Application of chlorogenic acid in preparation of medicine for resisting Zika virus
By using chlorogenic acid and its pharmaceutically acceptable salts, an anti-Zika virus drug was prepared, solving the problem of the lack of effective drugs in the prior art and achieving effective inhibition of Zika virus and protection of host cells.
Patent Information
- Application Number
- CN202511672209.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-14
- Publication Date
- 2026-02-17
AI Technical Summary
Currently, there are no effective specific anti-Zika virus drugs. Existing treatment strategies are mainly symptomatic treatments, and no vaccines or drugs have been approved for marketing.
Anti-Zika virus drugs were prepared by using chlorogenic acid and its pharmaceutically acceptable salts to inhibit Zika virus replication and activate host type I interferon.
Chlorogenic acid exhibits a half-maximal inhibitory rate (EC50) of 15.27 ± 1.59 μM at the cellular level, demonstrating a concentration-dependent effect. It is also safe for host cells and can effectively prevent and treat Zika virus infection.
Smart Images
Figure CN121534033A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of Zika virus treatment technology, and in particular to the use of chlorogenic acid in the preparation of anti-Zika virus drugs. Background Technology
[0002] Zika virus (ZIKV) is a single-stranded positive-sense RNA virus belonging to the Flaviviridae family and Flavivirus genus. Infection with Zika virus causes Zika virus disease, clinically characterized by fever and rash syndrome. Aedes albopictus and Aedes aegypti mosquitoes are the main vectors. Typical clinical manifestations of Zika virus infection include acute fever, accompanied by skin and mucous membrane symptoms such as maculopapular rash, joint pain, or conjunctivitis. In addition, infected individuals often experience systemic symptoms such as myalgia and headache. Zika virus infection is associated with Guillain-Barré syndrome and symptoms of autoimmune diseases, and is directly linked to an increased incidence of microcephaly in newborns. These clinical manifestations illustrate the potential threat of Zika virus to public health and the urgent need for effective prevention and control measures.
[0003] In the field of antiviral treatment for Zika virus (ZIKV) infection, no specific antiviral drugs have been approved for clinical use. Clinical practice primarily employs symptomatic treatment strategies, such as using acetaminophen to relieve fever and pain, and antihistamines to alleviate itchy rashes. In recent years, numerous lead compounds and drug candidates have been developed, some of which have entered clinical trials. These drugs can be divided into two main categories based on their mechanisms of action: drugs that act directly on the virus and drugs that act on host cells. Significant efforts have been invested in the development of vaccines and antiviral drugs, but to date, no vaccine or drug has been approved for market.
[0004] Honeysuckle (scientific name: Lonicera japonica Thunb Honeysuckle (Lonicera japonica), belonging to the genus Lonicera of the family Caprifoliaceae, is a perennial, semi-evergreen woody vine. Its flower buds are used as a medicinal material in traditional Chinese medicine, possessing the characteristic of being both food and medicine. One of the key active ingredients in honeysuckle is chlorogenic acid (CGA), which is present at approximately 0.5-1.5% in fresh flower buds and 2-4% in dried flower buds. Chlorogenic acid is not only the main active ingredient in honeysuckle extract, but also, according to the Pharmacopoeia of the People's Republic of China (2020 edition), is one of the standard indicators for evaluating the quality of honeysuckle medicinal materials and its extracts.
[0005] Currently, there are no clinically approved drugs specifically effective for the prevention and treatment of Zika virus infection. Therefore, finding drugs that can effectively combat Zika virus is of great significance. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of chlorogenic acid in the preparation of drugs against Zika virus.
[0007] The first objective of this invention is to provide the use of chlorogenic acid in the preparation of anti-Zika virus drugs.
[0008] A second objective of this invention is to provide the use of pharmaceutically acceptable salts of chlorogenic acid in the preparation of anti-Zika virus drugs.
[0009] A third objective of this invention is to provide the use of chlorogenic acid in the preparation of medicaments for treating diseases caused by the Zika virus.
[0010] A fourth object of the present invention is to provide the use of pharmaceutically acceptable salts of chlorogenic acid in the preparation of medicaments for treating diseases caused by the Zika virus.
[0011] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention discovered chlorogenic acid (CGA, It has a good inhibitory effect on Zika virus, with an EC50 of 15.27±1.59 μM at the cellular level, and the effect is concentration-dependent. At the same time, it has a mechanism of highly activating host type I interferon to exert antiviral infection, while being relatively safe for host cells, and can be used for the prevention and treatment of Zika virus infection.
[0012] Therefore, this invention claims protection for the following applications: Application of chlorogenic acid in the preparation of anti-Zika virus drugs; The application of pharmaceutically acceptable salts of chlorogenic acid in the preparation of anti-Zika virus drugs; Application of chlorogenic acid in the preparation of drugs against Zika virus-induced diseases; The use of pharmaceutically acceptable salts of chlorogenic acid in the preparation of drugs to combat diseases caused by the Zika virus.
[0013] Preferably, chlorogenic acid promotes the production of interferon in host cells.
[0014] Preferably, the interferon is a type I interferon.
[0015] Preferably, the half-maximal inhibitory concentration (EC50) of chlorogenic acid is... 50 The value was 15.27 ± 1.59 μM.
[0016] Preferably, the drug also contains pharmaceutically acceptable excipients.
[0017] Preferably, the drug also has a pharmaceutically acceptable carrier.
[0018] Preferably, the drug is in the form of tablets, oral liquids, oral pills, oral granules, oral powders, injections, eye drops, nasal drops, aerosols, or inhalers.
[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention has discovered that chlorogenic acid has a good inhibitory effect on Zika virus infection, with an EC50% (half-maximal inhibitory concentration) at the cellular level. 50 The concentration is 15.27 μM and exhibits a concentration-dependent effect; at the same time, it has a mechanism of highly activating host type I interferon to exert antiviral infection, while being relatively safe for host cells, and can be used for the prevention and treatment of Zika virus infection. Attached Figure Description
[0020] Figure 1 This diagram illustrates the cytotoxic effects of chlorogenic acid on Zika virus-susceptible cells C6 / 36 and U251.
[0021] Figure 2 The study investigated the inhibitory effect of chlorogenic acid on Zika virus viral particle production in C6 / 36 cells and its half-maximal inhibitory concentration (EC50). 50 .
[0022] Figure 3 Chlorogenic acid inhibits the expression of Zika virus NS1 protein in U251 cells.
[0023] Figure 4 This study aimed to investigate the protective effect of chlorogenic acid on the survival of U251 cells during Zika virus infection.
[0024] Figure 5 Chlorogenic acid can promote the production of type I interferon in U251 cells. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.
[0026] 1. Chlorogenic acid monomer (CGA) Chlorogenic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0027] 2. Viruses and cells The Zika virus strain was isolated and cultured by the inventor's research group. The virus was amplified and cultured using the Aedes albopictus cell line C6 / 36, which was preserved by the inventor's research group.
[0028] Zika virus-susceptible cells U251 (human glioma cell line) were preserved by the inventor's research group.
[0029] Example 1: Chlorogenic acid inhibits Zika virus replication I. Experimental Methods 1. Cell culture (1) Cell resuscitation Remove the cryovials containing Zika virus susceptible cells C6 / 36 or U251 from liquid nitrogen and immediately thaw them completely in a 37°C water bath. Centrifuge at 800 r / min for 5 min at room temperature. Discard the supernatant, add cell culture medium, gently pipette to resuspend the cells, and incubate in a 37°C, 5% CO2 cell culture incubator.
[0030] (2) Cell passage Once the cells have reached confluence, discard the culture medium, wash twice with PBS, add 0.25% trypsin, and incubate in a cell culture incubator at 37°C and 5% CO2 for 1 min. Discard the trypsin, add culture medium, pipette the cells until evenly dispersed, and incubate in a cell culture incubator at 37°C and 5% CO2.
[0031] (3) Cell cryopreservation Cells in the logarithmic growth phase were digested with trypsin, dispersed by pipetting in culture medium, centrifuged at 1000 r / min for 10 min, the supernatant was discarded, the cells were resuspended in cryopreservation solution, and transferred to cryovials. The cells were then subjected to a gradient cooling process: 4 ℃ for 20 min, -20 ℃ for 30 min, and -80 ℃ overnight, before being transferred to liquid nitrogen for storage.
[0032] 2. Detection of the toxic effects of CGA on ZIKV susceptible cells C6 / 36 and U251 The Aedes albopictus cell line C6 / 36 (1×10) was used. 4 / well) and human glioma cell line U251 (1×10 4 C6 / 36 and U251 cells were seeded in 96-well plates. After 24 hours, different concentrations of CGA compound (final concentrations of 0, 10, 20, 40, 80 and 160 μM) were added to treat the cells for 48 hours. Cell viability was measured by MTT assay to detect the effect of CGA on the growth of C6 / 36 and U251 cells.
[0033] 3. Detection of the effect of CGA on ZIKV virus replication C6 / 36 cells (2×10) 5Cells were seeded in 6-well plates and treated with different concentrations (final concentrations of 0, 1, 5, 10, 20, 40, 80, μM) of CGA compound for 2 h after 24 h. Then, the cells were infected with ZIKV virus solution (MOI=1) (CGA was added to maintain the previous treatment concentration). After 24 h, the cell culture supernatant was collected and the effect of different concentrations of CGA on the amount of live virus in the cell supernatant was determined by plaque assay.
[0034] U251 cells (2×10) 5 Cells were seeded in 6-well plates and treated with different concentrations (final concentrations of 0, 1, 5, and 15 μM) of CGA compound for 2 hours after 24 hours. Then, the cells were infected with ZIKV virus solution (MOI=1) (CGA was added to maintain the previous treatment concentration). Cell lysates were collected after 24 hours, and the nsP1 protein level of ZIKV virus in the cell lysates was detected by Western blotting.
[0035] 4. The protective effect of CGA on host cell survival during ZIKV virus infection. U251 cells were treated with different concentrations of CGA compound (final concentrations of 0, 1, 5, 10, 20, 40, and 80 μM) for 2 h, and then infected with ZIKV virus solution (CGA was supplemented to maintain the previous treatment concentration). After 24 h, the cell viability of each group was measured by MTT assay to detect the protective effect of CGA on host cell survival during ZIKV virus infection.
[0036] II. Experimental Results 1. Cytotoxic effects of CGA on ZIKV-susceptible cells The cell viability of each group was determined by MTT assay, and the results are as follows: Figure 1 As shown, up to the highest concentration of 160 μM, CGA did not have a significant growth inhibitory effect on C6 / 36 and U251 cells.
[0037] 2. CGA can strongly inhibit the replication of ZIKV virus. The results of the plaque test are as follows Figure 2 As shown, CGA has strong inhibitory activity against ZIKV virus replication, and the effect is concentration-dependent, with a half-maximal inhibitory concentration (EC50) of 15.27 ± 1.59 μM.
[0038] Immunoblotting results are as follows Figure 3 As shown, CGA has a strong inhibitory effect on the expression level of NS1 protein of ZIKV virus, and the effect is concentration-dependent.
[0039] The above experimental results all indicate that CGA has a strong ability to inhibit the replication of ZIKV virus.
[0040] 3. Detection of the protective effect of CGA on host cell survival during ZIKV virus infection. The results are as follows Figure 4 As shown, the survival rate of U251 cells increased in the CGA treatment group. At a CGA compound concentration of 10 μM, the survival rate of U251 cells after ZIKV infection exceeded 50%, and at a CGA compound concentration of 40 μM, the survival rate exceeded 80%. Furthermore, the protective effect of CGA on the survival of U251 cells under ZIKV infection was concentration-dependent. This indicates that CGA has a strong protective effect on host cells during ZIKV virus infection.
[0041] Example 2: Chlorogenic acid promotes the production of type I interferon in host cells. I. Experimental Methods The cervical cancer cell line U251 was selected and treated with the commonly used interferon inducer poly(I:C) to assess the production of type I interferon in the host cells. The specific procedures are as follows: U251 cells were treated with different concentrations (0, 1, 5, 15 μM) of CGA compound for 24 h. Cell lysates were collected, and the IFN-β-Luc dual reporter gene system (IFN-β-Luc) was used to detect whether CGA promoted IFN-β promoter transduction in cells. Simultaneously, cell culture supernatant was collected, and ELISA was used to detect the effect of CGA treatment on intracellular IFN-β expression levels.
[0042] II. Experimental Results The results of the detection using the IFN-β dual-luciferase reporter gene assay system (IFN-β-Luc) are attached. Figure 5 As shown in A, CGA can strongly promote the fluorescence signal of the IFN-β gene promoter activated by poly(I:C) in a concentration-dependent manner.
[0043] Meanwhile, the ELSIA experimental results are as follows Figure 5 As shown in Figure B, CGA strongly promotes the increase in the secretion level of IFN-β activated by poly(I:C). This suggests that CGA may have the function of promoting the production of type I interferon in host cells mediated by poly(I:C).
Claims
1. Use of chlorogenic acid in the preparation of a drug for resisting Zika virus.
2. Use of a pharmaceutically acceptable salt of chlorogenic acid in the preparation of a drug for resisting Zika virus.
3. Use of chlorogenic acid in the preparation of a drug for resisting diseases caused by Zika virus.
4. Use of a pharmaceutically acceptable salt of chlorogenic acid in the preparation of a drug for resisting diseases caused by Zika virus.
5. Use according to any one of claims 1 to 4, characterized in that, Chlorogenic acid promotes the production of interferon in host cells.
6. Use according to claim 5, characterized in that, The interferon is type I interferon.
7. Use according to any one of claims 1 to 4, characterized in that, The median effective inhibitory concentration EC50 of chlorogenic acid was 15.27 ± 1.59 μM. 50 The median effective inhibitory concentration EC50 of chlorogenic acid was 15.27 ± 1.59 μM.
8. Use according to any one of claims 1 to 4, characterized in that, The drug further comprises a pharmaceutically acceptable adjuvant.
9. Use according to any one of claims 1 to 4, characterized in that, The drug further comprises a pharmaceutically acceptable carrier.
10. Use according to any one of claims 1 to 4, characterized in that, The drug is in the form of a tablet, an oral liquid, an oral pill, an oral granule, an oral powder, an injection, an eye drop, a nose drop, an aerosol or an inhalant.