Use of yohimbine in the preparation of a drug for preventing respiratory syncytial virus infection

Abstract

The present invention relates to the technical field of antiviral drugs, and specifically discloses the application of yohimbine in the preparation of drugs for treating respiratory syncytial virus infection. The application of yohimbine in the preparation of drugs for treating respiratory syncytial virus infection provided by the present invention proves that yohimbine can inhibit the proliferation of respiratory syncytial virus and its infection of host cells, and can be used as a specific drug for treating diseases caused by respiratory syncytial virus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly to the application of yohimbine in the preparation of drugs for anti-respiratory syncytial virus (RSV) infection. Background Art

[0002] Respiratory syncytial virus (RSV) is one of the key pathogens causing lower respiratory tract infections. It is estimated that more than half of children will be infected with this virus within the first year after their birth, and almost all children will be infected within two years after birth. After infection with RSV, it can cause repeated wheezing, asthma, and decline in respiratory function in children, and increase the sensitivity of allergic reactions. These will lead to a significant decline in the quality of life of children and increase the cost of using healthcare resources. As people age, the RSV infection rate in adults will gradually increase. Research shows that the number of elderly people infected with RSV in some developed countries exceeds five million cases per year, and the death toll caused by RSV infection in developing countries is much higher than that in developed countries. However, no effective vaccine products have been developed yet, and its treatment methods have not yet met the demand. Therefore, it is crucial to find safe and effective anti-RSV drugs.

[0003] Phytochemicals refer to active substances extracted from plants. So far, more than a thousand phytochemicals have been discovered. They have diverse sources. Some important phytochemicals include carotenoids, bioflavonoids, glucosinolates, indoles, phenols and polyphenols, etc., which have strong antioxidant, antibacterial, antidiarrheal, anthelmintic, antiallergic, antispasmodic and antiviral activities.

[0004] Yohimbine is an indole substance derived from the bark of the West African yohimbe tree. It can selectively block the presynaptic α2 receptor and is a typical α2-adrenergic receptor antagonist. For decades, it has been used as a therapeutic agent for sexual difficulties and is a relatively safer drug with almost no known side effects. In addition, yohimbine has been reported to be involved in the antiviral process, including anti-herpes simplex virus, anti-HIV, etc. The latest research shows that yohimbine can play an important role in the treatment of coronavirus disease 2019 (COVID-19) by alleviating acute kidney injury and inhibiting the production of inflammatory factors. Generally speaking, yohimbine plays an important role in the treatment of myocardial dysfunction, antibacterial and antiviral, anti-inflammatory, antioxidant, antimalarial, etc. However, the relevant role of yohimbine in anti-respiratory syncytial virus has not been publicly reported. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art in anti-respiratory syncytial virus technology, the present invention provides the application of yohimbine in the preparation of drugs for anti-respiratory syncytial virus infection, and through experiments, it is proved that yohimbine has very excellent anti-respiratory syncytial virus effects and can be used as a specific drug for treating diseases caused by respiratory syncytial virus infection.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] Use of yohimbine in the preparation of a drug for preventing respiratory syncytial virus infection.

[0008] Preferably, the concentration of yohimbine in the anti-respiratory syncytial virus drug ≥ 0.01 wt%.

[0009] Preferably, the drug comprises a pharmaceutically acceptable carrier or excipient.

[0010] Preferably, the dosage form of the drug is at least one of tablets, granules, capsules, powders, injections, oral liquids or aerosols.

[0011] The use of yohimbine provided by the present invention in the preparation of a drug for preventing respiratory syncytial virus infection. Research shows that yohimbine has a very significant effect on anti-respiratory syncytial virus (strains A2 and B) activity, and yohimbine is derived from natural medicinal plant extracts, without chemical additives, is economically available, has low side effects, and high drug safety. Therefore, using it as an active ingredient in the preparation of drugs with anti-respiratory syncytial virus effects has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0014] Figure 1 It is the inhibitory effect diagram of yohimbine with different concentrations on RSV virus in the embodiments of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0016] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all conventional commercially available products, or can be prepared by conventional methods. The experimental methods without specific conditions are the conventional methods and conditions well-known in the art, or the conditions recommended by the manufacturer.

[0017] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0018] The sources of some raw materials used in the present invention are as follows:

[0019] Yohimbine was provided by Wuhan Botanical Garden, Chinese Academy of Sciences;

[0020] Respiratory syncytial virus RSV-A2 strain was provided by Guangzhou National Laboratory;

[0021] RSV-B strain was provided by Wuhan Institute of Virology, Chinese Academy of Sciences.

[0022] Example 1

[0023] 1. Prepare yohimbine extracts with different concentrations

[0024] Yohimbine was serially diluted with DMEM solution containing 2% FBS to obtain yohimbine extract dilutions with different concentrations (500 μM, 100 μM, 20 μM).

[0025] 2. Inhibitory experiment of yohimbine on RSV virus

[0026] 2.1 Cell culture

[0027] Human laryngeal carcinoma epithelial cells Hep-2 cells were seeded in a 96-well plate containing DMEM medium with 10% FBS, 1×10 4 cells per well, and cultured overnight for 24 hours in an incubator at 37 °C with 5% CO 2 .

[0028] 2.2 Prepare a mixture of yohimbine dilution and yohimbine virus

[0029] Hep-2 cells were infected at a multiplicity of infection of MOI = 0.1. The specific operation is as follows:

[0030] 1) In a sterile 96-well plate, different concentrations of yohimbine dilutions (500 μM, 100 μM, 20 μM) were added, 50 μl per well, and then 50 μl of RSV strain containing 2×10 3 PFU was added, and gently pipetted and mixed to make the final concentration of the drug: 250 μM, 50 μM, 10 μM;

[0031] 2) Prepare 4 groups of control wells with equal volume, including virus-only group (final concentration of 1×10 3 PFU of RSV-A2 strain), DMSO group with a concentration of 1% only, DMSO + virus group, and blank group with a concentration of DMEM containing 2% FBS (cell control group), and each group was repeated 3 times.

[0032] 2.3 Infection of Hep-2 cells with yohimbine at different working concentrations

[0033] Use a pipette to discard the culture medium in the 96-well plate containing Hep-2 cells, and add the mixed solution of yohimbine and virus prepared in step 2.2 respectively. Incubate in an incubator at 35°C with 5% CO 2 for 6 days.

[0034] 3. Test for the anti-respiratory syncytial virus activity of yohimbine

[0035] The inventors used a cck8 kit to determine the neutralizing ability of yohimbine at different concentrations against the virus. The more viable cells, the stronger the neutralizing ability of the traditional Chinese medicine at that concentration against RSV. The specific operation is as follows:

[0036] 1) Prepare the cck8 detection solution: Add 10 μl of cck8 solution to 100 μl of sterile PBS solution, mix well by pipetting and set aside for use;

[0037] 2) Use a pipette to aspirate the mixed solution of yohimbine and virus in the 96-well plate in step 2.3, and add 100 μl of cck8 detection solution to each well of the plate. Then incubate the culture plate at 35°C with 5% CO 2 in an incubator for 1.5 hours;

[0038] 3) Place the culture plate in an enzyme-linked immunosorbent assay (ELISA) reader, shake it slowly for 1 minute, and measure the absorbance at OD450.

[0039] 4) Calculate the inhibition rate of the drug against the virus based on the OD450 value:

[0040] Inhibition rate = [Absorbance (experimental group with different concentrations of drug) - Absorbance (virus group)] / [Absorbance (cell control group) - Absorbance (virus group)] * 100%. Draw the inhibition curve of yohimbine against respiratory syncytial virus.

[0041] Example 2

[0042] The operation steps are the same as those in Example 1, except that the virus strain used is the RSV-B strain.

[0043] The experimental results are as Figure 1As shown, for the RSV-A2 strain, compared with the control group without drug (Control-A2), yohimbine can effectively inhibit the activity of the virus in a concentration-dependent manner (IC50 = 36.2 μM); for the RSV-B strain, compared with the control group without drug (Control-B), although yohimbine at concentrations of 10 μM and 50 μM has certain antiviral ability, the antiviral ability at a concentration of 250 μM is more significant (IC50 = 96.54 μM). It can be seen that yohimbine has a significant effect on antiviral respiratory syncytial virus RSV and can be used to prepare drugs for respiratory syncytial virus RSV, providing more options for the clinical treatment of RSV.

[0044] The above embodiments are only the preferred technical solutions of the present invention and should not be regarded as limitations on the present invention. The protection scope of the present invention should be the technical solutions recorded in the claims, including the equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, the equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. The use of yohimbine in the preparation of a drug for resisting respiratory syncytial virus infection, characterized in that: The respiratory syncytial virus is RSV-A2 strain, and the IC of yohimbine to RSV-A2 strain 50 The value is 36.2μM.

2. The use of yohimbine according to claim 1 in the preparation of an anti-respiratory syncytial virus infection drug, characterized in that: The concentration of yohimbine in the anti-respiratory syncytial virus drug is ≥0.01wt%.

3. The use of yohimbine according to claim 1 in the preparation of a drug for resisting respiratory syncytial virus infection, characterized in that: The medicine includes pharmaceutically acceptable carriers or excipients.

4. The use of yohimbine according to any one of claims 1 to 3 in the preparation of a drug for resisting respiratory syncytial virus infection, characterized in that: The dosage form of the drug is at least one of tablets, granules, capsules, powders, injections, oral liquids or aerosols.

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