the nature of the drug. Bacteriostatic agent.

Advantageous effects of the invention

The inventors of the present application have found for the first time that Pulsatilla saponins can interfere with the infection process of EV71 virus, inhibit the apoptosis caused by EV71 virus infection, and have protective effects on EV71-infected mice, and have significant anti-EV71 virus activity. . Thus, Pulsatilla saponins can be used to treat diseases caused by EV71 infection or infection by EV71.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The various objects and advantageous aspects of the invention will be apparent to those skilled in the <

DRAWINGS

Figure 1 shows the results of analysis of RD cell apoptosis caused by EV71 virus infection using Annexin V/PI apoptosis assay in Example 2. Among them, the abscissa represents PI and the ordinate represents Annexin V. Result It is shown that with the prolonged infection time of EV71 virus, the proportion of apoptosis of RD cells increases.

Fig. 2 is a view showing the results of detecting the apoptosis ratio of RD cells by Annexin V/PI apoptosis assay after the treatment of RD cells with Pulsatilla saponin B4 and EV71 virus in Example 2. The results showed that Pulsatilla saponin B4 significantly interfered with the EV71 virus infection process and inhibited RD cell apoptosis caused by viral infection.

Fig. 3 shows the results of the cytopathic effect of the virus observed by an inverted fluorescence microscope after different concentrations of Pulsatilla saponin B4 were applied to RD cells infected with EV71 virus in Example 2. The results showed that Pulsatilla saponin B4 significantly ameliorated the cytopathic effect caused by EV71 virus infection.

Fig. 4 is a view showing the results of detecting the apoptosis ratio of RD cells by using Annexin V/PI apoptosis assay in different concentrations of Pulsatilla saponin B4 in RD cells infected with EV71 virus in Example 2. The results showed that Pulsatilla saponin B4 significantly inhibited apoptosis of RD cells caused by EV71 virus infection.

detailed description

The invention is described with reference to the following examples which are intended to illustrate, but not limit the invention.

The experiments and methods described in the Examples (e.g., molecular biology experimental methods and immunoassays) are carried out essentially according to conventional methods well known in the art and described in various references, unless otherwise indicated. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992). ), all of which are incorporated herein by reference. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially. The invention is described by way of example, and is not intended to limit the scope of the invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1. Preparation of Pulsatilla saponin B4.

Pulsatilla saponin B4 is commercially available or can be obtained by separation and purification from Pulsatilla chinensis. Methods for extracting Pulsatilla saponin B4 from Pulsatilla chinensis are known to those skilled in the art. In the present embodiment, the following exemplary methods are used. The Pulsatilla chinensis was pulverized into a coarse powder, and extracted twice with 50% ethanol under reflux for 1.5 h, filtered, and the filtrate was combined. The filtrate was concentrated under reduced pressure at 70 ° C to a non-alcoholic taste, filtered, and the filtrate was diluted with water to 0.5 g (raw dose) / mL, and adsorbed through D101 macroporous resin, followed by elution with water, 60% ethanol, 95% ethanol, and collected 60. % ethanol eluted fraction. The 60% ethanol eluted fraction was passed through an ODS reversed-phase silica gel column with a mixture of methanol and water (5:5-10:0). A gradient elution was carried out, and a 7:3 elution portion of methanol:water was collected to obtain a single component compound.

¹³ C-NMR data were as follows: ¹³ C-NMR (125 MHz, C ₅ D ₅ N) δ: aglycon moiety 39.4 (C-1), 26.6 (C-2), 81.3 (C-3), 43.8 (C- 4), 48.0 (C-5), 18.3 (C-6), 34.4 (C-7), 41.3 (C-8), 51.1 (C-9), 37.2 (C-10), 21.3 (C-11) ), 26.2 (C-12), 38.5 (C-13), 43.0 (C-14), 30.4 (C-15), 32.4 (C-16), 57.2 (C-17), 50.0 (C-18) , 47.6 (C-19), 150.2 (C-20), 31.0 (C-21), 37.0 (C-22), 64.1 (C-23), 13.9 (C-24), 17.1 (C-25), 16.6 (C-26), 15.1 (C-27), 175.1 (C-28), 110.2 (C-29), 19.6 (C-30); 3-linked sugar moiety 104.4 (Ara-1), 76.0 (Ara) -2), 74.7 (Ara-3), 69.4 (Ara-4), 65.6 (Ara-5), 101.8 (Rha-1), 72.5 (Rha-2), 72.7 (Rha-3), 74.3 (Rha- 4), 69.9 (Rha-5), 18.7 (Rha-6); 28-linked sugar moiety 95.4 (Glc-1), 74.3 (Glc-2), 78.5 (Glc-3), 71.0 (Glc-4), 78.2 (Glc-5), 69.6 (Glc-6), 105.3 (Glc-1'), 75.5 (Glc-2'), 76.6 (Glc-3'), 78.8 (Glc-4'), 77.3 (Glc- 5'), 61.5 (Glc-6'), 102.9 (Rha-1"), 72.7 (Rha-2"), 72.9 (Rha-3"), 74.1 (Rha-4"), 70.5 (Rha-5) ), 18.7 (Rha-6)).

Compared with the Pulsatilla saponin B4 standard (purchased in the China Food and Drug Control Institute, purity 99%, batch number 111766-200601), the compound was identified as Anemoside B4.

Example 2. Determination of anti-EV71 virus activity of Pulsatilla saponin B4 in vitro.

2.1 Establishment of an in vitro viral infection model

2.1.1 infection model

EV71 has significant muscle tropism and/or nerve tissue tropism and is susceptible to infection of muscle tissue and/or nerve tissue, so RD71-infected RD cells (human rhabdomyosarcoma cells) model can be used in vitro to simulate a hand-foot-mouth disease model.

2.1.2 Infection method

EV71 strain (strain name: SHAPHC695F/SH/CHN/10 (695F)), provided by Shanghai Public Health Clinical Center (SHPHC).

When the RD cells (purchased from ATCC) at a density of 1 × 10 ⁵ cells were seeded in 6-well plates until confluent cells (Confluence) about 80%, was added to each well EV71 virus (multiplicity of infection MOI = 1 RD cells were infected, and normal cultured RD cells not in contact with the virus were set as a control.

2.1.3 Detection of apoptosis induced by EV71 virus infection

The cytopathic effect (CPE) of the virus was observed by inverted fluorescence microscopy at 12h, 18h, 24h, 30h, and 36h after EV71 infection. After collecting the cells, Annexin V/PI staining was performed, and apoptosis of the cells was measured using a flow cytometer. The proportion of apoptosis is expressed as the proportion of Annexin V positive cells.

The test results are shown in Figure 1. In the multiplicity of infection of this example, as the infection time of EV71 virus is prolonged, the proportion of apoptosis of RD cells increases. By the 36th hour, most of the cells have been lysed, or contracted and rounded, or detached (not in apoptosis results). Calculate the cells that have been lysed). The results indicate that EV71 virus can cause significant apoptosis of RD cells.

2.2 Effect of Pulsatilla saponin B4 on EV71 virus infection process

Using the EV71 infection model described in 2.1.2, RD cells were seeded in 6-well plates, cells were infected with EV71 virus, and Pulsatilla saponin B4 was added to the culture system at a concentration of 50 ng/mL for 36 hours. . Among them, a total of four groups were established: the control group (normally cultured RD cells not exposed to virus), the B4 group (RD cells treated with Pulsatilla saponin B4 but not in contact with the virus), and the B4+EV71 group (contact with the virus) RD cells treated with Pulsatilla saponin B4 at the same time, and EV71 group (cells only in contact with virus). After the incubation, the cells were collected, subjected to Annexin V/PI staining, and the apoptosis of the cells was detected by flow cytometry, and the proportion of apoptosis was expressed as the proportion of Annexin V-positive cells.

The test results are shown in Figure 2. The addition of Pulsatilla saponin B4 to RD71 infected RD cells significantly inhibited RD cell apoptosis induced by viral infection, and Pulsatilla saponin B4 itself had no significant effect on the degree of apoptosis of normal cultured RD cells. The results showed that Pulsatilla saponin B4 could significantly interfere with the infection of RD cells by EV71 virus, thereby inhibiting the apoptosis caused by EV71 virus infection. Moreover, Pulsatilla saponin B4 had no obvious cytotoxicity to RD cells.

2.3 Effect of Pulsatilla saponin B4 on apoptosis induced by EV71 virus infection

Using the EV71 infection model described in 2.1.2, 18 hours after EV71 infection of RD cells, different concentrations (1ng/mL, 25ng/mL, 50ng/mL) of Pulsatilla saponin B4 were added to treat EV71-infected cells, and continue to cultivate. Among them, a total of 8 groups were established: 1) Blank/control group indicates: RD cells that were not normally contacted with virus and Pulsatilla saponin B4; 2) Groups B4-1, B4-25 or B4-50 respectively indicated: concentration used RD cells treated with 1 ng/mL, 25 ng/mL or 50 ng/mL of Pulsatilla saponin B4 but not in contact with virus; 3) B4-1+EV71, B4-25+EV71 and B4-50+EV71 groups respectively indicate: RD cells that have been contacted with virus treated with Pulsatilla saponin B4 at a concentration of 1 ng/mL, 25 ng/mL, or 50 ng/mL, respectively; 4) EV71 group: RD cells that were only contacted with the virus but not treated with Pulsatilla saponin B4. The cytopathic effect (CPE) of the virus was observed using an inverted fluorescence microscope. Then, the above groups of cells were collected, subjected to Annexin V/PI staining, and the apoptosis of the cells was detected by flow cytometry, and the proportion of apoptosis was expressed as the proportion of Annexin V-positive cells.

The test results are shown in Figure 3-4. RD cells appear after infection with EV71 virus compared to normal growth cells The typical cytopathic effects such as rounding, shedding, and aggregation are obvious, and the cytopathic effect caused by the virus is obviously alleviated after treatment with high concentration (25ng/mL and 50ng/mL) of Pulsatilla saponin B4. The cell morphology of uninfected cells treated with different concentrations of Pulsatilla saponin B4 was not significantly different from that of normal cultured RD cells. The above results indicated that Pulsatilla saponin B4 can effectively resist the cytopathic effect of EV71 virus infection, and There was no significant effect on cell viability (see Figure 3). Flow cytometry analysis showed consistent results with microscopic observation (see Figure 4). Middle and high concentrations (25 ng/mL and 50 ng/mL) of Pulsatilla saponin B4 significantly attenuated RD cell apoptosis caused by EV71 virus infection. It indicates that Pulsatilla saponin B4 has obvious protective effect on the infection of EV71 virus.

Example 3. Determination of anti-EV71 virus activity in vivo by Pulsatilla saponin B4.

3.1 Experimental animals: 2 day old ICR mice (Charles River Laboratories, Wilmington, MA, USA) with an average body weight of 1.6-1.8 grams.

3.2 Infection method: 2 days old mice were taken. After fasting for 5 hours, EV71 virus was infected by intraperitoneal injection. The virus titer was 10 ⁶ pfu, and the infectious dose was 10 μL/g bw (body weight, body weight).

3.3 Evaluation of the therapeutic effect of Pulsatilla saponin B4 on mice infected with EV71 virus

The 2-day-old mice were divided into 9 groups: negative group (uninfected virus group), model group (24 μl/g bw saline per day after infection), positive control group I (24 hours after infection) Peritoneal injection of ribavirin daily at a dose of 100 mg/kg bw/day), experimental group I (24 hours after infection with the virus, intraperitoneal injection of Pulsatilla saponin B 41 times a day at doses of 50, 100, 200 mg/kg bw/ Day), positive control group II (72 hours after infection with virus, daily intraperitoneal injection of ribavirin, the dose is 100mg/kg bw/day), experimental group II (72 hours after infection, daily intraperitoneal injection of Pulsatilla saponin B41 times The doses were 50, 100, 200 mg/kg bw/day, respectively. There were 12 experimental animals in each group, and the administration volume was 10 μL/g b.w. The day of infection with EV71 by intraperitoneal injection was recorded as day 0.

Two days after the virus infection, all the animals in the model group had obvious hindlimbs on one side of the hind limbs, crawling slightly around the circle, and the diet was normal. The symptoms of the experimental group I were not obvious. After 3 days of infection, all the animals in the model group and the experimental group II had stiff hind limbs, which were slightly difficult to crawl, slowed down, and the body weight was negatively decreased. The size of the individual was significantly different, and there were still food residues in the stomach. In the low-dose group (50 mg/kg bw/day), 5 of the hind limbs were stiff, and the symptoms of the other dose groups were not obvious. The above results indicate that Pulsatilla saponin B4 has obvious protective effect on EV71-infected mice, and can prevent or alleviate the symptoms of limb stiffness and even paralysis caused by EV71 virus infection.

The statistical results of the number of surviving mice in each group are shown in Tables 1 and 2. After 7 days of virus infection, all animals in the model group and the positive control group died; in the low dose group of experimental group I, 1 animal survived, but one side hind limb was stiff; in the middle dose group of experimental group I, 6 animals survived, but The average body weight was only half of the negative group. Four animals showed one side hind limb stiffness. The two animals had stiff hind limbs, the forelimbs were normal, and the amount of residual food in the stomach was small. In the high dose group of the experimental group I, 9 animals survived. There were no significant differences between the weight of the 6 animals and the negative group. Two animals showed mild stiffness on one side of the hind limbs and light weight. One animal showed obvious symptoms of hindlimb on one side. In the experimental group II, only the high dose group survived. There were 4 animals, and the hind limbs were obviously stiff, and the body weight was only about half of the negative group. The above results indicate that Pulsatilla saponin B4 can significantly improve the survival rate of mice infected with EV71 virus, and can be used for treating EV71 virus infection and diseases caused by EV71 virus infection.

Table 1: Viability statistics of mice in each group treated with Pulsatilla saponin B4 24 hours after viral infection (experimental group I)

Table 2: Viability statistics of mice in each group treated with Pulsatilla saponin B4 after 72 hours of viral infection (Experimental Group II)

While the invention has been described in detail, it will be understood by the . The invention is generally divided into the appended claims and any equivalents thereof.

Claims

The use of the compound of the formula (I) or a pharmaceutically acceptable salt or ester thereof for the treatment of a disease caused by EV71 infection or infection by EV71, or for the preparation of a disease caused by infection or infection by EV71 Use of the drug;

among them,

R 1 is hydrogen or a sugar residue consisting of n monosaccharide units;

R 2 is hydrogen or a sugar residue composed of m monosaccharide units;

n is an integer, and 1 ≤ n ≤ 7, for example, n is 1, 2, 3, 4, 5, 6, or 7;

m is an integer, and 1≤n≤7, for example, m is 1, 2, 3, 4, 5, 6, or 7;

n+m≤7;

Preferably, the monosaccharide units are each independently selected from the group consisting of glucose, rhamnose or arabinose.
The use of claim 1 wherein

R 1 is hydrogen or a sugar residue consisting of 1, 2 or 3 monosaccharide units;

For example, R 1 is hydrogen, or

For example, R 1 is an arabinose residue, or

For example, R 1 is a sugar residue consisting of one arabinose and one rhamnose monosaccharide unit; or

For example, R 1 is a sugar residue consisting of one arabinose, one rhamnose, and one glucose monosaccharide unit;

Preferably, R 1 is selected from the group consisting of H-, α-L-ara-, α-L-rha-(1→2)-α-L-ara- or

β-D-glc-(1→4)-[α-L-rha-(1→2)]-α-L-ara-.
The use of claim 1 or 2, wherein

R 2 is hydrogen or a sugar residue consisting of one or three monosaccharide units;

For example, R 2 is hydrogen, or

For example, R 2 is a glucose residue, or

For example, the sugar residue R 2 by one rhamnose and 2 units of glucose monosaccharide;

Preferably, R 2 is selected from H-, β-D-glc- or

α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-.
The use according to any one of claims 1 to 3, wherein R 1 and R 2 are a combination selected from the group consisting of:

1) R 1 is H-;

R 2 is H- or β-D-glc-;

2) R 1 is α-L-ara-;

R 2 is H- or β-D-glc-;

3) R 1 is α-L-rha-(1→2)-α-L-ara-;

R 2 is H- or α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-;

4) R 1 is β-D-glc-(1→4)-[α-L-rha-(1→2)]-α-L-ara-;

R 2 is H-.
The use according to any one of claims 1 to 4, wherein the compound has a chemical formula selected from the group consisting of:
The use according to any one of claims 1 to 5, wherein the disease caused by the infection with EV71 is hand, foot and mouth disease, central nervous system damage (for example, aseptic cerebrospinal meningitis, polio-like paralysis, or brain) Dry encephalitis) or neurogenic pulmonary edema.
A method for treating a disease caused by EV71 infection or infection by EV71, which comprises administering to a subject in need thereof an effective amount of a compound of the formula (I) or a pharmaceutically acceptable salt or ester thereof;

among them,

R 1 is hydrogen or a sugar residue consisting of n monosaccharide units;

R 2 is hydrogen or a sugar residue composed of m monosaccharide units;

n is an integer, and 1 ≤ n ≤ 7, for example, n is 1, 2, 3, 4, 5, 6, or 7;

m is an integer, and 1≤n≤7, for example, m is 1, 2, 3, 4, 5, 6, or 7;

n+m≤7;

Preferably, the monosaccharide units are each independently selected from the group consisting of glucose, rhamnose or arabinose;

Preferably, R 1 is hydrogen or a sugar residue consisting of 1, 2 or 3 monosaccharide units;

For example, R 1 is hydrogen, or

For example, R 1 is an arabinose residue, or

For example, R 1 by a arabinose and a rhamnose sugar residue of monosaccharide units; or

For example, R 1 is a sugar residue consisting of one arabinose, one rhamnose, and one glucose monosaccharide unit;

Preferably, R 1 is selected from the group consisting of H-, α-L-ara-, α-L-rha-(1→2)-α-L-ara- or

β-D-glc-(1→4)-[α-L-rha-(1→2)]-α-L-ara-;

Preferably, R 2 is hydrogen or a sugar residue consisting of 1 or 3 monosaccharide units;

For example, R 2 is hydrogen, or

For example, R 2 is a glucose residue, or

For example, R 2 is a sugar residue consisting of 1 rhamnose and 2 glucose monosaccharide units;

Preferably, R 2 is selected from H-, β-D-glc- or

α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-;

Preferably, R 1 and R 2 are a combination selected from the group consisting of:

1) R 1 is H-;

R 2 is H- or β-D-glc-;

2) R 1 is α-L-ara-;

R 2 is H- or β-D-glc-;

3) R 1 is α-L-rha-(1→2)-α-L-ara-;

R 2 is H- or α-L-rha-(1→4)-β-D-glc-(1→6)-β-D-glc-;

4) R 1 is β-D-glc-(1→4)-[α-L-rha-(1→2)]-α-L-ara-;

R 2 is H-;

Preferably, the compound has a chemical formula selected from the group consisting of:

Optionally, the method further comprises administering to the subject an effective amount of a drug having antiviral activity (eg, ribavirin, cleavage or shuanghuanglian);

Preferably, the disease caused by infection with EV71 is hand, foot and mouth disease, central nervous system damage (eg, aseptic cerebrospinal meningitis, polio-like paralysis, or brainstem encephalitis) or neurogenic pulmonary edema ;

Preferably, the subject is a mammal, such as a human.