Application of Huashi Baidu Granules in treatment of enterovirus-induced diseases

Abstract

The application is application of Huoshi Baiyin granules in treating diseases caused by enterovirus, and provides use of Huoshi Baiyin granules in preparing medicines for preventing, relieving and / or treating diseases caused by enterovirus. The inventor finds that Huoshi Baiyin granules have good inhibitory activity on enterovirus such as coxsackievirus A9 and enterovirus 71. Therefore, the technical scheme of the application has important clinical application value for treating diseases caused by enterovirus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Huashi Baidu Granules in the treatment of diseases caused by enteroviruses. Background Technology

[0002] Enteroviruses are a genus within the family Parvoviridae. Coxsackievirus A9 (CV-A9) and Enterovirus 71 (EV71) are typical examples that can cause hand, foot, and mouth disease. Because enteroviruses are classified as non-enveloped viruses in virology, they are highly resistant to their surrounding environment and tolerant to stomach acid and ethanol.

[0003] Specifically, Coxsackievirus belongs to the Human Enterovirus B (HEV-B) group. HEV-B presents with a wide range of acute clinical manifestations and is considered a major cause of aseptic meningitis. Coxsackieviruses can be divided into two types: A (CV-A) and B (CV-B). Coxsackievirus A9 is the only HEV-B virus among Coxsackievirus A (CV-A) and is considered one of the important pathogens of viral encephalitis. Furthermore, there is evidence that the pathogenesis of childhood diabetes may also be related to HEV-B, particularly CV-A9, CV-B1, CV-B3, and CV-B5, and the incidence of this disease is rising in Western countries. Additionally, Coxsackieviruses A16, A4, A5, A9, A10, as well as B2 and B5, can all cause hand-foot-and-mouth disease.

[0004] Enterovirus 71 (EV71) is a single-stranded, positive-sense RNA virus. The viral particles have an icosahedral spherical structure with a diameter of approximately 24–30 nm. The primary transmission route of EV71 is fecal-oral. Infection can cause low-grade fever, oral pain, vomiting, and diarrhea. Due to its high infectivity to the central nervous system, EV71 can also cause aseptic meningitis, myocarditis, and muscle rigidity. Furthermore, as the main pathogen of hand-foot-and-mouth disease, EV71 is frequently transmitted through close contact between people. Although asymptomatic or mildly symptomatic patients recover naturally and develop antibodies, those infected with EV71 who develop severe illness still have a very high risk of dying from cardiopulmonary failure and extensive brainstem damage.

[0005] The Huashi Baidu Granules are composed of 14 herbs: Ephedra, Apricot Kernel, Gypsum, Red Peony Root, Lepidium Seed, Licorice Root, Pinellia Rhizome, Poria Cocos, Amomum Fruit, Patchouli, Atractylodes Rhizome, Astragalus Root, Magnolia Bark, and Rhubarb. Developed by Academician Huang Luqi and his team based on traditional Chinese medicine formulas such as Ma Xing Shi Gan Tang, Huo Xiang Zheng Qi San, Xuan Bai Cheng Qi Tang, and Ting Li Da Zao Xie Fei Tang, the formula has been developed. Bioinformatics research in collaboration with the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, has revealed that four of the herbs—Gypsum, Red Peony Root, Lepidium Seed, and Licorice Root—primarily affect immunity, inflammation, and related signaling pathways. Modern research confirms that the Huashi Baidu Granules have two main functions: first, to eliminate the virus; and second, to enhance the patient's own immunity. Current treatment of severe COVID-19 patients shows that the use of Huashi Baidu Granules can significantly reduce the proportion of patients admitted to the ICU and requiring artificial oxygen therapy, and can also alleviate lung inflammation.

[0006] There are currently no reports of Huashi Baidu Granules inhibiting enteroviruses. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the use of Huashi Baidu Granules in the preparation of medicaments for the prevention, relief, and / or treatment of diseases caused by enteroviruses. According to an embodiment of the invention, the enterovirus is selected from Coxsackievirus and / or Enterovirus 71.

[0008] According to an embodiment of the present invention, the Huashi Baidu Granules are developed for the treatment of COVID-19 and are composed of 14 herbs, including ephedra, apricot kernel, gypsum, red peony root, lepidium seed, licorice, pinellia, poria, cardamom, agastache, atractylodes, astragalus, magnolia bark, and rhubarb.

[0009] According to an embodiment of the present invention, the Huashi Baidu Granules are produced by Guangdong Yifang Pharmaceutical Co., Ltd.

[0010] According to an embodiment of the present invention, the Huashi Baidu Granules inhibit Coxsackievirus, or improve diseases caused by Coxsackievirus infection, or improve symptoms caused by Coxsackievirus infection.

[0011] According to an embodiment of the present invention, the inhibition of Coxsackievirus may be performed at the organismal or cellular level.

[0012] According to embodiments of the present invention, the diseases caused by Coxsackievirus infection may include herpetic pharyngitis, acute hemorrhagic conjunctivitis, hand-foot-mouth disease, pleuralgia, myocarditis, pericarditis, and hepatitis. The symptoms caused by Coxsackievirus infection may include fever, sneezing, coughing, and rash.

[0013] In this invention, the Coxsackievirus may be Coxsackievirus A or other Coxsackieviruses. Further, the Coxsackievirus A may be Coxsackievirus A9 (CV-A9) or other types of Coxsackievirus A.

[0014] According to an embodiment of the present invention, the Huashi Baidu Granules inhibit enterovirus 71 (EV71), or improve diseases caused by EV71 infection, or improve symptoms caused by EV71 infection.

[0015] According to an embodiment of the present invention, the inhibition of enterovirus 71 can be performed at the organismal or cellular level to inhibit enterovirus types.

[0016] According to embodiments of the present invention, the diseases caused by enterovirus 71 infection may include hand-foot-mouth disease, herpetic pharyngitis, encephalitis, aseptic meningitis, acute paralysis of weak limbs, and acute hemorrhagic conjunctivitis. Symptoms caused by enterovirus 71 infection may include headache, fever, vomiting, and muscle rigidity.

[0017] In this invention, enterovirus 71 can be three subtypes, namely group A, group B, and group C. Further, enterovirus 71 group B and group C can each contain five subgroups, namely subgroups B1-B5 and C1-C5.

[0018] In a specific embodiment of the present invention, the Coxsackievirus A9 type is Coxsackievirus A9 strain BUCT01, which has the accession number CGMCCNo.20091 at the China General Microbiological Culture Collection Center.

[0019] In a specific embodiment of the present invention, the enterovirus 71 is EV71 (accession number: JN230523.1), a member of the group A of the genus Enterovirus in the family Parvoviridae.

[0020] The present invention also provides a pharmaceutical composition for preventing, alleviating and / or treating diseases caused by enteroviruses, comprising Huashi Baidu Granules.

[0021] The present invention also provides a pharmaceutical composition for preventing, alleviating and / or treating diseases caused by enteroviruses, the composition comprising Huashi Baidu granules, the diseases including: herpetic pharyngitis, acute hemorrhagic conjunctivitis, hand-foot-mouth disease, pleurisy, myocarditis, pericarditis, hepatitis, encephalitis, aseptic meningitis, acute paralytic limb paralysis and acute hemorrhagic conjunctivitis.

[0022] According to an embodiment of the present invention, the disease is caused by infection with Coxsackievirus and / or Enterovirus 71.

[0023] The present invention also provides the use of Huashi Baidu Granules in the preparation of medicaments for the prevention, relief and / or treatment of the following diseases: herpetic pharyngitis, acute hemorrhagic conjunctivitis, hand-foot-mouth disease, pleurisy, myocarditis, pericarditis, hepatitis, encephalitis, aseptic meningitis, acute paralysis of weak limbs and acute hemorrhagic conjunctivitis.

[0024] According to an embodiment of the present invention, the disease is caused by infection with Coxsackievirus and / or Enterovirus 71.

[0025] The present invention also provides a method for preventing, alleviating and / or treating diseases caused by enteroviruses, comprising applying Huashi Baidu Granules, or the above-mentioned pharmaceutical composition, to an individual in need of such treatment.

[0026] Beneficial effects

[0027] The inventors discovered that Huashi Baidu Granules exhibit good inhibitory activity against enteroviruses (preferably Coxsackievirus A9 and Enterovirus 71). Therefore, the technical solution of this invention has significant clinical application value for the treatment of diseases following enterovirus infection. Attached Figure Description

[0028] Figure 1 EC of Huashi Baidu Granules was obtained by culturing RD cells for 24 h with CV-A9 cells at MOI = 0.001 for 36 h. 50 CC 50 and SI.

[0029] Figure 2 EC of Huashi Baidu Granules was administered to RD cells cultured for 24 h with EV71 cells at MOI = 0.017 and infected for 48 h with Huashi Baidu Granules. 50 CC 50 and SI.

[0030] Figure 3 The results of the dosing time experiments for Huashi Baidu Granules at concentrations of 2 mg / ml, 1 mg / ml, and 0.5 mg / ml on CV-A9 were presented (where *** and **** indicate significant differences compared to the control group).

[0031] Figure 4 The results of the dosing time experiments for Huashi Baidu Granules at concentrations of 2 mg / ml, 1 mg / ml, and 0.5 mg / ml on EV71 were presented (*** indicates a significant difference compared to the control group). Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0033] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0034] Example 1

[0035] 1. Cell and virus culture

[0036] The human rhabdomyosarcoma cell line RD was obtained from the American Type Culture Collection (ATCC, CRL-3216TM) and cultured at 37°C and 5% CO2 in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen).

[0037] Coxsackievirus type A9 strain BUCT01 and Enterovirus genus A (Papillaviridae family) member EV71 were propagated in RD cells. Plaque assays were performed on RD cells (ATCC CCL-136) to determine the titer of Coxsackievirus type A9 strain BUCT01, and TCID50 was used to determine the titer of EV71. All infection experiments were performed in a biosafety level 2 (BLS-2) laboratory.

[0038] 2. Huashi Baidu Granules inhibit the EC50 of Coxsackievirus A9 strain BUCT01 (CV-A9) and Enterovirus 71 (EV71). 50 CC 50 Measurement

[0039] EC 50 Detection: 2.5×10 4RD cells were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. Then, a solution of Huashi Baidu Granules was added to the cell culture wells at final concentrations of 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, 0.0625 mg / ml, 0.0313 mg / ml, 0.0156 mg / ml, 0.0078 mg / ml, and 0.0039 mg / ml, respectively. The cells were pre-incubated at 37°C for 1 hour. Simultaneously, the virus was mixed with the solution at final concentrations of 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0039 mg / ml. A solution of Huashi Baidu Granules at concentrations of 0.0625 mg / ml, 0.0313 mg / ml, 0.0156 mg / ml, 0.0078 mg / ml, and 0.0039 mg / ml was pre-incubated at 4℃ for 1 h. After incubation, cells were infected with the incubated virus (CV-A9 final MOI = 0.001, EV71 final MOI = 0.017) and cultured in a 37℃, 5% CO2 incubator. Cytopathic effects were observed under a microscope 36 h after CV-A9 infection and 48 h after EV71 infection. The expression of viral RNA and the intracellular reference gene GAPDH was quantitatively detected by qRT-PCR. EC50 was calculated using GraphPad-Prism 8 software. 50 .

[0040] CC 50 Detection: CC was performed using the CellTiter-Blue method. 50 The detection was performed by seeding RD cells into 96-well cell culture plates. When the cell density reached 60%-80%, the drug was diluted and added after the RD cells were replaced with different media. The final concentrations were 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, 0.0625 mg / ml, 0.0313 mg / ml, 0.0156 mg / ml, 0.0078 mg / ml, and 0.0039 mg / ml. The cells were cultured at 37℃ with 5% CO2 for 48 h. The luminescence intensity at 593 nm was detected using CellTiter-Blue reagent, and the CC was calculated using GraphPad-Prism 8 software. 50 .

[0041] EC 50 This refers to the drug concentration that can effectively inhibit 50% of cell infection by the virus. The lower the value, the better the inhibitory effect on the virus.

[0042] CC 50It is the drug concentration at which 50% of cells become diseased; the higher the value, the lower the cytotoxicity.

[0043] SI: Selectivity Index, represented by CC 50 With EC 50 The ratio of .

[0044] 3. Dosing time experiment

[0045] Seed 2.5 × 10⁶ cells in a 24-well cell plate 5 RD cells were infected with CV-A9 strain BUCT01 (MOI=0.001 or EV71 strain MOI=0.017) 24 hours later. The drug was added at concentrations of 2 mg / ml, 1 mg / ml or 0.5 mg / ml at the time of virus addition and 2 h after virus incubation, before cell entry (at the time of virus addition), and after cell entry (2 h after virus incubation). Cytopathic effects were observed under a microscope. RNA was extracted from cells and supernatant in culture wells. The viral replication status in cells and supernatant and the expression of the internal reference gene GAPDH were measured by qRT-PCR.

[0046] 4. Virus sample processing and detection

[0047] RNA extraction was performed using the Axygen™ Multipurpose Total RNA Miniprep Kit (Axygene, catalog number AP-MN-MS-RNA-250G) according to the manufacturer's instructions. Reverse transcription was performed using the Hifair II 1st strand cDNA Synthesis Kit (Shanghai Yisheng Biotechnology, catalog number: 11121ES60) for digestible gDNA. qRT-PCR was performed using the Quantstudio Real-Time PCR Detection Kit (Applied Biosystems, Foster City, CA, USA). The sequence information of the primers used is shown in Table 2. SYBR-Green qPCR amplification (dye-based qPCR amplification program is shown in Table 1):

[0048] Table 1. Dye-based qPCR amplification program

[0049]

[0050] Homogenization is achieved by detecting the GAPDH gene.

[0051] Table 2 Primer sequences used in the study

[0052]

[0053] The half-maximal effective concentration (MCC) and half-maximal cytotoxicity assays showed that Huashi Baidu Granules had an effect on the EC50 of CV-A9 cells in RD cells.50 =0.1796mg / ml, CC 50 =8.4mg / ml, SI=46.77 ( Figure 1 ); The effect of Huashi Baidu Granules on EV71 EC on RD cells 50 =0.134mg / ml, CC 50 =8.4mg / ml, SI=62.69 ( Figure 2 For CV-A9, the drug showed good antiviral efficacy (99.98%, 99.98%, and 99.42%) at concentrations of 2 mg / ml, 1 mg / ml, and 0.5 mg / ml; for EV71, the drug showed good antiviral efficacy (99.92%, 99.85%, 99.62%, and 97.01%) at concentrations of 2 mg / ml, 1 mg / ml, 0.5 mg / ml, and 0.25 mg / ml, suggesting that Huashi Baidu Granules may serve as a potential antiviral inhibitor for enteroviruses CV-A9 and EV71.

[0054] The results of the drug administration time experiment showed that Huashi Baidu Granules had an effect on CV-A9 both before and after cell entry. Figure 3 )

[0055] The results of the drug administration time experiment showed that Huashi Baidu Granules had an effect on EV71 both before and after cell entry. Figure 4 )

[0056] In this invention, the inventors discovered that Huashi Baidu Granules exhibit good dose-dependent antiviral efficacy and low cytotoxicity against Coxsackievirus A9 strain BUCT01 and Enterovirus 71 infection in RD cells. Currently, there are no specific drugs targeting Coxsackievirus A9, Enterovirus 71, or other Enterovirus A and B groups. Therefore, this invention has important reference value for future clinical application of Coxsackievirus A9, Enterovirus 71, or other Enterovirus A and B groups.

[0057] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of Huashi Baidu Granules in the preparation of medicines for the prevention, relief and / or treatment of diseases caused by enteroviruses; The enteroviruses mentioned are Coxsackievirus A9 and / or Enterovirus 71.

2. The use according to claim 1, characterized in that, Diseases caused by Coxsackievirus A9 infection include herpetic pharyngitis, acute hemorrhagic conjunctivitis, hand-foot-mouth disease, pleuropneumonia, myocarditis, pericarditis, and hepatitis.

3. The use according to claim 1, characterized in that, Diseases caused by enterovirus 71 infection include hand-foot-mouth disease, herpetic pharyngitis, encephalitis, aseptic meningitis, acute paralysis of weak limbs, and acute hemorrhagic conjunctivitis.

4. The use according to claim 1, characterized in that, The Coxsackievirus A9 strain mentioned is BUCT01, which has the accession number CGMCCNo.20091 at the China General Microbiological Culture Collection Center.

5. The use according to claim 1, characterized in that, Enterovirus 71 is a member of the genus Enterovirus A of the family Parvoviridae, with accession number JN230523.1.

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