Application of ginsenoside Rg3 in preparation of medicine for resisting respiratory syncytial virus

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of ginsenoside Rg3 in preparation of drugs for resisting respiratory syncytial viruses. In-vivo and in-vitro experiments prove that the cell safety of the 20 (S)-ginsenoside and the inhibition effect of the 20 (S)-ginsenoside on RSV (Respiratory Syndrome Virus); the 20 (S)-ginsenoside Rg3 can inhibit invasion of viruses and reduce the virus load in animal bodies. The invention provides a theoretical basis for the development of 20 (S)-ginsenoside Rg3 as a drug for resisting respiratory syncytial virus.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of 20(S)-ginsenoside Rg3 in the preparation of anti-respiratory syncytial virus drugs. Background Art

[0002] Respiratory syncytial virus (RSV) infection poses a serious threat to human health, especially the health of infants and the elderly. According to relevant studies, about 3 million children under the age of 5 are hospitalized due to RSV-related lower respiratory tract infections each year, and 120,000 people die. After infection with RSV, bronchiolitis or pneumonia may occur in severe cases, and the risk of respiratory complications, asthma attacks and impaired lung function may be increased. At present, the development of RSV vaccines faces many challenges. Factors such as the genetic variation characteristics of the virus, the immune enhancement effect and the immune-compromised state of susceptible populations limit the effective response and application range of RSV vaccines. In terms of clinical treatment, the existing anti-RSV drugs mainly include ribavirin and palivizumab, but ribavirin has large side effects; and palivizumab is expensive, making it difficult for many patients to afford it, resulting in its inability to be widely popularized in clinical applications. Therefore, there is an urgent need to develop new, efficient and economical anti-RSV drugs.

[0003] Ginseng is a plant of the Araliaceae family, and its roots have been used as a natural tonic and medicinal plant in Asian countries for thousands of years. Ginsenosides are the main active ingredients of ginseng, among which 20(S)-ginsenoside Rg3 is a tetracyclic triterpenoid saponin. Modern pharmacological studies have found that it has a variety of significant pharmacological activities. In the field of anti-tumor, it can exert its effects through multiple mechanisms such as inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and inhibiting tumor angiogenesis; in terms of cardiovascular protection, it can regulate blood lipids, resist atherosclerosis, and protect myocardial cells; in addition, it also has immunomodulatory, anti-inflammatory, antioxidant, and anti-aging effects. These characteristics provide potential possibilities for its application in the development of anti-RSV drugs.

[0004] There is no relevant report on the anti-respiratory syncytial virus effect of 20(S)-ginsenoside Rg3. Summary of the invention

[0005] In order to solve the problems in the above background technology, the main purpose of the present invention is to provide the use of ginsenoside Rg3 in the preparation of anti-respiratory syncytial virus drugs. The 20(S)-ginsenoside Rg3 of the present invention has a good inhibitory effect on RSV virus (A2 strain and B strain).

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] Application of ginsenoside Rg3 in the preparation of anti-respiratory syncytial virus drugs.

[0008] Furthermore, the ginsenoside Rg3 is a ginsenoside monomer 20(S)-ginsenoside Rg3.

[0009] Furthermore, the drug is a preparation prepared with 20(S)-ginsenoside Rg3 as the only active ingredient and pharmaceutically acceptable excipients.

[0010] Furthermore, the content of the active ingredient in the preparation is >0.1 μM, preferably 0.1 to 100 μM, and more preferably 10 to 100 μM.

[0011] Furthermore, the drug dosage form is tablets, capsules, granules, suspensions, oral solutions or injections.

[0012] Furthermore, the drug is administered by injection, oral administration or external administration.

[0013] The beneficial effects of the present invention are:

[0014] 20(S)-ginsenoside Rg3 is derived from the traditional Chinese medicine ginseng, which has a wide source and low cost. Experimental verification shows that 20(S)-ginsenoside Rg3 has good biosafety and inhibitory effects on viruses. Developing it into a therapeutic drug for RSV virus has high economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described below in conjunction with the accompanying drawings:

[0016] Figure 1 is a graph showing the cytotoxic effects of different concentrations of 20(S)-ginsenoside Rg3 and its antiviral effects on RSV-A2;

[0017] Figure 2 is a graph showing the cytotoxic effects of different concentrations of 20(S)-ginsenoside Rg3 and its antiviral effects on RSV-B;

[0018] Figure 3 This is a graph showing the weight changes of mice in the normal group, model group, and treatment group over 5 days;

[0019] Figure 4 This is the viral load graph of the right lung of mice in the normal group, model group and treatment group after 5 days. DETAILED DESCRIPTION

[0020] The following examples further illustrate the technology of the present invention, but do not limit the protection scope of the present invention. Unless otherwise specified, the raw materials used in the present invention are all known products, which are obtained by purchasing commercial products.

[0021] The sources of some raw materials used in the present invention are as follows:

[0022] 20(S)-ginsenoside Rg3 was purchased from MedChemExpress (MCE);

[0023] Respiratory syncytial virus RSV-A2 strain was provided by Guangzhou National Laboratory;

[0024] Respiratory syncytial virus RSV-B strain was provided by Wuhan Institute of Virology, Chinese Academy of Sciences.

[0025] Embodiment 1:

[0026] S1. Cell culture and plating

[0027] Human laryngeal cancer epithelial cells Hep-2 were seeded in 96-well plates containing 10% FBSDMEM medium, with 1×10 4 The cells were placed in a 37°C, 5% CO2 incubator overnight to allow the cells to adhere to the wall and grow.

[0028] S2. Prepare different concentrations of 20(S)-ginsenoside dilutions and ginsenoside virus mixtures

[0029] Weigh 20(S)-ginsenoside Rg3 powder, dissolve it with DMSO, and prepare a 50mM stock solution. In the experiment, the drug stock solution was continuously diluted into 8 gradients of Chinese medicine dilutions (0.01μM, 0.1μM, 1μM, 10μM, 20μM, 50μM, 100μM, 250μM) with DMEM solution containing 2% FBS, and added to a 96-well plate (100μl / well). At the same time, an equal volume of DMSO group (final concentration 0.5%) control wells were set, and 3 replicate wells were set for each group.

[0030] S3. Infection of Hep-2 cells with different concentrations of 20(S)-ginsenoside

[0031] The culture supernatant in the 96-well plate was discarded, and the prepared 20(S)-ginsenoside dilutions of different concentrations were added to each well, and cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0032] S4. Cytotoxicity assay

[0033] After the drug acted on the cells for 48 hours, the cytotoxicity detection kit (CCK8 kit) was used to evaluate the toxicity of different concentrations of drugs on the cells. 50 The toxic concentration that caused 50% cell death is indicated.

[0034] Prepare the CCK8 detection solution according to the instructions of the kit, that is, add 10 μl / well of CCK8 solution to 100 μl / well of sterile PBS solution, pipette and mix well for use. Discard the drug dilution in the 96-well plate and aspirate it, and add 100 μl of CCK8 detection solution to each well in the plate. Place the culture plate in a 37°C, 5% CO2 incubator and incubate for 1.5 hours. Place the culture plate in an ELISA reader and shake it slowly for 1 minute, measure the optical density (OD value) at 450nm, and calculate the cell survival rate.

[0035] The cell viability calculation formula is:

[0036]

[0037] Embodiment 2:

[0038] S1. Cell culture and plating

[0039] Human laryngeal cancer epithelial cells Hep-2 were seeded in 96-well plates containing 10% FBSDMEM medium, with 1×10 4 The cells were placed in a 37°C, 5% CO2 incubator overnight to allow the cells to adhere to the wall and grow.

[0040] S2. Prepare different concentrations of 20(S)-ginsenoside dilutions and 20(S)-ginsenoside virus mixtures

[0041] Weigh 20(S)-ginsenoside Rg3 and positive drug ribavirin powder, dissolve with DMSO, and prepare a 50mM stock solution. Use DMEM solution containing 2% FBS to continuously dilute into 6 gradient drug dilutions, namely 0.2μM, 2μM, 20μM, 40μM, 100μM, and 200μM. Add 50μ to a 96-well plate, then add 50μl of the above drug dilution, and then add 50μl RSV-A2 strain or B strain virus liquid (infect Hep-2 cells with an infection multiplicity of MOI=0.1), gently pipet and mix, so that the final concentration of the drug is 0.1μM, 1μM, 10μM, 20μM, 50μM, and 100μM respectively; at the same time, set up 4 groups of control wells, including a virus-only group (final concentration of 1×10 3 PFU of RSV 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.

[0042] S3. Infection of Hep-2 cells with different concentrations of 20(S)-ginsenoside

[0043] The culture medium in the 96-well plate was discarded, and the prepared mixture of ginsenosides and viruses was added to each well, and the 96-well plate was placed in a 35°C, 5% CO2 incubator for 6 days.

[0044] S4. Antiviral effect detection

[0045] A cell viability test kit (CellTiter-Glo 2.0) was used to detect the neutralization ability of different concentrations of traditional Chinese medicine against viruses. The more cells survived, the stronger the neutralization ability of the traditional Chinese medicine at that concentration against RSV, and the better the antiviral effect. 50 It indicates the concentration required to inhibit virus replication by 50%. The specific operation is as follows:

[0046] Prepare according to the instructions: Thaw CellTiter-Glo 2.0 Reagent at room temperature. Discard the culture medium in the 96-well plate, rinse twice with sterile PBS and replace with 2% DMEM culture medium, then add CTG detection solution (100μl / well), pipette to mix, and incubate on a shaker at room temperature for 10 minutes. Transfer 50ul / well to an opaque white well plate, place the culture plate in an ELISA reader and shake slowly for 2 minutes, then measure the luminescence value. Calculate the inhibition rate of the drug on the virus.

[0047] The inhibition rate of the drug on the virus is expressed as:

[0048]

[0049] S5. Drug selection index (SI): SI is the safe range for judging the effect of drugs. The selection index (SI) is CC 50 / EC 50 .

[0050] The experimental results are as follows Figure 1-2 As shown in Figure 2, the CC of 20(S)-ginsenoside Rg3 in Hep-2 cells 50 The value is 73.07 μM; it inhibits the EC of RSV-A2 and B strains 50 The values ​​were 21.95 μM and 15.1 μM, respectively, indicating that virus replication can be effectively inhibited at lower concentrations. The selectivity index (SI) calculated according to the formula was 3.33 and 4.84, respectively, indicating that 20(S)-ginsenoside Rg3 has good anti-RSV activity and high safety in vitro.

[0051] Embodiment 3:

[0052] S1. RSV animal modeling and grouping

[0053] Twelve SPF grade female mice aged 6 to 8 weeks were prepared. The mice were randomly divided into 4 groups, with 3 mice in each group: except for the control group Vehicle, the other 3 groups were injected with 4×10 6 / 30μl RSV-A2 virus solution was used for infection. 1h after infection, mice began to be treated by gavage with an equal volume of drugs for 4 days. During the experiment, the weight of the mice was recorded. The groups and drug doses were as follows:

[0054]

[0055] S2. Determination of RSV-A2 viral load in lung tissue in vivo

[0056] On the fifth day of the experiment (the end point of the experiment), all mice were killed by cervical dislocation. The right lung tissue was quickly removed and the right lung suspension was prepared. The copy number of RSV-A2 virus in the mouse lung tissue was determined by QPCR to evaluate the inhibitory effect of 20(S)-ginsenoside Rg3 on RSV-A2 virus in vivo.

[0057] The experimental results are as follows Figure 3-4 As shown. Figure 3 It can be seen that except for the control group Vehicle, the body weight of all groups decreased significantly, confirming the successful construction of the mouse RSV infection model. Compared with the modeling group, the body weight of RSV-A2 mice treated with ribavirin and 20(S)-ginsenoside Rg3 increased significantly on the 4th day. This indicates that oral administration of 20(S)-ginsenoside Rg3 and ribavirin can alleviate the weight loss of RSV-A2 infected mice. Figure 4 It can be seen that compared with the normal group, the viral load in the lung tissue of the model group mice on the 4th day was significantly increased. In contrast, compared with the model group, the viral load in the lung tissue of mice treated with 20(S)-ginsenoside Rg3 and ribavirin was significantly reduced (P<0.0001). This further proves that 20(S)-ginsenoside Rg3 has a good anti-RSV-A2 virus effect in vivo. It shows that it has great potential as a drug for the treatment of RSV virus.

[0058] The above embodiments are only preferred technical solutions of the present invention and should not be regarded as limiting the present invention. The protection scope of the present invention shall be the technical solutions recorded in the claims, including equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. Application of ginsenoside Rg3 in the preparation of anti-respiratory syncytial virus drugs.

2. The use according to claim 1, characterized in that: The ginsenoside Rg3 is a ginsenoside monomer 20(S)-ginsenoside Rg3.

3. The use according to claim 2, characterized in that: The medicine is a preparation prepared by using 20(S)-ginsenoside Rg3 as the only active ingredient and adding pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The content of active ingredient in the preparation is >0.1 μM.

5. The use according to claim 3, characterized in that: The drug dosage form is tablet, capsule, granule, suspension, oral solution or injection.

6. The use according to claim 3, characterized in that: Furthermore, the drug is administered by injection, oral administration or external administration.

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