Application of Jujuboside B in the preparation of enterovirus inhibitors

Abstract

The present invention relates to the use of jujuboside B in the preparation of enterovirus inhibitors, belonging to the technical field of biopharmaceuticals. The main technical solutions are as follows: On the one hand, the present invention mainly provides the use of jujuboside B in the preparation of enterovirus inhibitors; on the other hand, the present invention provides an enterovirus inhibitor, the active ingredient of which is jujuboside B. The present invention mainly provides a potential drug with safety, high efficiency and low toxicity and side effects for the prevention or treatment of enterovirus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly to the application of jujuboside B in the preparation of enterovirus inhibitors. Background Art

[0002] Enteroviruses belong to the genus Enterovirus of the family Picornaviridae, including polioviruses, coxsackieviruses, enteric cytopathic human orphan viruses (ECHO viruses for short), and new enteroviruses, a total of 71 serotypes in four categories. Among them, human enteroviruses are mainly enteroviruses A - D, such as Enterovirus 71 (EV71), Coxsackievirus A16 (CVA16), Coxsackievirus A6 (CVA6), Enterovirus D68 (EVD68), etc.

[0003] Enteroviruses can cause various infectious diseases, including hand, foot and mouth disease (HFMD), encephalitis, myocarditis, acute flaccid myelitis, pneumonia, bronchiolitis, etc. Among them, EV71 and CA16 are the main pathogens causing hand, foot and mouth disease in infants and children under five years old. Although the disease is usually mild and self - limiting, it sometimes leads to serious complications, such as brainstem encephalitis, aseptic meningitis, acute flaccid paralysis, neurogenic pulmonary edema and other neurological diseases, and in severe cases, even death. EV71 was first isolated and identified in 1969, and then it has caused multiple pandemics globally, especially in the Asia - Pacific region. In recent years, it has been found that CVA6 and CVA10 can also cause hand, foot and mouth disease. EVD68 is relatively rare compared with EV71, but it can also cause severe respiratory diseases such as bronchiolitis and pneumonia after infection.

[0004] Since 2008, hand, foot and mouth disease (category C infectious disease) has ranked first in the incidence of legal infectious diseases in China. However, at present, there is no specific antiviral drug for enterovirus infection in clinical practice, but conventional antiviral and symptomatic treatments are used. For example, ribavirin and type I interferon are the most commonly used drugs for the treatment of EV71 infection. Although in 2016, an inactivated vaccine against EV71 C4 subtype was approved for marketing in China, the existence of 11 different EV71 subtypes indicates that the virus is still constantly mutating, and the vaccine has no cross - protection against other types of enteroviruses. Therefore, developing effective and broad - spectrum anti - enterovirus drugs has important clinical significance.

[0005] In summary, diseases caused by enterovirus infections pose a serious threat to human health. However, there are currently no clinically available specific drugs for the treatment of enterovirus infections. Therefore, there is an urgent need for broad-spectrum anti-enterovirus drugs to prevent / treat enterovirus infections. Summary of the Invention

[0006] In view of this, the present invention provides an application of jujuboside B in the preparation of an enterovirus inhibitor, with the main purpose of providing a safe, highly effective, and low-toxicity potential drug for the prevention or treatment of enterovirus infections.

[0007] To achieve the above object, the present invention mainly provides the following technical solutions:

[0008] On the one hand, an embodiment of the present invention provides an application of jujuboside B in the preparation of an enterovirus inhibitor, wherein the structural formula of the jujuboside B is as follows:

[0009]

[0010] Preferably, the enterovirus inhibitor can inhibit enteroviruses including EV71 (enterovirus A71), CVA10 (coxsackievirus A10), CVA16 (coxsackievirus A16), EVD68 (enterovirus D68), and CVA6, one or more of them.

[0011] Preferably, the enterovirus inhibitor can inhibit the replication of enteroviruses.

[0012] Preferably, the enterovirus inhibitor is a drug for preventing enterovirus infections or a drug for treating enterovirus infections.

[0013] On the one hand, an embodiment of the present invention provides an enterovirus inhibitor, wherein the active ingredient of the enterovirus inhibitor is jujuboside B.

[0014] Preferably, the enterovirus inhibitor can inhibit enteroviruses including EV71 (enterovirus A71), CVA10 (coxsackievirus A10), CVA16 (coxsackievirus A16), EVD68 (enterovirus D68), and CVA6, one or more of them.

[0015] Preferably, the enterovirus inhibitor is used to inhibit the replication of enteroviruses.

[0016] Preferably, the enterovirus inhibitor is a drug for preventing enterovirus infections or a drug for treating enterovirus infections.

[0017] Preferably, the enterovirus inhibitor is a drug for preventing or treating diseases caused by enteroviruses; wherein, the diseases caused by enteroviruses include hand, foot and mouth disease, encephalitis, myocarditis, acute flaccid myelitis, pneumonia, bronchiolitis and other diseases.

[0018] Preferably, the enterovirus inhibitor further comprises at least one pharmaceutically acceptable carrier.

[0019] Compared with the prior art, the application of jujuboside B in the preparation of enterovirus inhibitors has at least the following beneficial effects:

[0020] On the one hand, the present invention first discovers a new use of jujuboside B in the preparation of enterovirus inhibitors. Among them, jujuboside B can inhibit the replication of enteroviruses, and thus can be used as or prepared into a drug for preventing enterovirus infection or treating enterovirus infection, thereby providing a safe, highly effective, and low-toxicity potential drug for the prevention or treatment of enterovirus infection. In addition, the enteroviruses that jujuboside B can inhibit include EV71 (enterovirus A71), CVA10 (coxsackievirus A10), CVA16 (coxsackievirus A16), EVD68 (enterovirus D68), CVA6, but are not limited thereto.

[0021] On the other hand, the present invention also provides an enterovirus inhibitor, the active ingredient of which is jujuboside B, which can inhibit the transcriptional replication of enteroviruses, and is used as a drug for preventing enterovirus infection or treating enterovirus infection, with the effects of safety, high efficiency, and low toxicity. In addition, the enterovirus inhibitor is used to inhibit EV71 (enterovirus A71), CVA10 (coxsackievirus A10), CVA16 (coxsackievirus A16), EVD68 (enterovirus D68), CVA6, but is not limited thereto.

[0022] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following describes in detail with the preferred embodiments of the present invention in conjunction with the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the cytotoxicity detection results of jujuboside B on RD cells and HUVEC cells in Example 1. Among them, Figure 1 Figure A in 50 shows the toxicity of jujuboside B to RD cells, namely the CC Figure 1 Figure B in shows the toxicity of jujuboside B to primary cells HUVEC; Figure 1 Figure C in shows the toxicity of the positive drug ribavirin (Rb) to RD.

[0024] Figure 2 It is a schematic diagram of the anti-EV71 virus activity results of jujuboside B and ribavirin in Example 2. Among them Figure 2 Figure A in it shows a schematic diagram of the anti-EV71 virus activity results of jujuboside B; Figure 2 Figure B in it shows a schematic diagram of the anti-EV71 virus activity results of ribavirin (Rb).

[0025] Figure 3 It is a schematic diagram of the anti-other enterovirus activity results of jujuboside B in Example 3.

[0026] Among them, Figure 3 Figure A in it shows the EC 50 (half maximal effective concentration) of jujuboside B against EVD68 on RD cells; Figure 3 Figure B in it shows the EC 50 of jujuboside B against CVA10 on RD cells; Figure 3 Figure C in it shows the EC 50 of jujuboside B against CVA16 on RD cells.

[0027] Figure 4 It is a experimental result diagram of jujuboside B inhibiting the replication of EV71 virus; among them, Figure 4 Figure A and Figure B in it show that jujuboside B reduces the expression of VP1 protein of EV71 virus; Figure 4 Figure B in it is a statistical chart of the gray value analysis of the three repeated results of Figure A, Figure 4 Figure C in it shows that jujuboside B inhibits the EV71 virus RNA level; Figure 4 Figure D in it shows the change of virus titer in the corresponding cell supernatant in Figure A (in addition, **** in the figure represents P<0.0001), and Rb is the abbreviation of ribavirin. Detailed implementation manners

[0028] To further elaborate on the technical means and effects adopted by the present invention to achieve the intended invention purpose, the following combines the drawings and preferred embodiments to detail the specific implementation manners, structures, features and their effects of the application according to the present invention. In the following description, different "one embodiment" or "embodiment" do not necessarily refer to the same embodiment. In addition, the specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0029] The traditional Chinese medicine Semen Ziziphi Spinosae, which can be used both as medicine and food, is the dried mature seed of the plant Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow of the family Rhamnaceae. It has the effects of nourishing the heart and liver, calming the mind, arresting sweating, and promoting the production of body fluid, and is widely used in the treatment and health care of diseases related to the cardiovascular, nervous, and immune systems. Saponins are the key bioactive components of Semen Ziziphi Spinosae. It is reported that each kilogram of Semen Ziziphi Spinosae contains approximately 400 mg of jujuboside B (JuB). Due to its safety and various biological effects, JuB can be used as a potential candidate drug for the treatment and health care of various diseases. In addition, Semen Ziziphi Spinosae is widely distributed in China, including Shaanxi, Henan and other places, and is easy to obtain.

[0030] Jujuboside B (JuB) is one of the main active components of the dried mature seed Semen Ziziphi Spinosae of the plant Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow of the family Rhamnaceae. A number of in vitro studies and / or animal model experiments have shown that JuB has various pharmacological effects and application values, such as anti-inflammatory, antiplatelet aggregation, antitumor, sedative and hypnotic effects.

[0031] The molecular formula of jujuboside B is C52H84O21, and its molecular weight is 1045.223. Its structural formula is as follows:

[0032]

[0033] At present, the antiviral activity of jujuboside B has not been reported (the research on the antiviral activity of jujuboside B is a blank field). The present invention first discovers the new activity of JuB, that is, it can effectively inhibit a variety of enteroviruses (such as EV71, CVA10, CVA16, EVD68, CVA6), and has low cytotoxicity. Based on this activity, jujuboside B is used to prepare an enterovirus inhibitor (such as a drug for treating or preventing enterovirus infection), which will surely bring good news to its infected people.

[0034] The following is illustrated by specific experimental examples:

[0035] Example 1

[0036] This example mainly conducts the toxicity experiments of jujuboside B (25 mg / mL) on human rhabdomyoma cells RD and normal human umbilical vein endothelial cells HUVEC, and the toxicity experiment of ribavirin, an antiviral drug used clinically to treat EV71 infection, on human rhabdomyoma cells RD.

[0037] Experimental reagents: Jujuboside B was purchased from Baoji Chenguang Biotechnology Co., Ltd.; Ribavirin was purchased from selleck; DMSO was purchased from Beyotime Biotechnology Co., Ltd.; DMEM (high glucose) medium was purchased from Sangon Biotech Co., Ltd.; Penicillin-streptomycin was purchased from Nanjing Shenghang Biotechnology Co., Ltd.; Fetal bovine serum (FBS) was purchased from ZETA life; 0.25% Trypsin (containing EDTA) and CCK-8 were purchased from Suzhou Xinsaimi Biotechnology Co., Ltd. RD cells were donated by Shanghai Institute of Major Infectious Diseases and Biosafety, and then preserved after amplification. HUVEC cells (product number: CTCC-0804-PC) were purchased from Zhejiang Meisen Cell Technology Co., Ltd.

[0038] Experimental instruments: Epoch ultra-microplate spectrophotometer was purchased from BioTek Instruments, Inc. (USA)

[0039] Experimental methods: Human rhabdomyosarcoma cells RD or normal human umbilical vein endothelial cells HUVEC were seeded on 96-well cell culture plates overnight. When the cells proliferated to about 90%, serially diluted Jujuboside B (1.6 μg / mL - 50 μg / mL) or Ribavirin (12.5 μg / mL - 200 μg / mL) was added to the cell wells, and the cells were further cultured in a cell culture incubator at 37°C with 5% CO2. After 72 h, CCK-8 was used to detect cell viability. The main component of this reagent is WST-8 (water-soluble tetrazolium salt, chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt). In the presence of an electron coupling reagent, WST-8 can be reduced by some dehydrogenases in mitochondria to generate orange-yellow Formazan. For the same cells, the depth of color (the amount of Formazan generated) is linearly related to the number of cells, and it can be used for drug screening, cell proliferation and toxicity determination, tumor drug sensitivity and other tests. That is, after adding 10 μL to each well, it was placed in a cell culture incubator at 37°C with 5% CO2 for 1.5 h, and then the OD value was measured at a wavelength of 450 nm with an enzyme-labeling instrument. Three replicate wells were set for each concentration; at the same time, a medium blank control (only medium) and a cell control (cells without drug treatment) were set.

[0040] Cell survival rate (%) in the drug administration group = (OD of experimental well 450nm - OD of medium blank control well 450nm / OD of cell control well 450nm - OD of medium blank control well 450nm ) × 100%.

[0041] The results were used to calculate the mean and standard deviation by GraphPad Prism 5 software, and the CC 50 .

[0042] Experimental results: See the experimental results in Figure 1 shown in Figure 1 which is a schematic diagram of the cytotoxicity detection results of jujuboside B on RD cells and HUVEC cells. Among them, Figure 1 Figure A in 50 shows the toxicity of jujuboside B on RD cells, namely CC Figure 1 Figure B in Figure 1 shows the toxicity of jujuboside B on primary cells HUVEC (because RD is a tumor cell and JuB itself has anti-tumor cell activity, so the toxicity is relatively high, so the toxicity on primary cells (non-tumor cells) is detected);

[0043] From Figure 1 it can be seen that: The CC 50 (half cytotoxic concentration) of jujuboside B on human rhabdomyoma cells RD and normal human umbilical vein endothelial cells HUVEC are 42.15 μg / mL and 234.3 μg / mL respectively; The CC 50 of the positive drug ribavirin (Rb) on human rhabdomyoma cells RD is 285.8 μg / mL.

[0044] It can be concluded that: Although jujuboside B has certain toxicity on RD, the concentrations used in the activity study are all lower than CC 50 . In addition, the toxicity of jujuboside B to primary cells HUVEC is relatively low, which indicates that jujuboside B has relatively low toxicity and has a potential clinical use basis.

[0045] Example 2

[0046] This example mainly conducts the anti-EV71 virus activity experiment of jujuboside B and ribavirin on human rhabdomyoma cells RD.

[0047] Experimental reagents: Jujuboside B was purchased from Chenguang Biotechnology Co., Ltd. in Baoji City; Ribavirin was purchased from selleck company; DMSO was purchased from Beyotime Biotechnology Co., Ltd.; DMEM (high glucose) medium was purchased from Sangon Biotech Co., Ltd.; Penicillin-streptomycin was purchased from Nanjing Shenghang Biotechnology Co., Ltd.; Fetal bovine serum (FBS) was purchased from ZETA life company; 0.25% trypsin (containing EDTA) and CCK-8 were purchased from Suzhou Xinsaimi Biotechnology Co., Ltd. RD cells and EV71 virus strains were donated by the Shanghai Institute of Major Infectious Diseases and Biosafety, and were stored after amplification.

[0048] Experimental instruments: Epoch ultra-microplate spectrophotometer was purchased from BioTek, USA

[0049] Experimental method: RD cells were seeded in a 96-well cell culture plate overnight. When the cells proliferated to about 90%, the serially diluted jujuboside B (1.6 μg / mL - 50 μg / mL) or ribavirin (3.125 μg / mL - 100 μg / mL) was fully mixed with the virus dilution (M.O.I = 0.1), added into the cell wells, and cultured in a cell incubator at 37°C with 5% CO2 for 2 h. The cell supernatant was aspirated, and after washing the cells with PBS, fresh medium containing the corresponding concentration of jujuboside B or ribavirin was replaced, and the cells were further cultured for 72 h. The cell viability was detected using CCK-8. Three replicate wells were set for each sample, and cell control wells without drugs and virus infection control wells were also set.

[0050] Cell survival rate (%) of the drug-administered group = (OD of the experimental well 450nm - OD of the virus control well 450nm / OD of the cell control well 450nm - OD of the virus control well 450nm ) × 100%.

[0051] The results were used to calculate the mean and standard deviation by GraphPad Prism 5 software, and the EC 50 .

[0052] Experimental results: The experimental results are shown in Figure 2 as follows, Figure 2 which are the anti-EV71 virus activity results of jujuboside B and ribavirin. Among them, Figure 2 Figure A in Figure 2 shows the anti-EV71 virus activity results of jujuboside B;

[0053] From Figure 2 it can be seen that: The EC 50 of jujuboside B against EV71 virus on human rhabdomyosarcoma cells RD is 4.02 μg / mL; the EC 50 of ribavirin against EV71 on human rhabdomyosarcoma cells RD is 69.30 μg / mL. Combining the results of Example 1, the selectivity index (SI, calculation formula: SI = CC 50 / EC 50 ) of jujuboside B is 10.48, and the SI of ribavirin is 4.12, indicating that jujuboside B has a better anti-EV71 effect than ribavirin.

[0054] Example 3

[0055] This example mainly conducts an activity experiment of jujuboside B against other enteroviruses (EVD68 virus, CVA10 virus, CVA16 virus) on human rhabdoid tumor cells RD.

[0056] Experimental reagents: Jujuboside B was purchased from Baoji Chenguang Biotechnology Co., Ltd.; ribavirin was purchased from selleck; DMSO was purchased from Biyuntian Biotechnology Co., Ltd.; DMEM (high glucose) medium was purchased from Shenggong Biotechnology Co., Ltd.; penicillin and streptomycin were purchased from Nanjing Shenghang Biotechnology Co., Ltd., fetal bovine serum (FBS) was purchased from ZETA life; 0.25% trypsin (containing EDTA) and CCK-8 were purchased from Suzhou Xinsaimei Biotechnology Co., Ltd. RD cells, CVA10 / S0148b, CVA16 / SZ05, and EVD68 (US / MO / 14-18947) strains were donated by Shanghai Institute of Major Infectious Diseases and Biosafety, and then amplified and stored.

[0057] Experimental instruments: Epoch ultra-microplate spectrophotometer purchased from BioTek Instrument Co., Ltd., USA

[0058] Experimental method: RD cells were plated on a 96-well cell culture plate overnight, and when the cells proliferated to about 90%, the dilute jujuboside B (1.6 μg / mL-50 μg / mL) was fully mixed with the virus diluent (MOI = 0.1), added to the cell wells, and cultured in a cell culture incubator at 37°C and 5% CO2 for 2 hours, the cell supernatant was discarded, the cells were washed with PBS, and fresh culture medium containing the corresponding concentration of jujuboside B was replaced, and the culture was continued for 72 hours, and the cell activity was detected using CCK-8. Three replicate wells were set for each sample, and a cell control well without drug and a virus infection control well were set at the same time.

[0059] Cell survival rate of drug-treated group (%) = (OD 450nm -OD of virus control wells 450nm / cell control well OD 450nm -OD of virus control wells 450nm )×100%.

[0060] The mean and standard deviation of the results were calculated using GraphPad Prism 5 software, and the EC was calculated by fitting the curve. 50 .

[0061] Experimental results: The experimental results are as follows Figure 3 As shown, Figure 3 This is the result of the anti-enterovirus activity of jujuboside B. Figure 3 Figure A shows the EC of jujuboside B against EVD68 in RD cells. 50 ;Figure 3 Figure B in 50 ; Figure 3 Figure C in 50 .

[0062] From Figure 3 it can be seen that: the EC 50 of jujuboside B against EVD68 virus on RD cells is 5.208 μg / mL, and the EC 50 against CVA10 virus is 3.383 μg / mL; the EC 50 against CVA16 virus is 4.160 μg / mL. Combining the results of Example 1, the SI value of jujuboside B against EVD68 on RD cells is 8.09, the SI value against CVA10 is 12.46, and the SI value against CVA16 is 10.13. It shows that jujuboside B has broad-spectrum anti-enterovirus activity.

[0063] Example 4

[0064] This example mainly conducts an experiment on inhibiting the replication of EV71 virus by jujuboside B in human rhabdomyoma cells RD.

[0065] Experimental reagents: Jujuboside B was purchased from Chenguang Biotechnology Co., Ltd. in Baoji City; Ribavirin was purchased from selleck company; DMSO was purchased from Beyotime Biotechnology Co., Ltd.; DMEM (high glucose) medium was purchased from Sangon Biotech Co., Ltd.; Penicillin-streptomycin was purchased from Nanjing Shenghang Biotechnology Co., Ltd.; Fetal bovine serum (FBS) was purchased from ZETA life company; 0.25% Trypsin (containing EDTA) was purchased from Suzhou Xinsaimi Biotechnology Co., Ltd. The reverse transcription kit HiScript III RT SuperMix for qPCR (+gDNA wiper) was purchased from Novoprotein Scientific Inc. The 2×RealStar Fast SYBR qPCR Mix was purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd. The VP1 antibody was kindly provided by Shanghai Institute of Major Infectious Diseases and Biosafety.

[0066] Experimental instruments: The fluorescence quantitative PCR instrument (StepOnePlusTM) was purchased from Applied Biosystems; The Tianneng imaging equipment (Tanon 5200 Multi) was purchased from Shanghai Tianneng Technology Co., Ltd.

[0067] Experimental method: Seed RD cells in a 24-well cell culture plate overnight. When the cells have proliferated to about 90%, add the virus dilution (M.O.I. = 1) and incubate in a cell culture incubator at 37°C with 5% CO2 for 2 hours. Aspirate the cell supernatant, wash the cells twice with PBS, then add fresh medium containing serially diluted jujuboside B (5 μg / mL - 40 μg / mL), and continue culturing for 22 hours. Collect the cell supernatant and the cells, and extract intracellular RNA. After reverse transcribing the RNA into cDNA, perform quantitative PCR to measure the replication of the virus in the cells. Among them, the primers for quantitative PCR are sequences specific to the EV71 virus gene. Select the housekeeping gene GADPH as the internal reference control gene for calibration. Among them, the sequences of the primers are as follows:

[0068] EV71 virus upstream primer (EV71-F): 5'-CCCTGAATGCGGCTAATCC-3'

[0069] EV71 virus downstream primer (EV71-R): 5'-ATTGTCACCATAAGCAGCCA-3

[0070] Housekeeping gene upstream primer (GAPDH-F): 5'-ATCCCATCACCATCTTCCAGG-3'

[0071] Housekeeping gene downstream primer (GAPDH-R): 5'-CCTTCTCCATGGTGGTGAAGAC-3

[0072] Taking the relative expression level of EV71 in the virus-infected group as a control, compare it with the drug-treated groups to obtain the relative gene expression differences.

[0073] In addition, extract the total intracellular protein and use Western blotting (WB) to detect the expression of the viral protein VP1. Use ImageJ software to analyze the gray value of the WB bands, and repeat this experiment three times.

[0074] In addition, perform a virus titration experiment on the cell supernatant, that is, the above-collected supernatant is serially diluted and then used to infect RD cells for 72 hours. Observe the cytopathic effect and use the Reed-Muench method to calculate the 50% tissue culture infective dose TCID 50 .

[0075] Experimental results: See the experimental results graph of jujuboside B inhibiting EV71 virus replication as described in Figure 4 stated; Figure 4 Among them, Figure 4 Figures A and B in it show that jujuboside B reduces the expression of the VP1 protein of EV71 virus; Figure 4 Figure B in it is a statistical graph of the gray value analysis of the three repeated results of Figure A,Figure 4 Figure C in Figure 4 shows that jujuboside B inhibits the RNA level of EV71 virus; Figure D in

[0076] shows the change in virus titer in the corresponding cell supernatant in Figure A (****, P<0.0001). JuB and Rb are the abbreviations of jujuboside B and ribavirin respectively. Figure 4 As shown in Figure A in Figure 4 and Western blot (see Figures B and C in

[0077] and the virus titer results in the cell supernatant, it can be seen that jujuboside B inhibits the replication of EV71 virus in human rhabdomyoma cells RD in a dose-dependent manner. Moreover, the inhibitory effect of 40 μg / ml jujuboside B is significantly better than that of the positive drug ribavirin (100 μg / mL).

[0078] In summary, through the above experiments, it can be seen that jujuboside B has broad-spectrum anti-enterovirus replication activity in vitro. The specific effects are as follows: 50 1) The present invention first provides the toxicity of jujuboside B on human rhabdomyoma cells RD and normal human umbilical vein endothelial cells HUVEC, and its CC

[0079] are 42.15 μg / mL and 234.3 μg / mL respectively. This shows that jujuboside B has low toxicity and has a potential clinical use basis. 50 for anti-EV71 virus on RD cells is 4.021 μg / mL; the EC 50 for anti-CVA10 virus on RD cells is 3.383 μg / mL; the EC 50 for anti-CVA16 virus on RD cells is 4.160 μg / mL; the EC 50 for anti-EVD68 virus on RD cells is 5.208 μg / mL. Combining its CC 50 on RD, the therapeutic indices of jujuboside B against EV71 virus, CVA10 virus, CVA16 virus, and EVD68 virus on human rhabdomyoma cells RD are 10.48, 12.46, 10.13, and 8.09 respectively. This shows that jujuboside B has broad-spectrum anti-enterovirus activity.

[0080] 3) Again, it is provided that jujuboside B has antiviral activity by inhibiting EV71 virus replication. It significantly inhibits the viral RNA level and protein expression level of EV71 in RD cells in a dose-dependent manner. This indicates that jujuboside B has a significantly excellent inhibitory effect on enteroviruses.

[0081] In summary, jujuboside B has the effect of inhibiting enteroviruses, can inhibit the replication of enteroviruses, and thus jujuboside B can be used as or prepared into a drug for preventing enterovirus infection or treating enterovirus infection, thereby providing a safe, highly effective, and low-toxicity potential drug for the prevention or treatment of enterovirus infection.

[0082] As described above, it is only a preferred embodiment of the present invention, and there is no limitation in any form to the present invention. Any simple modification, equivalent change, and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An application of jujuboside B in the preparation of enterovirus inhibitors, wherein: The structural formula of the jujuboside B is as follows: ； Among them, the enterovirus inhibitor can inhibit one or more of the enteroviruses selected from EV71, CVA10, CVA16, EVD68, and CVA6.

2. The use of jujuboside B according to claim 1 in the preparation of enterovirus inhibitors, characterized in that: The enterovirus inhibitor can inhibit the replication of enterovirus.

3. The use of jujuboside B according to claim 1 or 2 in the preparation of enterovirus inhibitors, characterized in that: The enterovirus inhibitor is a drug for preventing enterovirus infection or a drug for treating enterovirus infection.

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