Application of isochlorogenic acid C in preparation of medicine for resisting respiratory syncytial virus type A strain

Abstract

The invention discloses application of isochlorogenic acid C in preparation of a medicine for resisting a respiratory syncytial virus A type strain, and belongs to the technical field of medicine. Experiments prove that the isochlorogenic acid C has an obvious inhibiting effect on the respiratory syncytial virus type A strain, so that the isochlorogenic acid C has an important application value when being used as an active ingredient for preparing the respiratory syncytial virus type A strain medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular to the application of isochlorogenic acid C in preparing drugs for resisting respiratory syncytial virus type A strains. Background Art

[0002] Respiratory syncytial virus (RSV) is a medium-sized single-stranded RNA virus of the genus Orthopneumovirus in the family Pneumoviridae. RSV is mainly divided into two subtypes, A and B. It is generally believed that the difference between these two subtypes does not constitute a difference in serotypes (Meng J, Stobart CC, Hotard AL, Moore ML. An overview of respiratory syncytial virus[J]. PLoS Pathog. 2014;10(4):e1004016.). The glycoprotein G of RSV is highly variable and has great differences, which is the main basis for the comparison of RSV phylogeny (Melero JA, Mas V, McLellan JS. Structural, antigenic and immunogenic features of respiratory syncytial virus glycoproteins relevant for vaccine development[J]. Vaccine. 2017;35(3):461-468.).

[0003] RSV is the most important viral pathogen for acute lower respiratory tract infection in children under 5 years of age worldwide, with more than 3 million RSV-infected children requiring hospitalization each year (Shi T, McAllister DA, O'Brien KL, et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study[J]. Lancet. 2017;390(10098):946-958.). RSV can also infect adults, causing colds, bronchitis, pneumonia and other diseases, and significantly increases the morbidity and mortality of the elderly and patients with underlying heart or lung diseases (Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory syncytial virus infection in elderly and high-risk adults[J]. N Engl J Med. 2005;352(17):1749-1759.). For more than 50 years, RSV has been the focus of vaccine and antiviral drug research and development. As a respiratory pathogen that seriously endangers the health of infants and young children, there is no effective vaccine to prevent RSV infection so far, and the existing therapeutic drugs still have many shortcomings. RSV still lacks specific drugs for the treatment or prevention of infection. The high price of palivizumab limits its scope of use, and the side effects caused by the use of ribavirin also discourage many patients from using it. Faced with the disease-enhancing effects caused by many RSV vaccines, they are helpless. In China, only one preventive monoclonal antibody, nicervir monoclonal antibody, has been approved, but nicervir monoclonal antibody can only maintain 4-6 months of protection against RSV infection, and cannot provide treatment and lifelong protection, so it is necessary to find and develop other drugs.

[0004] Isochlorogenic acid C, chemically known as 4,5-dicaffeoylquinic acid, is a dicaffeoylquinic acid compound composed of one quinic acid molecule and two caffeoyl molecules. This compound is widely found in a variety of medicinal plants, such as honeysuckle of the Caprifoliaceae family, stevia, Xanthium sibiricum, and Erigeron breviscapus of the Asteraceae family, and its natural resources are very abundant (DOI: 10.27257 / d.cnki.gnxhc.2019.000738). There are also many studies on isochlorogenic acid. For example, the Chinese invention patent application with application number 202310330920.9 discloses a traditional Chinese medicine composition, comprising active ingredients extracted and purified from Artemisia annua, honeysuckle and gardenia, the active ingredients including: more than 2‰ of neochlorogenic acid, more than 5‰ of chlorogenic acid, more than 2‰ of cryptochlorogenic acid, more than 0.07‰ of caffeic acid, more than 0.35‰ of isochlorogenic acid B, more than 0.17‰ of isochlorogenic acid A, more than 0.19‰ of isochlorogenic acid C, more than 5.97‰ of gardenia glycoside, more than 0.07‰ of broken-oxidized strychnosinoside, more than 0.17‰ of gardenia glycoside, more than 0.22‰ of geniposide, more than 0.76‰ of geniposide, and more than 2.44‰ of 7-hydroxyswertia; the composition contains more than 10.3‰ of total acid and more than 21.58‰ of total glycoside, and is used in the preparation of antipyretic and anti-inflammatory drugs. For example, Chinese invention patent application No. 200810121523.6 discloses the use of isochlorogenic acid compounds and different combinations in the treatment of hepatitis. For example, the patent application with application number 202110385137.3 discloses a Chinese medicine composition, comprising active ingredients extracted and purified from Artemisia annua, honeysuckle, and gardenia, wherein the active ingredients include: more than 2‰ of neochlorogenic acid, more than 5‰ of chlorogenic acid, more than 2‰ of cryptochlorogenic acid, more than 0.07‰ of caffeic acid, more than 0.35‰ of isochlorogenic acid B, more than 0.17‰ of isochlorogenic acid A, more than 0.19‰ of isochlorogenic acid C, more than 5.97‰ of gardenia glycoside, more than 0.07‰ of broken-oxidized strychnosyl glycoside, more than 0.17‰ of gardenia glycoside, more than 0.22‰ of geniposide, more than 0.76‰ of geniposide, and more than 2.44‰ of 7-hydroxysweettaria; the composition contains more than 10.3‰ of total acid and more than 21.58‰ of total glycoside, and is used to prepare antipyretic and anti-inflammatory drugs.

[0005] There are few reports on the antiviral research of isochlorogenic acid C. Only the Chinese invention patent application with application number 03129007.8 discloses chlorogenic acid and isochlorogenic acid, which have been shown to have significant anti-SARS virus activity through pharmacological tests. Chlorogenic acid, isochlorogenic acid and vitamin C with such activity are used as the main active ingredients, and excipients, adjuvants or carriers are added according to different requirements to prepare compositions of different dosage forms. They can be used in the preparation of drugs for the treatment and prevention of diseases caused by SARS virus. Summary of the invention

[0006] In response to the above problems, the present invention aims to provide an anti-respiratory syncytial virus type A strain drug with the advantages of low toxicity, small side effects, multi-target effects, and immune regulation through antiviral effect studies based on the cell level, infection studies at different concentrations, inhibition rate studies on respiratory syncytial virus type A strains, and antiviral effects based on the animal level.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: Application of isochlorogenic acid C in the preparation of drugs against respiratory syncytial virus type A strains.

[0008] Preferably, the concentration of isochlorogenic acid C in the anti-respiratory syncytial virus type A drug is ≥ 0.01 wt%.

[0009] Isochlorogenic acid C exhibits potent anti-RSV activity and is highly safe by directly inhibiting viral invasion and replication. Its multi-mechanism synergistic effect not only reduces the risk of drug resistance, but also provides an ideal candidate molecule for the development of new anti-RSV drugs, especially for the treatment of respiratory infections in children and immunocompromised people.

[0010] Compared with the prior art, the beneficial effects of the present invention are: (1) Unlike Western medicine and biological preparations, isochlorogenic acid C is widely found in a variety of medicinal plants. It is easy to obtain and has abundant resources. It has the advantages of low cost, oral administration, low toxicity, few side effects, multi-target effects, and immune regulation.

[0011] (2) The present invention proposes for the first time that isochlorogenic acid C has a strong ability to resist respiratory syncytial virus type A strains, and its unexpectedly strong inhibitory activity against respiratory syncytial virus type A strains provides more medical uses for isochlorogenic acid C. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure shows the toxic effect of different concentrations of isochlorogenic acid C on cells and the inhibitory effect on RSV A2 virus.

[0013] Figure 2 This is a graph showing the weight changes of mice in different groups during the animal experiment.

[0014] Figure 3 This is a graph showing the viral load in the right lung of mice in different groups during the animal experiment.

[0015] Figure 4 These are the results of left lung pathological sections of mice in different groups in the animal experiment. DETAILED DESCRIPTION

[0016] The technical scheme of the present invention is further described below by way of examples. The raw materials used in the examples can be commercially available or prepared by conventional methods. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0017] Example 1 Study on antiviral effects at the cellular level.

[0018] 1. Cytotoxicity assay (1) Cell culture and plating Human laryngeal cancer epithelial cells Hep-2 were seeded in 96-well plates containing 10% FBS DMEM medium, with 1x10 4 cells at 37°C, 5% CO 2 Incubate overnight in an incubator.

[0019] (2) Preparation of different concentrations of Chinese medicine dilutions and Chinese medicine-virus mixtures The isochlorogenic acid C powder was dissolved in DMSO to prepare a 50 mM stock solution. In the experiment, the drug stock solution was continuously diluted into 8 gradient dilutions of traditional Chinese medicine (0.01 μM, 0.1 μM, 1 μM, 10 μM, 20μ M, 50μM, 100 μM, 250 μM) with DMEM solution containing 2% FBS, and added to a 96-well plate (100 μl / well). At the same time, an equal volume of control wells was prepared, i.e., only DMSO group (final concentration 0.5%), and each group was repeated 3 times.

[0020] (3) Infection of Hep-2 cells with different concentrations of Chinese medicine The culture medium in the 96-well plate containing Hep-2 cells was discarded with a pipette, and the prepared drug dilutions were added respectively. 2 Culture in an incubator for 48 h.

[0021] (4) Drug cytotoxicity testing The cytotoxicity test kit (cck8 kit) was used to evaluate the toxic effects of different concentrations of drugs on cells. The optical density (OD value) of the solution was measured by a microplate reader. The OD value is proportional to the activity of the cells. The specific operation is as follows: prepare the cck8 test solution according to the instructions, that is, add 10 μl / well of sterile PBS solution to 100 μl / well of cck8 solution, pipette and mix well for use. Use a pipette to aspirate the Chinese medicine dilution in the 96-well plate, and add 100 μl of cck8 test solution to each well of the plate. The culture plate was placed at 37 °C, 5% C0 2Incubate in the incubator for 1.5 hours. Place the culture plate in an ELISA reader and shake slowly for 1 minute, then measure the optical density (OD value) at 450 nm. Cell viability is expressed as: Relative cell viability (%) = OD 450 (Experimental groups with different drug concentrations) / OD 450 (DMSO control group) × 100%.

[0022] 2. Research on the detection of drug inhibition rate on viruses.

[0023] (1) Cell plating Human laryngeal cancer epithelial cells Hep-2 were seeded in 96-well plates containing 10% FBS DMEM medium, with 1x10 4 cells at 37°C, 5% CO 2 Incubate overnight in an incubator.

[0024] (2) Preparation of different concentrations of Chinese medicine dilutions and Chinese medicine-virus mixtures Isochlorogenic acid C powder and positive control drug ribavirin were dissolved in DMSO to prepare a 50 mM stock solution. In the experiment, DMEM solution containing 2% FBS was used to continuously dilute the solution into 6 gradient drug dilutions (0.2 μM, 2 μM, 20 μM, 40 μM, 100 μM, 200 μM), and added to a 96-well plate (50 μl / well). Then, 50 μl of RSV-A2 virus solution (Hep-2 cells were infected at a multiplicity of infection of MOI=0.1) was added, and the mixture was gently pipetted to make the final concentration of the drug: 0.1 μM, 1 μM, 10 μM, 20μ M, 50 μM, 100 μM; at the same time, 4 groups of control wells were prepared with equal volumes, including a virus-only group (final concentration of 1×10 3 PFU of RSV-A2 virus solution), DMSO only group (final concentration 0.5%), DMSO+virus group, and 2% DMEM blank group (cell control group), each group was repeated 3 times.

[0025] (3) Infection of Hep-2 cells with different concentrations of Chinese medicine The culture medium in the 96-well plate containing Hep-2 cells was discarded with a pipette, and the prepared drug and virus mixtures were added respectively. 2 Culture in an incubator for 6 days.

[0026] (4) Antiviral testing The cytotoxicity detection kit (cck8 kit) was used to determine the neutralization ability of different concentrations of traditional Chinese medicine against viruses. The more cells survived, the stronger the neutralization ability of traditional Chinese medicine at that concentration against RSV. The specific operation is as follows: prepare the cck8 detection solution according to the instructions, that is, add 10 μl / well of sterile PBS solution to 100 μl / well of cck8 solution, pipette and mix well for use. Use a pipette to aspirate the mixture of traditional Chinese medicine and virus in the 96-well plate after 6 days of culture, and add 100 μl of cck8 detection solution to each well of the plate. The culture plate was placed at 35 ℃, 5% C0 2 Incubate in the incubator for 1.5 h. Place the culture plate in an ELISA reader and shake slowly for 1 min, then measure the optical density (OD value) at 450 nm. The inhibition rate of the drug on the virus is expressed as [OD 450 (Experimental groups with different drug concentrations) - OD 450 (Virus control group)] / [OD 450 (DMSO control group)-OD 450 (virus control group)]×100%.

[0027] The results are as follows Figure 1 As shown in the data, different concentrations of isochlorogenic acid C had very low toxic effects on cells, with the cell half-toxic concentration being greater than 250 μM. It had a significant inhibitory effect on RSV A2 virus in a concentration-dependent manner, with the drug half-effective concentration being 5.748 μM, and the drug SI value being >43.49, indicating that isochlorogenic acid C has both potent anti-RSV A2 viral activity and high cellular safety, and is a highly potential candidate anti-RSV-A2 drug.

[0028] Example 2 Antiviral effect study based on animal level.

[0029] Healthy 6-8 week old Balb / c female mice were selected and randomly divided into three groups according to their average body weight: A2 challenge experimental group, A2 challenge plus drug group, and control group. The experimental design is as follows: A2 challenge experimental group: 30 μl of 4 × 10 6 Mice were anesthetized with isoflurane for about two minutes and then their body weight was measured and then intranasal drip was administered. A2 challenge and administration group: mice were gavaged 1 h after being infected with RSV A2. The drug powder was dissolved in a solvent (10% DMSO+40% PEG 300+5% Tween-80+45% Saline, v:v:v:v). The concentrations of isochlorogenic acid C and the positive control drug ribavirin were 5 mg / mL. The dosages of isochlorogenic acid C and the positive control drug ribavirin were both 50 mg / kg. The mice were gavaged for 4 consecutive days.

[0030] Control group: Mice in the control group were given solvent (10% DMSO + 40% PEG 300 + 5% Tween-80 + 45% Saline, v:v:v:v) by gavage for 4 consecutive days.

[0031] The weight of mice was recorded every day. On the 4th day after infection, mice were euthanized and lungs were removed. The left lung was fixed in tissue fixative, embedded, sliced, and stained with HE to observe the pathological changes of lung tissue. After weighing the right lung, 400 μL PBS was added to the tube, ground with a grinder, and RNA was extracted from the tissue supernatant to determine the number of viral copies.

[0032] Figure 2 Indicates the weight changes of mice in different groups in the animal experiment. Compared with the first day, the weight of mice in the control group first increased and then decreased, while the weight of the other three groups decreased first and then increased. Compared with the RSV A2 challenge group, the weight of mice in the challenged and intragastricly gavaged isochlorogenic acid C group recovered the fastest, and the weight recovery effect of mice in the challenged and intragastricly gavaged ribavirin group was better than that of the RSV A2 challenge group from the third day. It is obvious from the weight of mice that isochlorogenic acid C is more conducive to the recovery of mice, and the effect is stronger than that of the ribavirin group.

[0033] Figure 3 In the animal experiment, the viral loads in the right lung of mice in the control group, the RSV A2-challenged group, the challenged and intragastricly administered isochlorogenic acid C group, and the challenged and intragastricly administered ribavirin group were significantly different from those in the RSV A2-challenged group, indicating that the compound can significantly reduce the viral load of RSV A2 in mice, and the effect is much higher than that of ribavirin.

[0034] Figure 4 The results of pathological sections of the left lungs of mice in the control group, the mouse RSV A2 challenge group, the challenge and intragastric administration of isochlorogenic acid C group, and the challenge and intragastric administration of ribavirin group in the animal experiment showed that the alveolar structure of the control group was honeycomb, with no interstitial thickening, no exudate or inflammatory cells. The alveolar cavity of the challenge group was filled with powdery exudate, a large number of inflammatory cells infiltrated the interstitium, and alveoli in some areas were fused. The inflammatory cell infiltration of the lung tissue in the challenge and intragastric administration of isochlorogenic acid C group was significantly reduced compared with the challenge group, the exudate in the alveolar cavity was absorbed, the alveolar wall structure was partially restored, and the interstitial edema was reduced. Compared with the challenge and intragastric administration of ribavirin group, the challenge and intragastric administration of isochlorogenic acid C group significantly alleviated the RSV A2 virus-induced lung tissue pathological damage, the exudate was reduced more, and the alveolar structure recovery effect was better, proving that isochlorogenic acid C has a significant anti-RSV A2 effect and is more effective than ribavirin.

[0035] It should be understood that the above embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. Application of isochlorogenic acid C in the preparation of drugs against respiratory syncytial virus type A strain.

2. The use of isochlorogenic acid C according to claim 1 in the preparation of an anti-respiratory syncytial virus type A drug, characterized in that: The concentration of the isochlorogenic acid C in the drug against respiratory syncytial virus type A strain is ≥0.01wt%.

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