Application of triterpenoid saponin component QS-21 in preparation of composition for preventing and / or treating RSV infected diseases

Abstract

The invention relates to the technical field of virus infection resistance, in particular to application of a triterpenoid saponin component QS-21 in preparation of a composition for preventing and / or treating RSV infection diseases. The triterpenoid saponin component QS-21 is disclosed to inhibit and / or treat respiratory syncytial virus infection through in-vivo tests (animal potential toxicity test and animal treatment test after challenge), and the application of the triterpenoid saponin component QS-21 in resisting respiratory syncytial virus is further disclosed through in-vivo test results.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of anti-viral infection, and particularly relates to application of triterpenoid saponin component QS-21 in preparation of a composition for preventing and / or treating a disease of RSV infection. BACKGROUND

[0002] Respiratory Syncytial Virus (RSV) is the most important viral pathogen causing acute lower respiratory tract infection in infants, immunocompromised individuals and the elderly. It can cause respiratory diseases including pneumonia, bronchopneumonia, otitis media, rhinitis. RSV was first isolated in 1956, which belongs to the single negative-strand RNA virus of the Paramyxoviridae family, and only one serotype, divided into A and B subtypes, two subtypes of respiratory syncytial virus are prevalent alone. The immunity generated after RSV infection is not durable, which is easy to cause repeated infection, leading to limited treatment. This is related to the immune escape mechanism of the virus. There are multiple glycoproteins on the surface of the virus, among which the fusion protein F and the adhesion protein G can undergo antigenic variation, thereby avoiding the immune recognition and clearance of the body.

[0003] Studies have shown that RSV can also infect immune cells such as dendritic cells and inhibit their immune function, further reducing the body's immune clearance ability to the virus. The virus causes severe pneumonia, asthmatic bronchitis and other diseases with high viral detection rate in lower respiratory tract diseases. However, in upper respiratory tract infections such as acute pharyngitis and acute tonsillitis, the viral detection rate is low, which may be due to the strong multiple defense mechanisms of the mucus barrier, cilia movement, and local immune response in the upper respiratory tract.

[0004] RSV infection is globally widespread, and the prevalence is affected by factors such as geographical location, temperature and humidity. In northern China, the RSV epidemic season starts in mid-October and ends in mid-May of the following year, while in southern China, it is more common in winter and spring, which is closely related to temperature.

[0005] Saponin is a substance contained in South American soapbark tree with a wide range of biological activities, mainly including anti-allergy, antibacterial, anti-cholesterol, anti-inflammatory, analgesic, antipyretic, and immune adjuvant activity. Triterpenoid saponin component QS-21, as a natural saponin product with significant immune stimulating effect, has been used in many clinical trials, and it is used as an adjuvant in combination with various vaccines. The research results show that triterpenoid saponin component QS-21 can stimulate the local and transient activation of the body's innate immune response by intramuscular injection, causing the recruitment and activation of dendritic cells with immune antigen presentation function.

[0006] Triterpenoid saponin component QS-21 is used as a substance to improve the immunogenicity of antigens in recombinant vaccines, but the effect and mechanism of triterpenoid saponin component QS-21 alone on respiratory syncytial virus have not been reported. SUMMARY

[0007] The present application provides the use of triterpenoid saponin component QS-21 in the preparation of a composition for preventing and / or treating the disease of RSV infection. The present application discloses that triterpenoid saponin component QS-21 inhibits and / or treats the infection of respiratory syncytial virus through in vivo test (animal potential toxicity test and post-challenge animal treatment test), and further discloses the use of triterpenoid saponin component QS-21 in the anti-respiratory syncytial virus through the result of in vivo test.

[0008] As shown in the following structural formula, it is the structural formula of triterpenoid saponin component QS-21. Figure 1

[0009] In the first aspect, the present application provides the use of triterpenoid saponin component QS-21 in the preparation of a composition for preventing and / or treating the disease of RSV infection.

[0010] Optionally, the triterpenoid saponin component QS-21 has no obvious toxicity to cells.

[0011] Optionally, the triterpenoid saponin component QS-21 can effectively inhibit the infection of respiratory syncytial virus A and B subtypes to cells at the concentration of 100 μM.

[0012] Optionally, the triterpenoid saponin component QS-21 can effectively reduce the virus titer in lung tissue.

[0013] In the second aspect, the present application provides a triterpenoid saponin component QS-21 liposome carrier drug.

[0014] Optionally, the pH value of the triterpenoid saponin component QS-21 liposome carrier drug is 6.10±0.1.

[0015] Optionally, the particle size of the triterpenoid saponin component QS-21 liposome carrier drug is between 94.4±2.4 nm, and the particle size distribution is between 0.12±0.07.

[0016] Optionally, the triterpenoid saponin component QS-21 liposome carrier drug comprises the following components in weight parts: triterpenoid saponin component QS-21 1-2 parts; soybean lecithin 8-10 parts; cholesterol 4-6 parts; sodium cholate 6-8 parts; isopropyl myristate 3-5 parts.

[0017] Optionally, the triterpenoid saponin component QS-21 liposome carrier drug comprises the following components in weight parts: triterpenoid saponin component QS-21 1 part; soybean lecithin 9 parts; cholesterol 5 parts; sodium cholate 7 parts; isopropyl myristate 4 parts.

[0018] In summary, the present application includes at least one of the following beneficial technical effects: ​The application carries out toxicity test of triterpene saponin component QS-21 on in-vitro cells (Hep-2 cells), and the result shows that triterpene saponin component QS-21 has no obvious toxicity to cells.

[0019] The application carries out virus inhibition activity test of triterpene saponin component QS-21, and the result shows that triterpene saponin component QS-21 can effectively inhibit respiratory syncytial virus A and B subtypes from infecting cells at a concentration of 100 μM.

[0020] The application prepares triterpene saponin component QS-21 liposome carrier drug, and explores potential toxicity of triterpene saponin component QS-21 liposome carrier drug by intramuscular injection, and the result shows that triterpene saponin component QS-21 liposome carrier drug has good safety. In order to further prolong the drug retention time in the body and improve the drug stability, selecting liposome as the preparation form is beneficial to improving the bioavailability.

[0021] The application also carries out treatment test of triterpene saponin component QS-21 liposome carrier drug on virus infection of mice, and the result shows that triterpene saponin component QS-21 can effectively reduce the virus titer in lung tissues. By evaluating possible toxic reaction of different strains of mice after being given triterpene saponin component QS-21 at different concentrations by single intramuscular injection, the drug concentration of triterpene saponin component QS-21 in the treatment test of respiratory syncytial virus infection of mice is determined, which provides a direction for prevention or treatment of respiratory syncytial virus by triterpene saponin component QS-21.

[0022] The application discloses that triterpene saponin component QS-21 can inhibit and / or treat respiratory syncytial virus infection through in-vivo test (animal potential toxicity test and treatment test of animals after challenge), and the application further discloses the use of triterpene saponin component QS-21 in resisting respiratory syncytial virus through the in-vivo test result. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The structural formula of triterpene saponin component QS-21.

[0024] Figure 2 The statistical result of cell viability of Hep-2 cells under different dosing concentrations of triterpene saponin component QS-21.

[0025] Figure 3 The statistical result of inhibition of respiratory syncytial virus A subtype by different dosing concentrations of triterpene saponin component QS-21.

[0026] Figure 4 The statistical result of inhibition of respiratory syncytial virus B subtype by different dosing concentrations of triterpene saponin component QS-21.

[0027] Figure 5Statistical results of body weight changes of BALB / c mice in potential toxicity test of intramuscular injection of each administration concentration of triterpene saponin component QS-21.

[0028] Figure 6 Statistical results of body weight changes of KM mice in potential toxicity test of intramuscular injection of each administration concentration of triterpene saponin component QS-21.

[0029] Figure 7 Statistical results of body weight changes of mice in mouse virus infection treatment test.

[0030] Figure 8 Results of lung virus load in mouse virus infection treatment test. DETAILED DESCRIPTION

[0031] Before describing the embodiments of the present application in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only. Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] It should be noted that the terms "first", "second" are used only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined as "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0033] The endpoints of the ranges and any values disclosed in the present application are not limited to the precise values stated. The ranges or values should be construed to be roughly about the ranges or values. For ranges of values, the endpoints of the ranges are combined with the single points to form new ranges, and the new ranges are to be considered as being specifically disclosed.

[0034] In the present application, the term "comprising" or "including" is an open-ended expression, i.e. including the contents indicated in the present application, but not excluding other aspects.

[0035] In order to make the purposes, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. The embodiments described below are exemplary and are used to explain the present application, and cannot be understood as limiting the present application.

[0036] Unless otherwise specified, the techniques or conditions in the examples were carried out according to the techniques or conditions described in the literature or according to the product manual. Unless otherwise specified, the reagents or instruments used were conventional products that can be obtained commercially.

[0037] Some reagents used in the following examples are as follows: The PBS solution was purchased from Solarbio with the product number P1020; the Hep-2 cells were purchased from Fenghui Biology with the product number CL0567; RSV-A was purchased from ATCC with the product number VR-1540; RSV-B was purchased from ATCC with the product number VR-3381; the triterpenoid saponin component QS-21 was purchased from Huawen Tai with the product number HA103; the CCK-8 kit was purchased from Solarbio with the product number CA1210.

[0038] The present application is further described in detail below in combination with the examples and test results.

[0039] Example 1

[0040] This example provides a cytotoxicity test of the triterpenoid saponin component QS-21.

[0041] Specifically includes the following steps: (I) Cell recovery (1) Preheat the cell culture reagents in a 37°C water bath, wipe the ultraviolet irradiated clean bench and hands with 75% alcohol, and strictly prepare for aseptic operation.

[0042] (2) Take out the preheated cell culture reagents, wipe them with alcohol cotton balls, and then place them in the clean bench. Take out the Hep-2 cells from the liquid nitrogen tank and quickly thaw the cells in a 37°C water bath.

[0043] (3) Absorb the thawed cells into a centrifuge tube containing 10 mL of complete medium, and centrifuge at 1000 rpm for 5 min.

[0044] (4) Absorb the supernatant, add 10 mL of complete medium to blow the cells apart, and transfer to a cell culture bottle for 37°C 5% CO2 culture.

[0045] (II) Cell plating (1) After the single-layer Hep-2 cells were digested and blown evenly, the cell concentration was adjusted to 1×10 5 6 / mL, 100 μL / well was plated in a 96-well plate.

[0046] (2) The triterpenoid saponin component QS-21 was prepared with different drug concentrations (3.13, 6.25, 12.5, 25, 50, 100 μM) using culture medium (commercially available MEM basal medium, manufacturer GIBCO) and added to the cell solution. At the same time, cell control wells and blank wells were set up and cultured in a 37℃, 5% CO2 incubator for 24h.

[0047] (3) Discard the culture medium in the cell culture plate, wash twice with PBS solution, add fresh culture medium, and add 10 μL of CCK-8 reagent. Incubate the cell culture plate in an incubator for 1-4 hours, measure the absorbance at 450 nm using a microplate reader, and calculate the cell viability according to the following formula: Cell viability (%) = [A(drug-treated) - A(blank)] / [A(cell control) - A(blank)] × 100.

[0048] Wherein, A (dosage): absorbance of wells containing cells, CCK-8 solution and drug solution; A (blank): absorbance of wells containing culture medium and CCK-8 solution but no cells; A (cell control): absorbance of wells containing cells and CCK-8 solution but no drug solution.

[0049] (III) Detection results of cytotoxicity test of triterpenoid saponin component QS-21 The absorbance values ​​of Hep-2 cells at various drug concentrations of the triterpenoid saponin component QS-21 are shown in Table 1. The statistical results of Hep-2 cell viability are as follows: Figure 2 As shown.

[0050] Table 1 Absorbance values ​​(OD) of Hep-2 cells at different concentrations of triterpenoid saponin component QS-21 450nm ) test results

[0051] From Table 1 and Figure 2 It can be seen that the cell viability of Hep-2 cells was higher than 90% at all concentrations of the triterpenoid saponin component QS-21, indicating that the triterpenoid saponin component QS-21 has no obvious toxicity to cells. Example

[0052] This embodiment provides an experiment demonstrating the antiviral activity of the triterpenoid saponin component QS-21. Specifically, the following steps are included: (a) Virus inhibition test (1) After digesting and mixing the Hep-2 cells that had grown into a monolayer, the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL were inoculated into 6-well plates and grown overnight.

[0053] (2) The next day, RSV-A and B subtype virus solution (1 x 10 5 TCID50 / well) was added to infect the cells for 1 h at 37°C in a 5% CO2 incubator, and the virus solution was discarded, and the cells were washed with PBS solution.

[0054] (3) The triterpene saponin component QS-21 was prepared in different concentrations (12.5, 25, 50, and 100 μM) in culture medium (commercially available MEM basic medium, manufacturer GIBCO) and added to the cell solution, and a virus control well was set up, and the cells were placed in a 37°C, 5% CO2 incubator for 24 h.

[0055] (4) The culture supernatant was collected, and the RSV-A and B subtype virus titers were detected using the microcytopathic method. The ability of the triterpene saponin component QS-21 at different concentrations to inhibit different virus subtypes of RSV was investigated.

[0056] (II) Detection results of the triterpene saponin component QS-21 virus activity inhibition test The inhibition of each concentration of the triterpene saponin component QS-21 on each subtype of respiratory syncytial virus is shown in Table 2. The statistical results of the inhibition of respiratory syncytial virus subtype A are shown in Table 3. Figure 3 The statistical results of the inhibition of respiratory syncytial virus subtype B are shown in Table 4. Figure 4

[0057] Table 2 Inhibition of each concentration of the triterpene saponin component QS-21 on each subtype of respiratory syncytial virus (LgTCID50).​

Claims

1. Use of triterpenoid saponin component QS-21 in the preparation of compositions for the prevention and / or treatment of RSV infection.

2. The application according to claim 1, characterized in that, The triterpenoid saponin component QS-21 has no obvious toxicity to cells.

3. The application according to claim 1, characterized in that, The triterpenoid saponin component QS-21 can effectively inhibit respiratory syncytial virus A and B subtypes from infecting cells at a concentration of 100 μM.

4. The application according to claim 1, characterized in that, The triterpenoid saponin component QS-21 can effectively reduce the viral titer in lung tissue.

5. A liposome-carrier drug containing a triterpenoid saponin component QS-21, characterized in that, The triterpenoid saponin component QS-21 is loaded onto a liposome-based drug using liposomes.

6. The triterpenoid saponin component QS-21 liposome carrier drug according to claim 6, characterized in that, The pH value of the triterpenoid saponin component QS-21 liposome carrier drug is 6.10±0.

1.

7. The triterpenoid saponin component QS-21 liposome carrier drug according to claim 6, characterized in that, The triterpenoid saponin component QS-21 liposome carrier drug has a particle size between 94.4±2.4 nm and a particle size distribution between 0.12±0.

07.

8. The triterpenoid saponin component QS-21 liposome carrier drug according to claim 6, characterized in that, The triterpenoid saponin component QS-21 liposome carrier drug comprises the following components in parts by weight: 1-2 parts of triterpenoid saponin component QS-21; 8-10 parts of soybean lecithin; 4-6 parts of cholesterol; 6-8 parts of sodium cholate; and 3-5 parts of isopropyl myristate.

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