Application of golden flower cold-clearing granules or active ingredients thereof in preparation of anti-enterovirus medicines

Abstract

The invention provides application of golden flower cold-clearing granules or active ingredients thereof in preparation of medicines for preventing, relieving and / or treating diseases caused by enterovirus infection. The golden flower cold-clearing granules and the effective components of the burdock phenol A and the 5, 7, 3 '-trihydroxy-6, 4', 5 '-trimethoxyflavone have good inhibitory activity on type 1 poliomyelitis virus, coxsackie virus group A type 9, enterovirus type 71, echovirus type 11 and coxsackie virus group B type 3, and the golden flower cold-clearing granules and the effective components of the burdock phenol A and the 5, 7, 3'-trihydroxy-6, 4 ', 5'-trimethoxyflavone have good inhibitory activity on type 1 poliomyelitis virus. Therefore, the golden flower cold-clearing granules or the effective components thereof have important clinical application value in treatment of diseases infected with enteroviruses.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to the use of Jinhua Qinggan granules or their active ingredients in the preparation of drugs for preventing, alleviating and / or treating diseases caused by enterovirus infection. Background Art

[0002] Enteroviruses are single-stranded, positive-sense RNA viruses belonging to the Picornaviridae family and the Enterovirus genus. They are highly infective and cause a high rate of morbidity. Enterovirus particles are spherical, icosahedral, and approximately 24 to 30 nm in diameter. They lack an envelope and protrusions, making them highly resilient to the environment and tolerant to both gastric acid and ethanol.

[0003] Enteroviruses primarily include poliovirus (PV), coxsackievirus (CV), echovirus, and the newer digital enteroviruses (including enteroviruses 68-71, 73-91, and 100-101). Enteroviruses are among the leading pathogens of childhood infection, causing a wide range of illnesses, ranging from mild symptoms such as respiratory and gastrointestinal infections, herpetic pharyngitis, and hand, foot, and mouth disease in children, to more severe conditions such as pleurisy, hepatitis, myocarditis, aseptic meningitis, and neonatal sepsis.

[0004] Poliovirus (PV) primarily affects children under five years of age, but anyone of any age who is not vaccinated can contract the disease. One in every 200 cases of infection causes irreversible paralysis. Of these cases, 5-10% of patients die from respiratory muscle paralysis. Despite the World Health Assembly's 1988 resolution to eradicate polio worldwide, two countries remain endemic.

[0005] Enterovirus 71 is a virus that can cause hand, foot, and mouth disease and central nervous system infections. It's primarily transmitted through fecal-oral transmission, and infection can result in low-grade fever, mouth pain, vomiting, and diarrhea. Because EV71 is highly infectious to the central nervous system, clinical symptoms include aseptic meningitis, myocarditis, and muscle stiffness. Although asymptomatic or mildly symptomatic patients recover naturally and develop antibodies, those who develop severe EV71 infection still have a high risk of death from cardiopulmonary failure and extensive brainstem damage.

[0006] CV-A9, CVB1-B6, and echoviruses are the primary pathogens of aseptic meningitis and several acute illnesses. Among them, coxsackievirus A9 (CV-A9) and coxsackievirus B3 (CVB3) can cause a variety of clinical symptoms, including aseptic meningitis, hand, foot, and mouth disease (HFMD), acute flaccid paralysis, persistent diarrhea, myocarditis, and aseptic meningitis. Furthermore, CVB3 can cause type 1 diabetes through persistent infection and inflammation of pancreatic beta cells. Echoviruses are classified into over 30 subtypes, with type 11 (E11) being the most common. E11 causes a wide spectrum of illnesses. Some individuals infected with E11 may not experience any clinical symptoms, representing an insidious infection. Those who do experience clinical symptoms generally have mild symptoms, such as rash, fever, and cough. In some individuals, infection can lead to hand, foot, and mouth disease (HFMD), and in very rare cases, enteroviral uveitis, aseptic meningitis, encephalitis, and acute flaccid paralysis. When newborns are infected with E11, the digestive, nervous and cardiovascular systems are easily affected, which can cause severe jaundice, liver failure, shock, multi-system bleeding and other symptoms.

[0007] The Jinhua Qinggan Recipe, developed by a group of experts based on references to classic Chinese medicine texts such as "Treatise on Febrile Diseases," "Treatise on Epidemic Diseases," and "Treatise on Warm Diseases," along with years of clinical experience, is a treatment for influenza A (H1N1). It primarily contains ingredients such as jasmine flower, honeysuckle, and forsythia. In preclinical and clinical trials, the Jinhua Qinggan Recipe demonstrated significant efficacy in suppressing influenza A (H1N1). Currently, Jinhua Qinggan Granules are approved for clinical use in the treatment of influenza.

[0008] Furthermore, Jinhua Qinggan Granules have demonstrated promising therapeutic effects in patients infected with the novel coronavirus. Clinical trial results have shown that Jinhua Qinggan Granules are a safe and effective traditional Chinese medicine formula that can alleviate symptoms in patients with mild COVID-19. They can be used to treat and alleviate symptoms such as fever, cough, and fatigue in patients infected with the novel coronavirus. As a complementary therapy, Jinhua Qinggan Granules can reduce hospitalization rates, shorten symptom recovery time, and improve patient psychological well-being.

[0009] However, there are currently no reports on the ability of Jinhua Qinggan Granules to inhibit enterovirus. Summary of the Invention

[0010] In order to solve the above technical problems, the primary purpose of the present invention is to provide the use of Jinhua Qinggan Granules in the preparation of a medicament for preventing, alleviating and / or treating diseases caused by enterovirus infection. Secondly, the present invention provides the use of arctiol A or 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone, the active ingredient of Jinhua Qinggan Granules, in the preparation of a medicament for preventing, alleviating and / or treating diseases caused by enterovirus infection.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides the use of Jinhua Qinggan Granules in any one of the following X1)-X4):

[0013] X1) Preparation of medicinal products for preventing and / or treating diseases or symptoms caused by enteroviruses;

[0014] X2) Preparation of medicaments for the prevention and / or treatment of enteroviral infections;

[0015] X3) Preparation of enterovirus inhibitors;

[0016] X4) preparing a medicament for inhibiting the proliferation of enterovirus;

[0017] The Jinhua Qinggan granules are composed of the following ingredients: honeysuckle, gypsum, honey ephedra, stir-fried bitter almonds, scutellaria, forsythia, thunbergia thunbergii, anemarrhena, burdock fruit, artemisia annua, mint, and licorice.

[0018] In one embodiment of the present invention, the enterovirus is enterovirus 71 (EV71), coxsackievirus A group 9 (CV-A9), coxsackievirus B group 3 (CVB3), poliovirus type 1 (PV1) or echovirus type 11 (E11).

[0019] In one embodiment of the present invention, the preparation method of the Jinhua Qinggan Granules is as follows: weighing Forsythia suspensa, Artemisia annua, and mint, adding water, distilling, and collecting volatile oil and the distilled aqueous solution respectively; weighing honeysuckle, gypsum, honey ephedra, stir-fried bitter almonds, Scutellaria baicalensis, Anemarrhena asphodeloides, and Licorice, decocting with water, filtering, collecting the filtrate, adding ethanol to precipitate, filtering and collecting the first filtrate; combining the first filtrate with the distilled aqueous solution, concentrating under reduced pressure to a clear paste with a relative density of 1.1 to 1.5, adding ethanol to precipitate, and filtering and collecting the supernatant; weighing Fritillaria thunbergii and Arctium lappa, adding ethanol for reflux extraction, filtering and collecting the second filtrate, combining the second filtrate with the supernatant, recovering ethanol under reduced pressure and concentrating to a clear paste with a relative density of 1.1 to 1.5, and drying to obtain a dry paste powder; and mixing the collected volatile oil and dry paste powder evenly.

[0020] In one embodiment of the present invention, Jinhua Qinggan Granules can be used as the sole active ingredient, or can be used as an active ingredient together with one, two or more other anti-enteroviral drugs.

[0021] Preferably, the other anti-enteroviral drugs are selected from acyclovir tablets, amoxicillin capsules, ribavirin capsules, oseltamivir, suramin sodium, pediatric Jiebiao oral solution, Xiyanping injection or berberine.

[0022] In one embodiment of the present invention, the diseases caused by enterovirus are hand, foot and mouth disease, herpangina, encephalitis, aseptic meningitis, acute weak limb paralysis, acute hemorrhagic conjunctivitis, myositis, muscle calcification, motor end plate abnormalities, pleurisy, myocarditis, pericarditis and hepatitis, etc.

[0023] In one embodiment of the present invention, the symptoms caused by enterovirus are headache, fever, vomiting, sneezing, coughing, rash or muscle stiffness.

[0024] In a second aspect, the present invention provides the use of arctiol A or 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone in any one of the following X1) to X4):

[0025] X1) Preparation of medicinal products for preventing and / or treating diseases or symptoms caused by enteroviruses;

[0026] X2) Preparation of medicaments for the prevention and / or treatment of enteroviral infections;

[0027] X3) Preparation of enterovirus inhibitors;

[0028] X4) preparing a medicament for inhibiting the proliferation of enterovirus;

[0029] The structural formula of the arctiol A is:

[0030] The structural formula of the 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone is:

[0031]

[0032] In one embodiment of the present invention, the enterovirus is enterovirus 71, coxsackievirus A group 9, coxsackievirus B group 3, poliovirus type 1, or echovirus type 11.

[0033] In one embodiment of the present invention, arctiol A or 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone can be used as the sole active ingredient, or can be used as an active ingredient together with one, two or more other anti-enteroviral drugs.

[0034] Preferably, the other anti-enteroviral drugs are selected from acyclovir tablets, amoxicillin capsules, ribavirin capsules, oseltamivir, suramin sodium, pediatric Jiebiao oral liquid, Xiyanping injection or berberine.

[0035] In one embodiment of the present invention, the diseases caused by enterovirus are hand, foot and mouth disease, herpangina, encephalitis, aseptic meningitis, acute weak limb paralysis, acute hemorrhagic conjunctivitis, myositis, muscle calcification, motor end plate abnormalities, pleurisy, myocarditis, pericarditis and hepatitis, etc.

[0036] In one embodiment of the present invention, the symptoms caused by enterovirus are headache, fever, vomiting, sneezing, coughing, rash or muscle stiffness.

[0037] In a third aspect, the present invention provides a pharmaceutical composition comprising Jinhua Qinggan granules or arctiol A or 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone for use in any one of the following X1) to X4):

[0038] X1) Preparation of medicinal products for preventing and / or treating diseases or symptoms caused by enteroviruses;

[0039] X2) Preparation of medicaments for the prevention and / or treatment of enteroviral infections;

[0040] X3) Preparation of enterovirus inhibitors;

[0041] X4) preparing a medicament for inhibiting the proliferation of enterovirus;

[0042] The structural formula of the arctiol A is:

[0043] The structural formula of the 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone is:

[0044]

[0045] The Jinhua Qinggan granules are composed of the following ingredients: honeysuckle, gypsum, honey ephedra, stir-fried bitter almonds, scutellaria, forsythia, thunbergia thunbergii, anemarrhena, burdock fruit, artemisia annua, mint, and licorice.

[0046] In one embodiment of the present invention, the enterovirus is enterovirus 71, coxsackievirus A group 9, coxsackievirus B group 3, poliovirus type 1, or echovirus type 11.

[0047] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0048] Preferably, the carrier includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred.

[0049] Preferably, conventional cosolvents, buffers, pH regulators, etc. may also be added to the pharmaceutical composition.

[0050] Preferably, colorants, preservatives, spices, flavorings, sweeteners or other materials are added to the pharmaceutical composition.

[0051] Preferably, the pharmaceutical composition is in the form of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, and lyophilized powder injections. The above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field. These dosage forms can be administered by injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; cavitary administration, such as rectal and vaginal; respiratory tract administration, such as nasal; and mucosal administration.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The present invention discovered for the first time that Jinhua Qinggan Granules have good inhibitory activity against enterovirus 71, coxsackievirus A group 9, coxsackievirus B group 3, poliovirus type 1, and echovirus type 11. Jinhua Qinggan Granules have an inhibitory effect on EV71, PV1, and CVB3 both before and after endocytosis; in addition, oral gavage treatment with Jinhua Qinggan Granules can reduce the viral load in various parts of the body (such as serum, heart, colon, liver, hind limb muscles, and spleen) of suckling mice infected with CV-A9, and can alleviate inflammatory responses. Therefore, Jinhua Qinggan Granules have important clinical application value for the treatment of diseases caused by infection with enterovirus 71, coxsackievirus A group 9, coxsackievirus B group 3, poliovirus type 1, and echovirus type 11.

[0054] Furthermore, the present invention also discovered that two compounds contained in Jinhua Qinggan Granules, arctiol A or 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone, exhibited an inhibition rate of greater than 90% at 20 μM against enterovirus 71, coxsackievirus A group 9, coxsackievirus B group 3, poliovirus type 1, and echovirus type 11, with no significant cytotoxicity. Furthermore, intraperitoneal injection of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone reduced viral loads throughout the body (e.g., serum, heart, colon, liver, hind limb muscle, and spleen) in suckling mice infected with CV-A9, and alleviated inflammatory responses. Therefore, these compounds have important implications for the future clinical use of enterovirus 71, coxsackievirus A group 9, coxsackievirus B group 3, poliovirus type 1, echovirus type 11, or other enteroviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The cells were cultured for 24 hours and infected with EV71 at MOI = 0.1 for 24 hours. The EC of Jinhua Qinggan Granules against EV71 in RD cells was 50 、CC 50 and SI.

[0056] Figure 2After 24 h of cell culture and 36 h of infection with CV-A9 at MOI = 0.001, the EC of Jinhua Qinggan Granules against CV-A9 in RD cells was 50 、CC 50 and SI.

[0057] Figure 3 The cells were cultured for 24 hours and infected with PV1 at MOI = 0.003 for 24 hours. The EC of Jinhua Qinggan Granules against PV1 in RD cells was 50 、CC 50 and SI.

[0058] Figure 4 The cells were cultured for 24 hours and infected with E11 at MOI = 0.005 for 24 hours. The EC of E11 in RD cells was 50 、CC 50 and SI.

[0059] Figure 5 The cells were cultured for 24 h and infected with CVB3 at MOI = 0.1 for 24 h. The EC of Jinhua Qinggan granules in Vero E6 cells was 50 、CC 50 and SI.

[0060] Figure 6 These are the experimental results of the dosing time of Jinhua Qinggan Granules at 1.25 mg / mL for EV71.

[0061] Figure 7 These are the experimental results of the dosing time of Jinhua Qinggan Granules at 2.5 mg / mL for CV-A9.

[0062] Figure 8 These are the experimental results of the dosing time of Jinhua Qinggan Granules at 1.25 mg / mL for PV1.

[0063] Figure 9 These are the experimental results of the dosing time of Jinhua Qinggan Granules at 2.5 mg / mL for E11.

[0064] Figure 10 These are the experimental results of the dosing time of Jinhua Qinggan Granules at 1.25 mg / mL for CVB3.

[0065] Figure 11 It is the inhibition rate of the effective anti-EV71 virus components in Jinhua Qinggan Granules.

[0066] Figure 12 It is the inhibition rate of the effective anti-CV-A9 virus components in Jinhua Qinggan Granules.

[0067] Figure 13 It is the inhibition rate of the effective anti-PV1 virus components in Jinhua Qinggan Granules.

[0068] Figure 14 It is the inhibition rate of the effective anti-E11 virus components in Jinhua Qinggan Granules.

[0069] Figure 15 It is the inhibition rate of the effective anti-CVB3 virus components in Jinhua Qinggan Granules.

[0070] Figure 16 The EC value of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone against EV71 in RD cells was calculated after 24 hours of cell culture and infection with EV71 at MOI = 0.1. 50 、CC 50 and SI.

[0071] Figure 17 After 24 h of cell culture and 36 h of infection with CV-A9 at MOI = 0.001, the EC value of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone on CV-A9 in RD cells was 50 、CC 50 and SI.

[0072] Figure 18 The EC value of arctiol A against CV-A9 in RD cells was 0.001 after 24 h of cell culture and 36 h after infection with CV-A9 at MOI = 0.001. 50 、CC 50 and SI.

[0073] Figure 19 The cells were cultured for 24 h and infected with PV1 at MOI = 0.003 for 24 h. The EC value of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone against PV1 in RD cells was 50 、CC 50 and SI.

[0074] Figure 20 The cells were cultured for 24 h and infected with E11 at MOI = 0.005 for 24 h. The EC value of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone on E11 in RD cells was 50 、CC 50 and SI.

[0075] Figure 21 The EC of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone in Vero E6 cells was 24 hours after infection with CVB3 at MOI = 0.1. 50 、CC 50 and SI.

[0076] Figure 22These are the experimental results of the dosing time of EV71 at 25 μM 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone.

[0077] Figure 23 These are the experimental results of the dosing time of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone at 25 μM for CV-A9.

[0078] Figure 24 The results of the arctiinol A dosing time experiment on CV-A9 at 30 μM.

[0079] Figure 25 These are the experimental results of the dosing time of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone at 12.5 μM on PV1.

[0080] Figure 26 These are the experimental results of the dosing time of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone at 12.5 μM for E11.

[0081] Figure 27 These are the experimental results of the dosing time of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone at 12.5 μM on CVB3.

[0082] Figure 28 The results are as follows: the effects of oral administration of 750 mg / kg Jinhua Qinggan granules and intraperitoneal injection of 6.25 mg / kg 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone on the body weight of mice.

[0083] Figure 29 These are the results of the inhibition of viral load in various tissues of mice by oral administration of 750 mg / kg Jinhua Qinggan Granules and intraperitoneal injection of 6.25 mg / kg 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone.

[0084] Figure 30 The results of the inhibition of inflammatory responses in various tissues of mice by oral administration of 750 mg / kg Jinhua Qinggan Granules and intraperitoneal injection of 6.25 mg / kg 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone. DETAILED DESCRIPTION

[0085] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0086] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0087] The Jinhua Qinggan granules used in the examples of the present invention are from Juxiechang (Beijing) Pharmaceutical Co., Ltd., exist in the form of granules, and are stored at room temperature. When used, they are prepared into a solution using a cell maintenance medium (DMEM medium containing 2% FBS).

[0088] The Genbank accession number of the amino acid sequence encoded by the full gene of enterovirus 71 used in the embodiment of the present invention is AEU10773.1, the Genbank accession number of the amino acid sequence of the protein encoded by the full gene of coxsackievirus group A type 9 is UJG81396.1, the Genbank accession number of the amino acid sequence of the protein encoded by the full gene of coxsackievirus group B type 3 is AEH42467.1, the Genbank accession number of the amino acid sequence encoded by the full gene of echovirus type 11 is QNS36366.1, and the Genbank accession number of the amino acid sequence encoded by the full gene of poliovirus type 1 is CAA24465.1.

[0089] Corresponding to the genome, the Genbank accession number of the full genome sequence of enterovirus type 71 is JN230523.1, the Genbank accession number of the full genome sequence of coxsackievirus group A type 9 is MW192795.1, the Genbank accession number of the full genome sequence of coxsackievirus group B type 3 is JN048468.1, the Genbank accession number of the full genome sequence of echovirus type 11 is MN817130.1, and the Genbank accession number of the full genome sequence of poliovirus type 1 is V01150.1.

[0090] The human rhabdomyosarcoma cell line RD was obtained from the American Type Culture Collection (ATCC, CRL-3216™).

[0091] Vero E6 African green monkey kidney cells were obtained from the American Type Culture Collection (ATCC, No. 1586).

[0092] The experimental mice C57BL / 6 were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. (certificate of conformity: SCXK (Beijing) 2019-0010). During the experiment, they were kept in an environment with a temperature of 22-24°C and a humidity of 45-55%, with alternating light / darkness (12 / 12 h). The mother mice had free access to food and water, and the pups were raised under the care of their mothers.

[0093] The following examples were processed using GraphPad-Prism 8.3.0 statistical software. The experimental results are expressed as mean values and T-test was used. ns indicates no difference (P>0.5), * indicates a significant difference (P<0.1), ** indicates a significant difference (P<0.01), *** indicates a very significant difference (P<0.001), and **** indicates an extremely significant difference (P<0.0001).

[0094] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0095] Example 1 Cell culture and virus culture

[0096] The human rhabdomyosarcoma cell line RD was cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen) at 37°C in a 5% CO2 incubator.

[0097] Vero E6 African green monkey kidney cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen) at 37°C in a 5% CO2 incubator.

[0098] EV71, CV-A9, PV1, and E11, members of the Picornaviridae family Enterovirus, were propagated in RD cells, and CVB3, a member of the Picornaviridae family Enterovirus, was propagated in Vero E6 cells. Plaque assay and TCID 50 Virus titer was determined by immunohistochemistry. All infection experiments were performed in a biosafety level 2 (BSL-2) laboratory.

[0099] Example 2 Real-time fluorescence quantitative PCR

[0100] In the present invention, the primers used in real-time fluorescence quantitative PCR are shown in Table 1, the SYBR-Green method amplification procedure is shown in Table 2, and the reaction system is shown in Table 3.

[0101] Table 1. Primer sequences used in the present invention

[0102]

[0103] Table 2. Dye-based qPCR amplification procedure

[0104]

[0105] Table 3. Dye method reaction system

[0106]

[0107] In the present invention, GraphPad-Prism 8.3.0 software was used for data analysis.

[0108] Example 3: Jinhua Qinggan Granules inhibit enterovirus EC 50 and CC 50 Determination

[0109] EC 50 Detection: When testing EV71, CV-A9, PV1 and E11, 2.5×10 4 RD cells were seeded into 96-well plates. When testing CVB3, 2.5×10 4 Vero E6 cells were seeded into 96-well plates and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0110] Jinhua Qinggan Granules were dissolved to 10 mg / mL in DMEM medium containing 2% FBS (i.e., maintenance medium), heated in a metal bath above 65°C for 10 minutes, and filtered using a 0.22 μM filter membrane. The drug was then diluted twice using cell maintenance medium to obtain 10 concentrations of Jinhua Qinggan Granules solution. Each concentration was inoculated into 2 wells (50 μl / well), and the virus dilution was added to the cell wells (50 μl / well) at the same time, so that the MOI of EV71 after entry was 0.1, the MOI of CV-A9 was 0.001, the MOI of PV1 was 0.003, the MOI of CVB3 was 0.1, and the MOI of E11 was 0.005.

[0111] Test EC 50 The drug inlet concentrations were 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.312 mg / mL, 0.156 mg / mL, 0.078 mg / mL, 0.039 mg / mL, 0.019 mg / mL, and 0.010 mg / mL, respectively. The test continued to be cultured for about 24 hours until the negative control group (i.e., the group to which the virus was added but no reagents were added) showed obvious lesions under the microscope and photographed. A blank group was also set up. The expression of intracellular viral RNA and the intracellular reference gene GAPDH was quantitatively detected by RT-qPCR. GraphPad-Prism 8 software was used for data analysis and calculation of EC 50 .

[0112] CC 50 Detection: CC was performed using the Cell Titer-Blue method 50Detection. RD cells and Vero E6 cells were seeded into 96-well cell culture plates, and the test was carried out when the cell density reached 60%-80%. After the RD cells and Vero E6 cells were replaced with the diluted drugs, the final concentrations were 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.312 mg / mL, 0.156 mg / mL, 0.078 mg / mL, 0.039 mg / mL, 0.019 mg / mL, and 0.010 mg / mL, and 2 wells were inoculated for each concentration. The cells were cultured at 37°C with 5% CO2 for 24 hours, and the luminescence intensity at 593 nm was detected using Cell Titer-Blue reagent. The data were analyzed and calculated using GraphPad-Prism8 software. 50 .

[0113] EC 50 It 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.

[0114] CC 50 It is the drug concentration that causes 50% of cells to become diseased. The higher the value, the lower the toxicity to cells.

[0115] SI: Selectivity index, CC 50 With EC 50 The larger the value, the higher the possibility of drug development.

[0116] Effect of Jinhua Qinggan Granules on EC of EV71 50 and CC 50 The results of the test are as follows Figure 1 As shown, the EC of CV-A9 50 and CC 50 The results of the test are as follows Figure 2 As shown, the EC of PV1 50 and CC 50 The results of the test are as follows Figure 3 As shown, EC of E11 50 and CC 50 The results of the test are as follows Figure 4 As shown, the EC of CVB3 50 and CC 50 The results of the test are as follows Figure 5 The left vertical axis in the figure represents the inhibition rate of Jinhua Qinggan Granules on viruses, the right vertical axis represents the toxicity of Jinhua Qinggan Granules on RD cells or Vero E6 cells, and the horizontal axis represents the concentration of Jinhua Qinggan Granules Log 10 (μM).

[0117] Figure 1 The results showed that Jinhua Qinggan Granules had an effect on the EC of EV71 in RD cells.50 =0.13mg / mL, CC 50 =5.21 mg / mL, SI=40.08; Figure 2 The results showed that Jinhua Qinggan Granules had an effect on the EC of CV-A9 on RD cells. 50 =0.27mg / mL, CC 50 =5.21 mg / mL, SI=19.30; Figure 3 The results showed that Jinhua Qinggan Granules could effectively inhibit the EC of PV1 in RD cells. 50 =0.15mg / mL, CC 50 =5.21 mg / mL, SI=37.73; Figure 4 The results showed that Jinhua Qinggan Granules had an effect on the EC of E11 in RD cells. 50 =0.33mg / mL, CC 50 =5.21 mg / mL, SI=15.79; Figure 5 The results showed that Jinhua Qinggan Granules could inhibit the EC of CVB3 in Vero E6 cells. 50 =0.33mg / mL, CC 50 =2.71 mg / mL, SI=8.24. For EV71, the drug concentration ranged from 0.31 to 5 mg / mL, exhibiting a strong antiviral effect (99%-90%); for CV-A9, the drug concentration ranged from 0.62 to 5 mg / mL, exhibiting a strong antiviral effect (99%-90%); for PV1, the drug concentration ranged from 0.62 to 5 mg / mL, exhibiting a strong antiviral effect (99%-90%); for E11, the drug concentration ranged from 1.25 to 5 mg / mL, exhibiting a strong antiviral effect (99%-90%); for CVB3, the drug concentration ranged from 1.25 to 2.5 mg / mL, exhibiting a strong antiviral effect (99%-90%). These results suggest that Jinhua Qinggan Granules are potential inhibitors against EV71, CV-A9, PV1, E11, and CVB3-infected cells.

[0118] Example 4: Experiment on the Dosage Time of Jinhua Qinggan Granules in Inhibiting Enterovirus

[0119] 2.5×10 cells were seeded in 24-well plates. 5 RD cells were infected with EV71 at an MOI of 0.1, CV-A9 at an MOI of 0.001, PV1 at an MOI of 0.003, and E11 at an MOI of 0.005 after 24 hours. Or 2.5×10 5Vero E6 cells were infected with CVB3 at an MOI of 0.1 24 hours later. Corresponding concentrations of Jinhua Qinggan Granules (Jinhua Qinggan Granules with a final concentration of 1.25 mg / mL for EV71, 2.5 mg / mL for CV-A9, 1.25 mg / mL for PV1, 2.5 mg / mL for E11, and 1.25 mg / mL for CVB3) were added to the three experimental groups during the entire time course (when the virus was added and after incubation for 2 hours), before entry into the cells (when the virus was added, the cells were treated with drugs for 2 hours, and then washed with PBS three times after 2 hours to absorb the unadsorbed free viruses and drugs), and after entry into the cells (after incubation for 2 hours). The cells were cultured for a further 12 hours, and the RNA in the cells in the culture wells was extracted. The viral replication in the cells and the expression of the cellular reference gene GAPDH were determined by RT-qPCR. At the same time, a drug-treated control group was also set up, in which no drug treatment was added during the entire virus infection process, and only a corresponding volume of DMEM culture medium containing 2% fetal bovine serum was added.

[0120] The results are as follows Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 and Figure 10 As shown. The bar graphs from left to right are the relative expression levels of viral mRNA in the control group, the entire time course, before and after EV71 entry. The results of the drug addition time experiment showed that Jinhua Qinggan Granules had an inhibitory effect on EV71 both before and after EV71 entry. Figure 6 ); Jinhua Qinggan Granules have an inhibitory effect on CV-A9 after it enters the cell ( Figure 7 ); Jinhua Qinggan Granules have an inhibitory effect on PV1 both before and after its entry into the cell ( Figure 8 ); Jinhua Qinggan Granules have an inhibitory effect on E11 mainly after it enters the cell ( Figure 9 ); Jinhua Qinggan Granules have an inhibitory effect on CVB3 both before and after its entry into the cell ( Figure 10 ).

[0121] Example 5: Screening of Anti-Enteroviral Effects of Monomeric Components of Jinhua Qinggan Granules

[0122] The compounds in Jinhua Qinggan Granules were analyzed by mass spectrometry, and 62 compounds with high content were obtained. 23 of these compounds were purchased as powder from Shanghai Taosu Biochemical Technology Co., Ltd. Each compound was diluted with DMSO to an initial concentration of 10mM and then diluted with PBS to 1mM for later use. 1×10 4RD cells were infected with EV71 at an MOI of 0.1, CV-A9 at an MOI of 0.001, PV1 at an MOI of 0.003, and E11 at an MOI of 0.005 after 24 hours. Or 1×10 4 Vero E6 cells were infected with CVB3 at an MOI of 0.1 24 hours later. Various known compounds were added to a final concentration of 20 μM (2 μL of a 1 mM compound was added to 100 μL). RT-qPCR was used to measure viral replication and the expression of the cellular reference gene GAPDH 24 hours after drug administration.

[0123] like Figure 11 As shown in Figure 2, there is an unreported compound with an inhibition rate of more than 90% against EV71 and no obvious cytotoxicity at 20 μM; Figure 12 As shown in Figure 2, there are two unreported compounds with an inhibition rate of greater than 90% on CV-A9 and no obvious cytotoxicity at 20 μM; Figure 13 As shown in Figure 2, an unreported compound has an inhibition rate of more than 90% on PV1 and has no obvious cytotoxicity at 20 μM; Figure 14 As shown in Figure 2, an unreported compound has an inhibition rate of more than 90% on E11 and has no obvious cytotoxicity at 20 μM; Figure 15 As shown, one unreported compound exhibited an inhibition rate greater than 90% against CVB3 and showed no significant cytotoxicity at 20 μM. The inhibition rates were as follows: 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone against EV71 (99.52%), 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone against CV-A9 (99.99%), arctiol A against CV-A9 (99.99%), 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone against PV1 (100%), 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone against E11 (100%), and 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone against CVB3 (99.93%). These results suggest that 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone or arctiol A is a potential potent inhibitor against EV71, CV-A9, PV1, E11 and CVB3 infected cells.

[0124] Example 6: Monomeric components inhibit enterovirus EC 50 and CC 50 Determination

[0125] The EC values of the unreported compounds 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone and arctiol A were determined according to the method of Example 3. 50 and CC 50 The ten final concentrations of the potential antiviral monomer were: 0.195 μM, 0.39 μM, 0.78 μM, 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM.

[0126] like Figure 16 As shown, the EC of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone against EV71 was 50 =3.06μM, CC 50 >100μM, SI>32.68, and has a good antiviral effect (99% to 90%) in the concentration range of 12.5μM to 100μM; Figure 17 As shown, the EC value of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone on CV-A9 was 50 =1.64μM, CC 50 >100μM, SI>60.98, and has a good antiviral effect (99% to 90%) in the concentration range of 3.125μM to 100μM; Figure 18 As shown, the EC of arctiinol A to CV-A9 was 50 =2.80μM, CC 50 >100μM, SI>35.77, and has a good antiviral effect (99% to 90%) in the concentration range of 12.5μM to 100μM; Figure 19 As shown, the EC of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone to PV1 50 =2.94μM, CC 50 >100μM, SI>34.01, and has a good antiviral effect (99% to 90%) in the concentration range of 25μM to 100μM; Figure 20 As shown, the EC of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone to E11 50 =1.15μM, CC 50 >100μM, SI>86.96, and has a good antiviral effect (99% to 90%) in the concentration range of 12.5μM to 100μM; Figure 21 As shown, the EC of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone on CVB3 50 =0.65μM, CC 50The results showed that the concentration of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone or arctiol A was >100μM, SI>153.85, and it had a good antiviral effect (99%-90%) in the concentration range of 6.25μM to 100μM. This suggests that 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone or arctiol A is a potential strong inhibitor against EV71, CV-A9, PV1, E11 and CVB3 infected cells.

[0127] Example 7: Experiment on the Dosage Time of Monomer Inhibition of Enterovirus

[0128] According to the method of Example 4, the enterovirus inhibition stage of the above-mentioned unreported compounds 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone and arctiol A was preliminarily determined. For EV71, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone was added at a final concentration of 25 μM, for CV-A9, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone was added at a final concentration of 25 μM or arctiol A was added at a final concentration of 30 μM, for PV1, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone was added at a final concentration of 12.5 μM, for E11, and for CVB3, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone was added at a final concentration of 12.5 μM.

[0129] The results are as follows Figure 22 、 Figure 23 、 Figure 24 、 Figure 25 、 Figure 26 and Figure 27 As shown. The bar graphs from left to right are the relative mRNA expression levels of the virus after the addition of the drug after the poisoning control group, the whole time course, before the entry into the cell, and after the entry into the cell. The results of the drug addition time experiment showed that 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone has an inhibitory effect on EV71 mainly in the post-entry stage ( Figure 22 ); 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone has an inhibitory effect on CV-A9 before and after its entry into the cell ( Figure 23 ); Arctium phenol A has an inhibitory effect on CV-A9 after it enters the cell ( Figure 24 ); 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone has an inhibitory effect on PV1 mainly after it enters the cell ( Figure 25 ); 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone has an inhibitory effect on E11 mainly after it enters the cell ( Figure 26); 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone has an inhibitory effect on CVB3 mainly after it enters the cell, and has a partial inhibitory effect before it enters the cell ( Figure 27 ).

[0130] Example 8: In vivo evaluation of the efficacy of Jinhua Qinggan granules and their monomeric components in treating enterovirus infection

[0131] Three-day-old C57BL / 6 suckling mice (6) and their mothers were grouped into 5 groups, which were set as blank control group (not challenged and treated with placebo), negative control group (treated with placebo after challenge), positive control group (treated with 6.25 mg / kg positive control drug YM-201636 by intraperitoneal injection after challenge, refer to Inhibition of PIKFYVE kinaseinterferes ESCRT pathway to suppress RNA virus replication.doi:10.1002 / jmv.28527.), Jinhua Qinggan Granule treatment group (treated with 750 mg / kg Jinhua Qinggan Granule by gavage after challenge), and 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone treatment group (treated with 6.25 mg / kg 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone by intraperitoneal injection after challenge). One day before the challenge, different groups were given the above drugs and administration methods for prevention. On the second day, 20 μL 1×10 8 pfu / mL (i.e. 2×10 6 pfu) CV-A9 virus, and 1 hour after infection, the mice were treated with the corresponding drugs by intraperitoneal injection (5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone and YM-201636) or gavage (Jinhua Qinggan Granules). Thereafter, the drugs were administered once a day, and the weight changes of the mice were observed.

[0132] The mice in each group were weighed daily for 3 days and weight changes were recorded. Three days after infection, serum, heart, colon, liver, hind limb muscle, and spleen tissues were collected from three suckling mice in each group.

[0133] Pathology: Tissues including the heart, colon, liver, hindlimb muscles, and spleen were collected from mice in each experimental group and fixed with 4% paraformaldehyde (purchased from Beijing Solebow Technology Co., Ltd., Cat. No. P1110) for 3 days. All samples were then sent to Wuhan Sewell Biotechnology Co., Ltd. for paraffin embedding, sectioning, and H&E staining. Pathological sections were examined for the presence of severe tissue necrosis, including visible muscle bundle rupture and myofibrolysis in the hindlimb muscles.

[0134] Viral load test: A portion of the collected tissues was weighed and fully lysed in sterile PBS using a freeze lysator to extract viral RNA from the tissues. RT-qPCR experiments were performed to detect the viral load in different tissues, expressed as log 10 Copies / mg.

[0135] like Figure 28 、 Figure 29 and Figure 30 As shown in the results, although there was no significant difference in the body weight of the mice, oral administration of Jinhua Qinggan Granules and injection of 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone could reduce the viral load in the whole body of CV-A9 infected suckling mice (such as serum, heart, colon, liver, hind limb muscles and spleen), and alleviate the inflammatory response.

[0136] In the present invention, the inventors discovered that Jinhua Qinggan Granule and 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone exhibited strong dose-dependent antiviral effects and low cytotoxicity against infection with enterovirus 71, coxsackievirus A group 9 strain BUCT01, poliovirus type 1, echovirus 11, and coxsackievirus B group 3 in RD cells. Arctiol A also exhibited strong dose-dependent antiviral effects and low cytotoxicity against infection with coxsackievirus A group 9 strain BUCT01. Currently, there are no specific drugs for enterovirus 71, coxsackievirus A group 9, poliovirus type 1, echovirus 11, coxsackievirus B group 3, or other enteroviruses. Therefore, the present invention provides important insights into the future clinical use of enterovirus 71, coxsackievirus A group 9, poliovirus type 1, echovirus 11, coxsackievirus B group 3, or other enteroviruses.

[0137] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Use of Jinhua Qinggan Granules in any of the following: X1)-X4): X1) Preparation of medicinal products for preventing and / or treating diseases or symptoms caused by enteroviruses; X2) Preparation of medicaments for the prevention and / or treatment of enteroviral infections; X3) Preparation of enterovirus inhibitors; X4) preparing a medicament for inhibiting the proliferation of enterovirus; The Jinhua Qinggan granules are composed of the following ingredients: honeysuckle, gypsum, honey ephedra, stir-fried bitter almonds, scutellaria, forsythia, thunbergia thunbergii, anemarrhena, burdock fruit, artemisia annua, mint, and liquorice; Preferably, the enterovirus is enterovirus 71 (EV71), coxsackievirus A group 9 (CV-A9), coxsackievirus B group 3 (CVB3), poliovirus type 1 (PV1) or echovirus type 11 (E11).

2. The use according to claim 1, characterized in that Jinhua Qinggan Granules as the sole active ingredient, or together with one, two or more other anti-enteroviral drugs as active ingredients; Preferably, the other anti-enteroviral drugs are selected from acyclovir tablets, amoxicillin capsules, ribavirin capsules, oseltamivir, suramin sodium, pediatric Jiebiao oral liquid, Xiyanping injection or berberine.

3. The use according to claim 1, characterized in that The diseases caused by enterovirus include hand, foot and mouth disease, herpangina, encephalitis, aseptic meningitis, acute weakness and paralysis of limbs, acute hemorrhagic conjunctivitis, myositis, muscle calcification, motor end plate abnormalities, pleural ulcer, myocarditis, pericarditis and hepatitis; The symptoms caused by the enterovirus include headache, fever, vomiting, sneezing, coughing, rash or muscle stiffness.

4. Use of arctiol A or 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone in any of the following X1)-X4): X1) Preparation of medicinal products for preventing and / or treating diseases or symptoms caused by enteroviruses; X2) Preparation of medicaments for the prevention and / or treatment of enteroviral infections; X3) Preparation of enterovirus inhibitors; X4) preparing a medicament for inhibiting the proliferation of enterovirus; The structural formula of the arctiol A is: The structural formula of the 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone is: Preferably, the enterovirus is enterovirus type 71, coxsackievirus group A type 9, coxsackievirus group B type 3, poliovirus type 1 or echovirus type 11.

5. The use according to claim 4, characterized in that Arctiol A or 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone as the sole active ingredient or together with one, two or more other anti-enteroviral drugs as active ingredients; Preferably, the other anti-enteroviral drugs are selected from acyclovir tablets, amoxicillin capsules, ribavirin capsules, oseltamivir, suramin sodium, pediatric Jiebiao oral liquid, Xiyanping injection or berberine.

6. The use according to claim 4, characterized in that The diseases caused by enterovirus include hand, foot and mouth disease, herpangina, encephalitis, aseptic meningitis, acute weakness and paralysis of limbs, acute hemorrhagic conjunctivitis, myositis, muscle calcification, motor end plate abnormalities, pleural ulcer, myocarditis, pericarditis and hepatitis; The symptoms caused by the enterovirus include headache, fever, vomiting, sneezing, coughing, rash or muscle stiffness.

7. Use of a pharmaceutical composition comprising Jinhua Qinggan Granules, arctiol A, or 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone in any of the following X1) to X4): X1) Preparation of medicinal products for the prevention and / or treatment of diseases or symptoms caused by enteroviruses; X2) Preparation of medicaments for the prevention and / or treatment of enterovirus infections; X3) Preparation of enterovirus inhibitors; X4) preparing a medicament for inhibiting the proliferation of enterovirus; The structural formula of the arctiol A is: The structural formula of the 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone is: The Jinhua Qinggan granules are composed of the following ingredients: honeysuckle, gypsum, honey ephedra, stir-fried bitter almonds, scutellaria, forsythia, thunbergia thunbergii, anemarrhena, burdock fruit, artemisia annua, mint, and liquorice; Preferably, the enterovirus is enterovirus type 71, coxsackievirus group A type 9, coxsackievirus group B type 3, poliovirus type 1 or echovirus type 11.

8. The use according to claim 7, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier; Preferably, the carrier includes but is not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), insoluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.), among which water-soluble carrier materials are preferred.

9. The use according to claim 7, characterized in that Conventional cosolvents, buffers, pH regulators, colorants, preservatives, spices, flavorings, sweeteners or other materials may also be added to the pharmaceutical composition.

10. The use according to claim 7, characterized in that The dosage forms of the pharmaceutical composition include, but are not limited to, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, suppositories, freeze-dried powder injections, and the like.

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