Active ingredient composition of common goldenrop decoction and application of active ingredient composition in prevention and treatment of human respiratory syncytial virus

Abstract

The invention belongs to the technical field of medicines, and discloses a common goldenrop decoction active ingredient composition and application thereof in prevention and treatment of human respiratory syncytial virus. The invention relates to application of an active ingredient composition (isoliquiritigenin, baicalein, 6-shogaol, schisandrin B, ferulic acid and methyl salicylate) of Xiaochaihu decoction and / or a derivative of the active ingredient or a pharmaceutically acceptable salt thereof in preparation of medicines for preventing, relieving and / or treating RSV (Respiratory Syndrome Virus) infectious diseases. According to the invention, by establishing a mouse RSV infection model, the effect and the action mechanism of the composition in preventing and treating RSV infection are evaluated. Results show that each dose group can inhibit replication of RSV in mouse lung tissues, relieve pulmonary edema, improve pathological changes of the lung, inhibit expression of inflammatory factors and improve humoral immunity, and the effect is equivalent to that of ribavirin. The radix bupleuri granules have better effects on relieving pulmonary edema and inhibiting virus proliferation compared with small radix bupleuri granules. The composition of the effective components of the common goldenrop can provide a new drug treatment scheme for clinical RSV infectious diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to application of a composition of effective components of Xiaochaihu Decoction LG0414 in preparation of a medicine for preventing and treating human respiratory syncytial virus (RSV) infection. BACKGROUND

[0002] Human respiratory syncytial virus (RSV) is a virus widely prevalent in the world, causing a serious disease burden. Almost all children will be infected with RSV once before the age of 5, and cannot form long-term immunity, and repeated infection is common throughout their lives. In children, the elderly and immunodeficient people, it can cause serious respiratory diseases and even death. At present, there is a great lack of clinical treatment drugs for RSV infection, and the use of single antiviral drugs is often ineffective due to viral genetic mutations, and establishing a combination of antiviral drugs is an effective way to develop new antiviral drugs.

[0003] Xiaochaihu Decoction is derived from Shanghan Lun of Zhang Zhongjing of the Eastern Han Dynasty, and is composed of seven Chinese medicines, including Radix Bupleuri, Radix Scutellariae, Rhizoma Pinelliae, Radix et Rhizoma Ginseng, Radix Glycyrrhizae, Rhizoma Zingiberis Recens and Fructus Jujubae, and has the effect of resolving exterior-interior, and is a main prescription of traditional Chinese medicine for preventing and treating exogenous diseases such as colds and epidemics. The composition LG0414 is derived from six natural products in the effective components of Xiaochaihu Decoction, including isofraxidin, baicalein, 6-gingerol, schisantherin B, ferulic acid and methyl salicylate, has antiviral and anti-inflammatory activities, and has the advantages of low preparation cost, good oral bioavailability, good in-vivo safety and the like, and has advantages in the development of antiviral combination drugs. SUMMARY

[0004] In view of the current lack of clinical drugs for RSV infection, the application aims to provide application of a composition or a derivative or a pharmaceutically acceptable salt thereof derived from effective components of Xiaochaihu Decoction in preparation of a medicine for preventing, alleviating and / or treating RSV infectious diseases.

[0005] In order to achieve the above-mentioned purpose, the following technical scheme is adopted in the patent:

[0006] The first aspect of the technical scheme of the application is to provide a composition LG0414 containing effective components of Xiaochaihu Decoction, characterized in that the composition contains two or more of the effective components of Xiaochaihu Decoction and derivatives or pharmaceutically acceptable salts thereof, and the effective components containing Xiaochaihu Decoction are selected from the following compounds of formula I-VI;

[0007]

[0008] The pharmaceutically acceptable salt is prepared by reacting the effective components of formula I-VI or derivatives thereof with inorganic acids, organic acids, amino acids, sulfonic acids, alkali metal or ammonium ions.

[0009] The composition contains all compounds of formula I-VI, and the mass ratio of formula I-VI is 1:1:1:1:1:1.

[0010] The second aspect of the technical scheme of the present application provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the composition of the first aspect or a pharmaceutically acceptable carrier or excipient.

[0011] The dosage form of the pharmaceutical composition is selected from tablets, capsules, pills, powders, mixtures, granules.

[0012] The third aspect of the technical scheme of the present application provides the use of the composition of the first aspect or the pharmaceutical composition of the second aspect in the preparation of a medicament for preventing, alleviating and / or treating RSV infectious diseases.

[0013] The RSV includes RSV A and B strains.

[0014] The human respiratory virus infectious disease includes upper respiratory tract infection and / or lower respiratory tract infection.

[0015] The upper respiratory tract infection includes rhinitis, pharyngitis, sinusitis, otitis media, laryngitis and / or epiglottitis; and the lower respiratory tract infection includes tracheitis, bronchitis and / or pneumonia.

[0016] The medicament is a medicament for reducing the activity of RSV.

[0017] The examples analyze the anti-RSV efficacy and mechanism of action of the LG0414 combination compound, i.e., glycyrrhizin, baicalein, 6-gingerol, schisantherin B, ferulic acid and methyl salicylate (formula I-VI) in the mass ratio of 1:1:1:1:1:1.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The effective component combination of Xiaochaihu Decoction provided by the present application is a combination medicament for reducing the activity of RSV, which can inhibit the replication of viruses in vivo, reduce lung edema and inflammation, and has an effect on pathological changes equivalent to that of ribavirin, and is better than Xiaochaihu Granules, and has a unique advantage for respiratory infectious diseases caused by RSV.

[0020] The compound provided by the present application is a medicament for preventing, alleviating and / or treating upper respiratory tract infection, including a medicament for preventing, alleviating and / or treating rhinitis, pharyngitis, sinusitis, otitis media, laryngitis and pharyngitis; and the medicament for preventing, alleviating and / or treating lower respiratory tract infection includes a medicament for preventing, alleviating and / or treating tracheitis, bronchitis and pneumonia. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1Effects of Xiao-Chaihu-Tang and LG0414 on body weight curve of RSV-infected mice.

[0022] Figure 2 Effects of Xiao-Chaihu-Tang and LG0414 on lung, thymus and spleen organ index of RSV-infected mice.

[0023] Figure 3 Effects of Xiao-Chaihu-Tang and LG0414 on lung tissue viral replication of RSV-infected mice.

[0024] Figure 4 Effects of Xiao-Chaihu-Tang and LG0414 on lung pathological changes of RSV-infected mice.

[0025] Figure 5 Effects of Xiao-Chaihu-Tang and LG0414 on lung RSV F protein expression of RSV-infected mice.

[0026] Figure 6 Effects of Xiao-Chaihu-Tang and LG0414 on inflammatory cytokine expression of mice.

[0027] Figure 7 Effects of Xiao-Chaihu-Tang and LG0414 on total splenocytes of RSV-infected mice.

[0028] Figure 8 Effects of Xiao-Chaihu-Tang and LG0414 on total CD3+T cells of RSV-infected mice.

[0029] Figure 9 Effects of Xiao-Chaihu-Tang and LG0414 on CD4+T cells / CD8+T cells ratio of RSV-infected mice.

[0030] Figure 10 Effects of Xiao-Chaihu-Tang and LG0414 on CD19+B cells of RSV-infected mice. DETAILED DESCRIPTION

[0031] Embodiments of the present application will be described in detail with reference to the drawings, but the skilled person will appreciate that the following examples are for illustration only and should not be taken as limiting the scope of the present application. Where specific conditions are not noted in the examples, they were carried out under conventional conditions or as recommended by the manufacturer. Where the reagents or instruments used are not noted by the manufacturer, they are all conventional products that can be obtained commercially.

[0032] Unless otherwise defined, all terms (including technical and scientific terms) used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined in the present application.

[0033] The present application provides the application of Xiaochaihu Decoction effective component composition LG0414 in the preparation of drugs for preventing and treating RSV infection. The compound combination in the example is: isofraxidin, baicalein, 6-gingerol, schizandrin B, ferulic acid, methyl salicylate, and the mass ratio is 1:1:1:1:1:1. The present application provides a scientific experimental basis for the research and development of RSV drugs, and has a broad prospect in the field of clinical respiratory viral infectious diseases.

[0034] The virus used in the following examples is stored at -80℃ by the China Disease Prevention and Control Center Virus Disease Prevention and Control (Key Laboratory of Medical Viruses and Viral Diseases, National Health Commission).

[0035] Experimental method of animal experiment

[0036] 1. Animal grouping

[0037] SPF male Balb / c mice, 15-17g, 6-8 weeks, were randomly divided into 9 groups, namely normal group, virus group, Xiaochaihu granules (XCHKL, Hualun Sanjiu Pharmaceutical Co., Ltd.) low, medium and high dose groups, LG0414 low, medium and high dose groups and positive drug ribavirin group, 6 in each group.

[0038] 2. Animal modeling

[0039] The mouse RSV infection pneumonia model was established by the method of nasal instillation of virus liquid: the RSV-Long strain virus was diluted with normal saline, and the mice were anesthetized with 200mg / kg tri- bromoethanol intraperitoneally, and 2×10 6 PFU / mouse of virus liquid was instilled into the nose to establish the model. The normal group of mice was instilled with the same amount of normal saline by the same method.

[0040] 3. Animal administration

[0041] Each group of mice was administered once a day by gavage 2 hours before modeling and from the second day after modeling (Xiaochaihu granules XCHKL low, medium and high dose groups were 0.965 g / kg, 1.93 g / kg and 3.86 g / kg respectively; LG0414 low, medium and high dose groups were 25 mg / kg, 50 mg / kg and 100 mg / kg respectively, prepared with 0.5% CMC-Na), the normal group and the virus group were given the same amount of 0.5% CMC-Na by gavage, and the positive drug group was given 50 mg / kg of ribavirin by gavage, for 5 consecutive days.

[0042] Example 1: Effect of LG0414 on the body weight and organ index of mice

[0043] The body weight of mice was monitored daily. On the 6th day, the lung, thymus and spleen tissues of mice were weighed after being decapitated, and the organ index was calculated. The body weight curve results showed that the body weight recovery of mice in the three dose groups of Xiaochaihu granules and the three dose groups of LG0414 was faster than that of the virus group Figure 1 ). The organ index results showed that the lung index of the virus group mice was significantly increased (P<0.0001), the thymus index was significantly decreased (P<0.01), and the spleen index was significantly decreased (P<0.01). Only the high dose group (XCHKL-H) of Xiaochaihu granules could significantly reduce the lung index of mice, while the three dose groups of LG0414 could significantly reduce the lung index of mice and improve the symptoms of pulmonary edema. The thymus index and spleen index of mice in each dose group of Xiaochaihu granules and LG0414 were improved to some extent Figure 2 ), improving the immune ability of mice.

[0044] Example 2: Inhibitory effect of LG0414 on RSV virus titer in lung tissue of mice

[0045] The lung tissue of mice was taken, 10 mL of normal saline was added per gram of lung tissue, and 10% tissue homogenate was prepared. 100 μl of homogenate was used to extract RNA using FreeZol reagent, cDNA was synthesized using HiScript II 1st Strand cDNA Synthesis Kit, and Quantitative Real-time PCR was performed using AceQ Universal SYBR qPCR Master Mix. After 2 -ΔΔCt The relative transcription level of RSV M gene mRNA was calculated, and GAPDH was used as an internal reference gene. The primer sequences are as follows:

[0046] RSV-M forward primer: 5'-GGCAAATATGGAAACATACGTGAA-3';

[0047] RSV-M reverse primer: 5'-TCTTTTTCTAGGACATTGTAYTGAACAG-3';

[0048] GAPDH forward primer: 5'-AGCTTCGGCACATATTTCATCTG-3';

[0049] GAPDH reverse primer: 5'-CGTTCACTCCCATGACAAACA-3'.

[0050] Quantitative Real-time PCR results showed that compared with the virus group, only the medium dose group of Xiaochaihu granules (XCHKL-M) significantly reduced the virus replication in the lung tissue of mice, while the three dose groups of LG0414 could significantly inhibit the virus proliferation in the lung tissue of mice, which was better than each dose group of Xiaochaihu granules, and was equivalent to the positive drug ribavirin group Figure 3 ).

[0051] Example 3: Effect of LG0414 on pathological changes in lung tissue of mice

[0052] The left lung of the mouse was taken and completely immersed in 4% paraformaldehyde solution for fixation. After 48 h, routine dehydration was performed, and paraffin embedding was used for sectioning, HE staining and RSV F protein immunohistochemical staining. The pathological changes and RSV F protein expression in the lung tissue were observed. The results showed that Xiaochaihu granules and LG0414 could significantly improve the lung tissue lesions of mice, reduce inflammatory cell infiltration and alveolar fusion phenomenon. The lung status of the XCHKL-H group and the XCHKL-M group was significantly better than that of the XCHKL-L group. The effect of the three dose groups of LG0414 was better than that of the XCHKL-L group, and was equivalent to the positive drug ribavirin group Figure 4 ). In addition, the immunohistochemical results showed that Xiaochaihu granules and LG0414 could significantly inhibit the expression of RSV F protein in mice Figure 5 ).

[0053] Example 4: Effect of LG0414 on the expression of inflammatory factors in mice

[0054] The contents of IL-6 and IL-8 in the lung tissue homogenate supernatant and the contents of TNF-α and IFN-γ in the serum sample of mice were determined by ELISA kit. The results showed that each dose group of Xiaochaihu granules and LG0414 could significantly inhibit the levels of IL-1β, IL-6, TNF-α and IFN-γ induced by RSV infection, which was equivalent to the effect of the positive drug ribavirin, indicating that Xiaochaihu granules and LG0414 had significant anti-inflammatory activity Figure 6 ).

[0055] Example 5: Effect of LG0414 on the changes of spleen lymphocytes in mice

[0056] The mouse spleen was ground to prepare a lymphocyte suspension, and flow cytometry antibody staining (FITC anti-CD3, APC anti-CD4, PE anti-CD8a and PE / Cyanine7 anti-CD19) was performed, followed by detection of the changes in the spleen lymphocytes of the mice in each group by flow cytometry.

[0057] The flow cytometry results showed that the Xiaochaihu granules and LG0414 in each dose group could significantly inhibit the increase in the total spleen lymphocytes and CD3+T cells of the infected mice ( Figure 7 , Figure 8 ), to a certain extent, improve the ratio of CD4+T cells to CD8+T cells in the spleen of the mice ( Figure 9 ), and significantly increase the number of CD19+B cells in the spleen of the mice ( Figure 10 ). This indicated that the Xiaochaihu granules and LG0414 could regulate the proliferation and differentiation of T lymphocytes, promote the proliferation of B lymphocytes, regulate the cellular immunity of the mice and improve the humoral immunity of the mice.

[0058] In summary, the mice in each dose group of the effective component composition LG0414 of Xiaochaihu decoction and the Xiaochaihu granules recovered faster than the mice in the virus group; each dose group could inhibit the replication of RSV in the lung tissues of the infected mice, relieve pulmonary edema, improve the pathological changes in the lungs, inhibit the expression of inflammatory factors, and improve the humoral immunity of the mice, and the effect was comparable to that of ribavirin. The composition LG0414 was better than the Xiaochaihu granules in terms of relieving pulmonary edema and inhibiting the proliferation of viruses in the lungs, and had the effect of treating viral pneumonia, and was expected to be used to treat the existing RSV infectious diseases and respiratory viral infectious diseases or multi-virus cross-infectious diseases.

[0059] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A composition LG0414 containing effective components of Sho-saiko-to, characterized in that, The composition contains two or more of effective components of Xiaochaihu Decoction and derivatives or pharmaceutically acceptable salts thereof, and the effective components of Xiaochaihu Decoction are selected from the following compounds of formula I-VI:

2. Composition according to claim 1, characterized in that The pharmaceutically acceptable salt is prepared by reacting the effective components of formula I-VI or derivatives thereof with inorganic acids, organic acids, amino acids, sulfonic acids, alkali metal or ammonium ions.

3. The composition according to claim 1, characterized in that, The composition contains all the compounds of formula I-VI, and the mass ratio of the compounds of formula I-VI is 1:1:1:1:1:

1.

4. A pharmaceutical composition, characterized by, The pharmaceutical composition contains the composition of any one of claims 1-3 or pharmaceutically acceptable carriers or excipients.

5. Pharmaceutical composition according to claim 4, characterized in that The dosage form of the pharmaceutical composition is selected from tablets, capsules, pills, powders, mixtures, granules.

6. Use of the composition of any one of claims 1-3 or the pharmaceutical composition of claim 4 in the preparation of a medicament for preventing, alleviating and / or treating human respiratory syncytial virus infectious diseases.

7. Use according to claim 6, characterised in that, The human respiratory syncytial virus includes human respiratory syncytial virus type A and type B.

8. Use according to claim 6, characterised in that, The human respiratory virus infectious disease includes upper respiratory tract infection and / or lower respiratory tract infection.

9. Use according to claim 8, characterised in that, The upper respiratory tract infection includes rhinitis, pharyngitis, sinusitis, otitis media, laryngitis and / or epiglottitis; the lower respiratory tract infection includes tracheitis, bronchitis and / or pneumonia.

10. Use according to claim 6, characterised in that, The drug is a drug for reducing the activity of human respiratory syncytial virus.

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