Traditional Chinese medicine composition and application

Abstract

The invention relates to the field of medicines, in particular to a traditional Chinese medicine composition and a pharmacodynamic effect and an in-vivo anti-inflammatory effect of the traditional Chinese medicine composition in an anti-H3N2 influenza virus model. The traditional Chinese medicine composition provided by the invention has an inhibition effect on proliferation of influenza virus H3N2 in cells; the high dosage of the compound has an inhibition effect on the weight change, lung index and lung tissue virus titer of a mouse infected by the H3N2 virus, and has a better improvement effect on lung lesions caused by virus infection; a good inhibition effect on mouse ear swelling caused by medium and high doses of p-xylene is achieved; the pharmaceutical composition has a good intervention effect on prophylactic administration and therapeutic administration of LPS-induced mouse acute pneumonia, can effectively reduce lung indexes, improve lung pathology, inhibit inflammatory mediator level and IgM level and regulate T cell immune function, and has a good dose-effect relationship. In conclusion, the traditional Chinese medicine composition has an antagonistic effect and an anti-inflammatory effect on influenza virus, and has a good treatment prospect on influenza and complicated inflammation thereof clinically.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to a Chinese medicine composition and application thereof. Background Art

[0002] Influenza viruses are divided into three types: A, B, and C. Influenza A viruses are most likely to mutate. Flu pandemics are caused by the emergence of new subtypes or the reappearance of old subtypes of influenza A viruses. The surface antigens of influenza A viruses often undergo small mutations, which are called "drift". Figuratively speaking, "drift" means that the virus disguises itself through small changes to avoid recognition by the human immune system. The result of "drift" of influenza A viruses is that the strains that cause influenza may be different every year, and people need to be re-vaccinated with influenza vaccines every year for prevention. "Shift" refers to the mutation of influenza A viruses, resulting in the emergence of a new virus "subtype". Because the human body has almost no antibodies to resist this new virus, the result of "shift" often leads to a global outbreak of influenza. Influenza viruses usually rely on the combination of a part of the viral protein with a specific protein in the human body to invade the human body, because through such a combination, the influenza virus can suppress the human body's own natural defense system against viral infection, paving the way for the virus to effectively replicate in the human body.

[0003] Influenza viruses are divided into three types: A, B, and C. Type A influenza viruses are divided into many subtypes based on the differences in H and N antigens. H can be divided into 17 subtypes (H1 to H17), and N has 10 subtypes (N1 to N10). Among them, only H1N1, H2N2, and H3N2 mainly infect humans, and the natural hosts of many other subtypes are various poultry and animals. Among them, the most harmful to poultry are the H5, H7, and H9 subtype strains. Generally speaking, avian influenza viruses do not infect animals other than birds and pigs.

[0004] The most important mutation of influenza virus is type A, which is often closely related to global pandemics. Generally speaking, the antigenic variation of influenza virus refers to the change of H and N antigen structure. Small variations (quantitative changes) often occur within the subtype, which is called antigenic drift. Although it is only a small variation, it can make the virus easily evade the host's immune system. Compared with the previously discovered strains, if a mutation occurs on the hemagglutinin molecule-specific antigenic determinant cluster (antigenic epitope), the new strain is considered to be a heterologous variant type of the previous strain, which has epidemiological significance and can cause influenza epidemics.

[0005] Antigenic variation only occurs in type A viruses. It may be caused by genetic reassortment between viruses when two viruses from humans and animals infect the same cell. The resulting viral hemagglutinin and neuraminidase undergo a completely new combination, making the population immune. Antigenic shift is the cause of the global influenza pandemic. Type A influenza viruses undergo a major mutation approximately every ten years. Since 1933, type A viruses have undergone four antigenic shifts: H0N1 (original type A, A0) from 1933 to 1946, H1N1 (subtype A, A1) from 1946 to 1957, H2N2 (Asian type A, A2) from 1957 to 1968, and H3N2 (Hong Kong type, A3) after 1968. Generally, there is a clear alternation between the new and old subtypes. After a new subtype appears and spreads to a region, the old subtype can no longer be isolated.

[0006] The influenza A viruses currently prevalent in the global population are mainly influenza A H1N1pdm09 virus and influenza A H3N2 virus. Among them, influenza epidemics caused by H3N2 virus are associated with higher morbidity and mortality in the elderly, and H3N2 can also cause higher morbidity and mortality in children when different degrees of antigenic drift (influenza virus mutation) occur. Influenza viruses can invade the lower respiratory tract, causing complications and developing into serious illness. Pneumonia is the most common influenza-related lower respiratory tract disease.

[0007] Therefore, providing a drug that can effectively treat influenza A virus has important practical significance. Summary of the invention

[0008] In view of this, the present invention provides a Chinese medicine composition and its application. The Chinese medicine composition provided by the present invention has antagonistic and anti-inflammatory effects on influenza virus, indicating that the Chinese medicine composition provided by the present invention has a good therapeutic effect on influenza and its concurrent inflammation in clinical practice.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a Chinese medicine composition, comprising the following components in parts by weight:

[0011]

[0012] In some specific embodiments of the present invention, the Chinese medicine composition comprises the following components in parts by weight:

[0013]

[0014] This prescription is caused by the exogenous wind-heat and dampness, or the body is damp and the wind-heat is affected. The clinical symptoms are fever, or accompanied by aversion to cold, dry mouth, sore throat or even pain like a knife, headache or body aches, or cough, sputum, upset and nausea, decreased taste and smell, etc. The treatment should be to dispel wind and detoxify, clear away plague and dampness. The two herbs of honeysuckle vine and forsythia in the prescription are combined, taking the meaning of honeysuckle and forsythia in the pungent and cool Yinqiao powder, and changing honeysuckle to honeysuckle vine, reducing the bitter and cold dampness, increasing the power of clearing and removing dampness, and combining bamboo leaves and bupleurum with pungent bitter and slightly cold taste to dispel wind and heat. The three herbs are the main ones. The assistant drugs are divided into three groups. Wind, heat and dampness evil block the Wei Qi. Patchouli, Pueraria lobata and Artemisia annua are used to lightly and clearly penetrate, help the main drugs to dispel wind and heat, and also transform dampness and turbidity; loquat leaves, reed roots and isatis indigotica can descend the lung Qi, and combined with the main drugs' light-dispersing properties, they can restore the lung's function of dispersing and descending, help expel evil Qi, and stop coughing. Loquat leaves can descend the Qi of the lungs and stomach. "Compendium of Materia Medica" says: "Loquat leaves can treat lung and stomach diseases...those who vomit will not vomit anymore, those who are thirsty will not be thirsty anymore, and those who cough will not cough anymore"; Sichuan Shegan, Isatis Root, Paris polyphylla and Taraxacum mongolicum go to the throat, clear Qi and divide heat and toxicity, and combined with the main drugs to resolve both the exterior and interior, and relieve sore throat, dispel evil and stop pain. Peppermint is pungent and cool, penetrating the surface, clearing the head and eyes, and Schizonepeta is light and penetrating, dispelling wind and dampness. The two herbs assist the monarch and minister, dispersing the evil spirits; Angelica dahurica and Perilla leaf are pungent and warm in nature, and are added to the cold and cool herbs to assist in the control, exerting the effects of dispelling wind and relieving pain, harmonizing the stomach and relieving the surface, and at the same time guiding the herbs upward. The above four herbs are the adjuvants. The comprehensive formula uses both dispersing and descending, one ascending and one descending, and focuses on dispersing to disperse the wind, clear the heat, and remove the dampness; it is mainly light, clear, penetrating and dispersing, reflecting the principle of using medicine for the initial onset of exogenous wind-heat evil or damp-heat evil qi, "treating the upper part of the body like a feather, it must be light to lift it."

[0015] In a second aspect, the present invention also provides the use of the traditional Chinese medicine composition in the preparation of drugs for improving, preventing and / or treating anti-inflammatory and / or antiviral effects.

[0016] In some specific embodiments of the present invention, the anti-inflammatory agent includes any of the following:

[0017] (I) inhibit ear swelling;

[0018] (II), reduce lung index;

[0019] (III), reducing TNF-α content;

[0020] (IV), reducing IL-6 and IL-1β levels;

[0021] (V) Reduce IgM content;

[0022] (VI), increased percentage of CD3+ cell population;

[0023] (VII), increase the percentage of CD4+T cell population;

[0024] (VIII), reduce the percentage of CD8+T cell population;

[0025] (IX) Reducing the degree of inflammatory cell infiltration, perivascular edema and / or alveolar structural destruction in lung tissue;

[0026] (X), inhibiting the decrease in body weight of mice induced by LPS; and / or

[0027] (XI) Inhibits the increase of lung index caused by LPS.

[0028] In some specific embodiments of the present invention, the antiviral agent includes any of the following:

[0029] (I) inhibiting viral proliferation;

[0030] (II), inhibiting weight loss;

[0031] (III) inhibiting the increase of lung index caused by viral infection;

[0032] (IV) inhibiting the upregulation of lung virus titers caused by viral infection;

[0033] (V), improving lung tissue damage; and / or

[0034] (VI) Reduce the overexpression of inflammatory factors.

[0035] In some specific embodiments of the present invention, the lung tissue damage includes interstitial pneumonia, edema fluid visible in the alveolar cavity, bleeding, lymphocyte infiltration, formation of hyaline membrane, interstitial lymphocyte infiltration, degeneration / necrosis and shedding of adjacent bronchiolar mucosa or bronchial mucosa.

[0036] In some specific embodiments of the present invention, the inflammatory factors include one or more of IL-6, IL-8, IP-10, TNF-α, MCP-1 or IL-1β.

[0037] In some specific embodiments of the present invention, the antiviral agent includes anti-influenza virus H3N2 strain.

[0038] In a third aspect, the present invention also provides a medicine, including the Chinese medicine composition and pharmaceutically acceptable adjuvants / excipients, and a pharmaceutically acceptable preparation.

[0039] The present invention provides a Chinese medicine composition and its pharmacodynamic effect against H3N2 influenza virus model, as well as the in vivo anti-inflammatory effect of xylene-induced ear swelling model and lipopolysaccharide-induced pneumonia model. The results show that the Chinese medicine composition provided by the present invention has an inhibitory effect on the proliferation of influenza virus H3N2 in cells; the high dose of the Chinese medicine composition provided by the present invention (39.8g slices / kg) has an inhibitory effect on the weight change, lung index and lung tissue virus titer of mice infected with H3N2 virus, and has a good improvement effect on lung lesions caused by viral infection; the Chinese medicine composition provided by the present invention at a medium dose (19.9g slices / kg) and a high dose (39.8g slices / kg) has a good inhibitory effect on xylene-induced mouse ear swelling; the Chinese medicine composition provided by the present invention has a good intervention effect on LPS-induced acute pneumonia in mice by both preventive and therapeutic administration, can effectively reduce the lung index, improve lung pathology, inhibit the level of inflammatory mediators and IgM levels, regulate T cell immune function, and has a good dose-effect relationship. The above data show that the Chinese medicine composition provided by the present invention has a certain antagonistic effect and anti-inflammatory effect on influenza virus, suggesting that the Chinese medicine composition provided by the present invention has a potential therapeutic effect on influenza and its concurrent inflammation in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0041] Figure 1 The figures show the effect of the Chinese medicine composition provided by the present invention on the body weight of male mice (a) and female mice (b) in the xylene-induced ear swelling model;

[0042] Figure 2 The figures show the effect of the Chinese medicine composition provided by the present invention on the ear swelling degree of male mice (a) and female mice (b) induced by xylene;

[0043] Figure 3 The figures show the effect of the Chinese medicine composition provided by the present invention on the body weight of male mice (a) and female mice (b) in the LPS-induced acute pneumonia model - preventive administration;

[0044] Figure 4 The effect of the Chinese medicine composition provided by the present invention on the body weight of male mice (a) and female mice (b) in the LPS-induced acute pneumonia model - therapeutic administration;

[0045] Figure 5 The effect of the Chinese medicine composition provided by the present invention on the lung index of male mice (a) and female mice (b) in the LPS-induced acute pneumonia model - preventive administration;

[0046] Figure 6The effect of the Chinese medicine composition provided by the present invention on the lung index of male mice (a) and female mice (b) in the LPS-induced acute pneumonia model - therapeutic administration;

[0047] Figure 7 The effect of the Chinese medicine composition provided by the present invention on serum IL-6 in male mice (a) and female mice (b) with acute pneumonia - preventive administration;

[0048] Figure 8 The effect of the Chinese medicine composition provided by the present invention on serum IL-1β in male mice (a) and female mice (b) with acute pneumonia - preventive administration;

[0049] Fig. 9 The effect of the Chinese medicine composition provided by the present invention on serum TNF-α of male mice (a) and female mice (b) with acute pneumonia - preventive administration;

[0050] Fig.10 The effect of the Chinese medicine composition provided by the present invention on serum IL-6 in male mice (a) and female mice (b) with acute pneumonia - therapeutic administration;

[0051] Fig.11 The effect of the Chinese medicine composition provided by the present invention on serum IL-1β in male mice (a) and female mice (b) with acute pneumonia - therapeutic administration;

[0052] Fig.12 The effect of the Chinese medicine composition provided by the present invention on serum TNF-α in male mice (a) and female mice (b) with acute pneumonia - therapeutic administration;

[0053] Fig.13 The effect of the Chinese medicine composition provided by the present invention on the serum IgM content of male mice (a) and female mice (b) with acute pneumonia - preventive administration;

[0054] Fig.14 The effect of the Chinese medicine composition provided by the present invention on the serum IgM content of male mice (a) and female mice (b) with acute pneumonia - therapeutic administration;

[0055] Fig.15 Figure 2 shows the effect of the Chinese medicine composition provided by the present invention on HE staining of lung tissue of male mice (a) and female mice (b) in the LPS-induced acute pneumonia model - preventive administration (100×); Note: Pathological pictures of lung tissue of mice with pneumonia; except for the Sham group, each group had varying degrees of perivascular edema (↑) and inflammatory infiltration (▲), and eosinophils were occasionally seen in the alveolar cavity.

[0056] Fig.16The figures show the lung pathology scores of male mice (a) and female mice (b) in the LPS-induced acute pneumonia model of the Chinese medicine composition provided by the present invention - preventive administration;

[0057] Fig.17 Figure 2 shows the results of HE staining of lung tissues of male mice (a) and female mice (b) in the LPS-induced acute pneumonia model using the Chinese medicine composition provided by the present invention (100×) - therapeutic administration; Note: Pathological images of lung tissues of mice with pneumonia; except for the sham operation group, each group had varying degrees of perivascular edema (↑) and inflammatory infiltration (▲), and eosinophils were occasionally seen in the alveolar cavity.

[0058] Fig.18 The figures show the lung pathology scores of male mice (a) and female mice (b) in the LPS-induced acute pneumonia model of the Chinese medicine composition provided by the present invention - therapeutic administration;

[0059] Fig.19 The antiviral effect of the Chinese medicine composition provided by the present invention on influenza virus H3N2 is shown;

[0060] Fig. 20 The effect of the Chinese medicine composition provided by the present invention on the weight change of mice infected with influenza virus is shown;

[0061] Fig.21 The results of the test on the change of lung index of mice infected with influenza virus by the Chinese medicine composition provided by the present invention are shown; compared with the virus group: *** P < 0.001, ** P<0.01, * P < 0.05;

[0062] Fig. 22 The results of the test of the Chinese medicine composition provided by the present invention on the virus titer of the lung tissue of mice infected with influenza virus are shown; compared with the virus group: *** P < 0.001, ** P<0.01, * P < 0.05;

[0063] Fig.23 The results of pathological detection of lung tissue of mice infected with influenza virus by the Chinese medicine composition provided by the present invention are shown;

[0064] Fig.24 The results of the pathological test of the lung tissue of mice infected with influenza virus by the Chinese medicine composition provided by the present invention are shown; compared with the virus group: *** P < 0.001, ** P<0.01, * P < 0.05;

[0065] Fig.25The results of the Chinese medicine composition provided by the present invention on the expression of inflammatory factors in mice infected with influenza virus are shown; compared with the Virus group, * P<0.05, ** P<0.01, *** P<0.001. DETAILED DESCRIPTION

[0066] The present invention discloses a Chinese medicine composition and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0067] Anti-inflammatory Effects:

[0068] A mouse ear swelling model and an acute pneumonia mouse model induced by exposed tracheal injection of lipopolysaccharide (LPS) were used to comprehensively evaluate the intervention effects of the preventive and therapeutic administration of the traditional Chinese medicine composition provided by the present invention on acute pneumonia in mice, thereby providing a pharmacological basis for the treatment of acute pneumonia caused by influenza.

[0069] The mouse ear swelling model was prepared by smearing the right ear of the mouse with the topical inflammatory agent xylene (0.05 mL). Female and male mice were given continuous drug intervention in a preventive manner, and the weight changes of the mice were monitored. After the last drug administration, the left and right ear pieces at the same site were prepared with an ear piece puncher, and the swelling rate was calculated.

[0070] The mouse acute pneumonia model was established by exposed tracheal injection of lipopolysaccharide (LPS). Female and male mice were continuously intervened by preventive and therapeutic administration. The weight changes of mice were monitored, and blood samples were collected at different time points after modeling. The levels of serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were detected by enzyme-linked immunosorbent assay (ELISA). The level of serum immunoglobulin M (IgM) was detected by animal blood biochemical analyzer. The peripheral blood CD4+ / CD5+ / CD6+ / CD7+ / CD8+ / CD9+ / CD10+ / CD1 ... 3+ , CD 4+ and CD 8+ The percentage of T cell population and lung index were determined at the experimental endpoint, and the pathological morphology of lung tissue was examined.

[0071] The results of the mouse ear swelling experiment showed that: in male mice, the ear swelling of the Chinese medicine composition-H provided by the present invention and the Chinese medicine composition-M group provided by the present invention was significantly reduced, and the ear swelling inhibition rates were 88.24% and 60.29%, respectively; in female mice, the ear swelling of the Chinese medicine composition-H provided by the present invention and the Chinese medicine composition-M group provided by the present invention was also significantly reduced, and the ear swelling inhibition rates were 88.96% and 74.48%, respectively.

[0072] The results of the preventive administration experiment for acute pneumonia in mice showed that: for male mice, compared with the LPS group, the lung index of the Chinese medicine composition-M provided by the present invention and the Chinese medicine composition-H group provided by the present invention was significantly reduced (p<0.001); for female mice, compared with the LPS group, the lung index of each group of the Chinese medicine composition provided by the present invention was significantly reduced (p<0.001). Compared with the LPS group, the serum TNF-α content of each group of male mice was significantly reduced (p<0.01), and the serum IL-6 and IL-1β contents of the Chinese medicine composition-H group provided by the present invention were significantly reduced (p<0.001); compared with the LPS group, the serum IL-6 content of each group of female mice (Chinese medicine composition provided by the present invention) was significantly reduced (p<0.05), and the serum IL-1β content of DEX and Chinese medicine composition-H groups provided by the present invention was significantly reduced (p<0.05 or p<0.001). Compared with the LPS group, the IgM content in the serum of female mice DEX and the Chinese medicine composition-H group provided by the present invention was significantly reduced (p<0.01). The percentage of CD3+ cell group in the peripheral blood of male and female mice in each administration group of the Chinese medicine composition provided by the present invention was significantly increased (p<0.05); in male mice, compared with the LPS group, the percentage of CD4+T cell group in the Chinese medicine composition-H dosage group provided by the present invention was significantly increased (p<0.05), and the percentage of CD8+T cell group in the Chinese medicine composition-L and Chinese medicine composition-M dosage groups provided by the present invention was significantly reduced (p<0.01 or p<0.05); in female mice, the percentage of CD4+T cell group in the peripheral blood of mice in each administration group of the Chinese medicine composition provided by the present invention was significantly increased (p<0.01), and the percentage of CD8+T cell group was significantly reduced (p<0.05). Compared with the LPS group, the inflammatory cell infiltration, perivascular edema, and alveolar structural destruction of the lung tissue in the male (Chinese medicine composition provided by the present invention) administration group were alleviated to varying degrees, and the pathological score was significantly reduced (p<0.05); after treatment with the Chinese medicine composition provided by the present invention, the lung structure of female mice was restored, the eosinophil infiltration in the alveolar space and the bronchial goblet cell metaplasia were reduced, the perivascular edema phenomenon was weakened, and the pathological score was significantly lower than that of the LPS group (p<0.05).

[0073] In the therapeutic administration test, after 3 days of treatment, the Chinese medicine composition-H group provided by the present invention can significantly inhibit the weight loss of mice caused by LPS. In the administration of male mice, the Chinese medicine composition-M provided by the present invention and the Chinese medicine composition-H group provided by the present invention significantly inhibited the increase in lung index caused by LPS (p<0.05); in the administration of female mice, the Chinese medicine composition-H group provided by the present invention significantly inhibited the increase in lung index caused by LPS (p<0.05). In the administration of male mice, compared with the LPS group, the IL-6 content in the serum of each group of mice of the Chinese medicine composition provided by the present invention was significantly reduced (p<0.05), and the IL-1β content in the serum of mice of the Chinese medicine composition-H group provided by the present invention was significantly reduced (p<0.05); in female mice, compared with the LPS group, the IL-6 and IL-1β contents in the serum of each group of mice of the Chinese medicine composition provided by the present invention were significantly reduced (p<0.05), and the TNF-α content in the Chinese medicine composition-H group provided by the present invention and the Chinese medicine composition-L group provided by the present invention was significantly reduced (p<0.05). Compared with the LPS group, the IgM content in the serum of female mice DEX and the Chinese medicine composition-H group provided by the present invention was significantly reduced (p<0.01). Compared with the LPS group, the percentage of CD3+ cell group in the peripheral blood of male and female mice in each administration group of the Chinese medicine composition provided by the present invention was significantly increased (p<0.01), and the percentage of CD8+T cell group was significantly reduced (p<0.05); the percentage of CD4+T cell group, all groups of female mice were significantly increased (p<0.01), while only the Chinese medicine composition-M and Chinese medicine composition-H groups provided by the present invention were significantly increased in male mice (p<0.05). In therapeutic administration, after treatment with the Chinese medicine composition provided by the present invention, the infiltration of eosinophils in the alveolar space and the metaplasia of bronchial goblet cells were reduced, the edema around blood vessels was weakened, and the pathological score was significantly lower than that of the LPS group (p<0.001).

[0074] The Chinese medicine composition provided by the present invention has a good inhibitory effect on xylene-induced ear swelling in mice; the Chinese medicine composition provided by the present invention has a good intervention effect on LPS-induced acute pneumonia in mice by both preventive and therapeutic administration, can effectively reduce the lung index, improve lung pathology, inhibit the level of inflammatory mediators and IgM level, regulate T cell immune function, and has a good dose-effect relationship. The effect of the Chinese medicine composition provided by the present invention at a clinically equivalent dose on intervening in LPS acute pneumonia is equivalent to that of 1.82 mg of dexamethasone, and there is no obvious gender difference in antagonizing LPS acute pneumonia in male and female mice.

[0075] Antiviral Effects:

[0076] The present invention has conducted in vitro and in vivo studies on the efficacy of the Chinese medicine composition against influenza virus H3N2 strains, aiming to evaluate the effect of the Chinese medicine composition provided by the present invention against influenza virus H3N2 through in vitro and in vivo experiments, and to provide a basis for its clinical research. In vitro experiments were conducted to test the anti-influenza virus H3N2 strain; in vivo experiments used influenza virus H3N2 to infect BALB / c mice, and administered the Chinese medicine composition provided by the present invention by oral gavage for 5 consecutive days, recorded the changes in body weight, collected the lung tissue of the infected mice, weighed the lung tissue of the mice, detected the virus titer of the lung tissue, performed lung tissue pathology detection, and detected the effects on the expression of inflammatory factors. The specific experimental results are summarized as follows:

[0077] (1) In vitro antiviral test results: The antiviral test results show that the IC value of the Chinese medicine composition provided by the present invention against influenza virus H3N2 is 50 It is 1331μg / mL.

[0078] (2) In vivo experimental results: ①② Effect on weight changes of infected mice: After influenza virus infection, the weight of mice showed a downward trend to varying degrees. The high-dose group of the Chinese medicine composition provided by the present invention (39.8 g slices / kg) had a certain inhibitory effect on the weight loss of infected mice; ③ Effect on lung index: After influenza virus infection, the lung index of the model group was significantly increased. After intervention administration, the high-dose group of the Chinese medicine composition provided by the present invention had a significant inhibitory effect on the increase in lung index induced by virus infection; ④ Effect on virus titer in lung tissue: After influenza virus infection, the virus titer in the lung tissue of the infected mice in the model group was significantly increased. After administration of the Chinese medicine composition provided by the present invention, the 39.8 g slices / kg group had a significant inhibitory effect on the increase in virus titer in the lungs of infected mice; ⑤ Effect on pathological damage to lung tissue: Compared with the blank group, the lung tissue of the animals in the model group showed interstitial pneumonia, edema fluid was visible in the alveolar cavity, bleeding, lymphocyte infiltration, hyaline membrane formation, interstitial lymphocyte infiltration, adjacent bronchiolar mucosa, bronchial mucosa degeneration / necrosis, shedding and other changes. After the intervention administration of the Chinese medicine composition provided by the present invention, the 39.8g slices / kg administration group was compared with the model group, and the mouse lung tissue damage was significantly improved, especially interstitial pneumonia, bronchiolar mucosal epithelial degeneration / necrosis, and shedding had a good improvement effect. ⑥ Effect on the expression of inflammatory factors: After influenza virus infection of mice, compared with the normal group, the expression of IL-6, IL-8, IP-10, TNF-α, MCP-1 and IL-1β in the virus model group was significantly increased; compared with the virus model group, the Chinese medicine composition provided by the present invention can reduce the overexpression of inflammatory factors to a certain extent after intervention, among which the 39.8g slices / kg administration group had a significant inhibitory effect on the overexpression of IL-8, and the 19.9g slices / kg administration group had a significant inhibitory effect on the overexpression of MCP-1.

[0079] In summary, under the conditions of this study, a high dose of the Chinese medicine composition provided by the present invention (39.8 g / kg) has an inhibitory effect on the proliferation of influenza virus H3N2 in cells, and has an inhibitory effect on the weight change, lung index and lung tissue virus titer of virus-infected mice, and has a good improvement effect on lung lesions caused by virus infection, especially interstitial pneumonia, degeneration / necrosis and shedding of bronchiolar mucosal epithelium.

[0080] The Chinese medicine composition provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0081] The present invention will be further described below in conjunction with embodiments:

[0082] Preparation Example 1 Preparation of Chinese medicine composition

[0083]

[0084] 140g of the above 17 herbs, crushed forsythia, added 500ml of water and boiled three times, each time for 2 hours. The distilled volatile oil was collected during the first and second decoctions and encapsulated with beta-cyclodextrin; filtered, the filtrate was centrifuged, the supernatant was concentrated to a clear paste with a relative density of about 1.10-1.15 (60°C), spray-dried, and 3.98g of slices / g of powder was prepared for use. Before use, it was prepared into a low (0.25g extract / ml, 0.995g slices / ml), medium (0.5g extract / ml, 1.99g slices / ml), and high (1g extract / ml, 3.98g slices / ml) concentration of Chinese medicine composition.

[0085] Example 1

[0086] 1 Experimental Purpose

[0087] (1) A mouse ear swelling model was prepared by the xylene smearing method to observe the anti-inflammatory effect of the Chinese medicine composition provided by the present invention (prepared in Preparation Example 1).

[0088] (2) An acute pneumonia model in mice was prepared by injecting lipopolysaccharide (LPS) through exposed trachea, and the anti-inflammatory effects of the preventive and therapeutic administration of the Chinese medicine composition provided by the present invention were observed.

[0089] 2 Experimental Materials

[0090] 2.1 Experimental drugs

[0091] Table 1 Experimental drug information

[0092]

[0093] 2.2 Experimental reagents and consumables

[0094] Table 2 Information of main experimental reagents and consumables

[0095]

[0096] Table 2: Information of main experimental reagents and consumables

[0097]

[0098]

[0099] 2.3 Experimental animals

[0100] Kunming mice (KM), SPF grade, body weight 20 ± 2g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., animal production license number: SCXK (Xiang) 2019-0004, animal certificate numbers: No. 430727230102182451, No. 430727230102071925, No. 430727230102146686, No. 430727230102155684 and No. 430727230102181025. All animals were kept at a temperature of 23 ± 1 °C, humidity of 50 ± 5%, and a 12-h light-dark cycle (8:00-20:00 light cycle, 20:00-8:00 dark cycle). According to the guidelines of the National Institutes of Health (USA), all animals were protected by the Animal Care and Use Committee of the Institute of Integration and Translation of Chinese Medicine for Brain Diseases, Chengdu University of Traditional Chinese Medicine (animal ethics approval numbers are: IBD2023008, IBD 2023009, IBD 2023010, IBD 2023011, and IBD 2023012).

[0101] 2.4 Experimental instruments

[0102] Table 3 Main experimental instrument information

[0103]

[0104] 2.5 Test Site

[0105] Level 3 Laboratory of State Administration of Traditional Chinese Medicine - Level 3 Laboratory of Traditional Chinese Medicine Pharmacology of Chengdu University of Traditional Chinese Medicine (TCM-2019-315). Experimental operators strictly wear masks, laboratory coats, gloves and hats.

[0106] 3 Animal grouping and drug dosage design

[0107] 3.1 Grouping and drug dosage design of mouse ear swelling model

[0108] The mice were adaptively fed for 3 days and randomly divided into 5 groups according to body weight stratification, namely, a xylene model group (Xylenes), a dexamethasone group (Dexamethasone, DEX, 1.82 mg / kg), a low-dose group of the Chinese medicine composition provided by the present invention (the Chinese medicine composition-L provided by the present invention, 2.5 g extract / kg, 9.95 g decoction pieces / kg), a medium-dose group of the Chinese medicine composition provided by the present invention (the Chinese medicine composition-M provided by the present invention, 5 g extract / kg, 19.9 g decoction pieces / kg), and a high-dose group of the Chinese medicine composition provided by the present invention (the Chinese medicine composition-H provided by the present invention, 10 g extract / kg, 39.8 g decoction pieces / kg), with 6 males and 6 females in each group. DEX, the Chinese medicine composition-L provided by the present invention, the Chinese medicine composition-M provided by the present invention, and the Chinese medicine composition-H provided by the present invention are all prepared into suspensions of corresponding concentrations using 0.5% CMC-Na solution containing 26.5mg / ml β-cyclodextrin as solvent (the auxiliary material β-cyclodextrin accounts for 2.65% of the total amount of the material); the mice in each administration group of the Chinese medicine composition provided by the present invention and the dexamethasone group are respectively gavaged once a day at the corresponding concentration for 3 consecutive days. 2h after the last administration, 0.05mL of xylene is evenly applied to both sides of the right ear auricle of each group of mice to induce ear swelling, and the left ear is used as a control. The mice are killed 30min later, and the left and right ear pieces of the same part are taken with a puncher and weighed.

[0109] 3.2 Grouping and drug dosage design for preventive administration in the mouse acute pneumonia model

[0110] The mice were adaptively fed for 3 days and randomly divided into 6 groups according to body weight stratification, namely, a sham operation group (Sham), an LPS model group (LPS), a dexamethasone group (Dexamethasone, DEX, 1.82 mg / kg), a low-dose group of the Chinese medicine composition provided by the present invention (the Chinese medicine composition-L provided by the present invention, 2.5 g extract / kg, 9.95 g decoction pieces / kg), a medium-dose group of the Chinese medicine composition provided by the present invention (the Chinese medicine composition-M provided by the present invention, 5 g extract / kg, 19.9 g decoction pieces / kg), and a high-dose group of the Chinese medicine composition provided by the present invention (the Chinese medicine composition-H provided by the present invention, 10 g extract / kg, 39.8 g decoction pieces / kg), with 7 males and 7 females in each group. DEX, the Chinese medicine composition-L provided by the present invention, the Chinese medicine composition-M provided by the present invention, and the Chinese medicine composition-H provided by the present invention are all prepared into suspensions of corresponding concentrations using 0.5% CMC-Na solution containing 26.5 mg / ml β-cyclodextrin as a solvent (the auxiliary material β-cyclodextrin accounts for 2.65% of the total amount of the material); each administration group of the Chinese medicine composition provided by the present invention and the DEX group of mice were gavaged once a day at the corresponding concentration, and the administration volume was 10 ml / kg, and the administration was continued for 3 days. The mice in the Sham group and the LPS group were given an equal volume of CMC-Na solution (the auxiliary material β-cyclodextrin accounts for 2.65% of the total amount of the material). 2 hours after the last administration, the mice were anesthetized with isoflurane, the trachea was exposed, and LPS solution (5 mg / mL, the administration volume was 10 μl / 10 g) was slowly dripped in within 3 minutes. [1-2] ) to prepare a mouse acute pneumonia model. After the model was established, blood was collected from the eye sockets at different time points according to the pharmacokinetic results of inflammatory factors (determined according to the kinetic results of the detection indicators). [3] ). Blood was collected from the orbit at 2h (to be tested for IL-6), and blood was collected at 8h (to be tested for IL-1β, TNF-α, and immunoglobulin IgM); blood was collected 8h after modeling and the mice were killed, lungs were collected under sterile conditions, lung index was determined, lung tissues were fixed with 4% neutral formaldehyde, and HE staining was performed for pathological morphology.

[0111] 3.3 Grouping and drug dosage design for therapeutic administration in the mouse acute pneumonia model

[0112] The mice were adaptively fed for 3 days, and the grouping, modeling method and drug preparation were the same as in 3.2. The difference was that the mice were adaptively fed for 3 days and the acute pneumonia model of mice was first prepared. 30 minutes after modeling, the mice were given the corresponding drugs by gavage for the first time in a conscious state. The dosage and volume were the same as in 3.2, and the drugs were given by gavage once a day for 3 consecutive days. 8 hours after the administration on the 3rd day, blood was collected from the orbits (to be tested for IL-6, IL-1β and TNF-α, immunoglobulin IgM) and the mice were killed. The lungs were taken under sterile conditions, the lung index was determined, and the pathological morphology was detected by HE staining after fixation with 4% neutral formaldehyde.

[0113] 3.4 Detection indicators

[0114] 3.4.1 Ear swelling

[0115] The animals were sacrificed and ear pieces were prepared. The left and right ear pieces were weighed, and the swelling degree and swelling inhibition rate were calculated.

[0116] Swelling degree = right ear weight (swollen ear) - left ear weight (control ear)

[0117]

[0118] 3.4.2 Preventive and therapeutic administration

[0119] (1)Weight

[0120] The animals were weighed and recorded 2 h before modeling, and then weighed and recorded once a day.

[0121] (2) Lung Index

[0122] After the animals were sacrificed, lung tissues were obtained aseptically, weighed immediately, and lung index was calculated.

[0123]

[0124] (3) Serum inflammatory indicators

[0125] Blood was collected from the mouse orbits, separated and prepared for serum, and the levels of IL-6, IL-1β, and TNF-α in serum were detected using ELISA kits.

[0126] (4) Serum immunoglobulin M (IgM)

[0127] Blood was collected from the mouse orbits, and serum was separated and prepared. The IgM content in the serum was determined by immunoturbidimetry.

[0128] (5) Peripheral blood T cell subsets

[0129] Flow cytometry detection of CD 3+ , CD 4+ and CD 8+ T cell percentage.

[0130] (6) Lung tissue pathomorphology

[0131] After the lung tissue was weighed, it was immediately fixed with 4% neutral formaldehyde for 48 h. The lung tissue paraffin blocks were prepared by conventional methods and then sliced ​​(4 μm) by an automatic microtome. HE staining was performed routinely, and the films were read and photographed under a 40× field of view, and pathological scores were performed according to Table 4. The total score (range 0 to 10) was the sum of the individual scores and was scored by a pulmonary pathologist in a blinded manner (Table 4).

[0132] Table 4 Lung tissue pathological morphology scoring table

[0133]

[0134] 3.5 Data processing and statistical analysis

[0135] The measurement data were normal distribution and the mean ± standard deviation was used. "One-way ANOVA was used to compare the significance among the groups. p<0.05 was considered statistically significant.

[0136] Effect Example 1 Ear swelling test

[0137] 1.1 Effect of the Chinese medicine composition provided by the present invention on the body weight of mice with ear swelling

[0138] The results are shown in Table 5 and Figure 1 Compared with the Xylenes group, there was no significant difference in the body weight changes of female and male mice in the DEX and Chinese medicine composition groups at the same time point (p>0.05).

[0139] Table 5 Effect of the Chinese medicine composition provided by the present invention on the body weight of mice with ear swelling

[0140]

[0141]

[0142] Note: Compared with the Xylenes group, * p<0.05

[0143] 1.2 Effect of the Chinese medicine composition provided by the present invention on xylene-induced ear swelling in mice

[0144] The results are shown in Table 6 and Figure 2 . It can be seen from the results that, compared with the swelling degree of each group, in male mice, the ear swelling degrees of the DEX, the Chinese medicinal composition-M provided by the present invention, and the Chinese medicinal composition-H provided by the present invention were significantly lower than those of the Xylenes group (p<0.05 or p<0.001), and the swelling inhibition rates were 93.38%, 60.29%, and 88.24%, respectively; in female mice, the ear swelling degrees of the DEX, the Chinese medicinal composition-M provided by the present invention, and the Chinese medicinal composition-H provided by the present invention were significantly lower than those of the Xylenes group (p<0.01), and the swelling inhibition rates were 95.17%, 74.48%, and 88.96%, respectively; the Chinese medicinal composition provided by the present invention showed a good dose-effect relationship in the inhibitory effect on the ear swelling of mice.

[0145] Table 6 Effect of the Chinese medicine composition provided by the present invention on xylene-induced ear swelling in mice

[0146]

[0147] Note: Compared with the Xylenes group, *p<0.05; **p<0.01; ***p<0.001

[0148] Effect Example 2 Effect of preventive and therapeutic administration of the Chinese medicine composition provided by the present invention on acute pneumonia in mice

[0149] 2.1 Effect of the Chinese medicine composition provided by the present invention on the body weight of mice with acute pneumonia

[0150] In the preventive administration, compared with the Sham group, at the same time point of preventive administration, there was no significant difference in the body weight changes of male and female mice among the groups. The results are shown in Tables 7 and Figure 3 .

[0151] Table 7 Effect of the Chinese medicine composition provided by the present invention on the body weight of mice with acute pneumonia - preventive administration

[0152]

[0153] Note: Compared with the Sham group, # p<0.05, compared with LPS group, * p<0.05.

[0154] In the therapeutic administration, compared with the Sham group, the body weight of both male and female mice in the LPS modeling group decreased on the second day, and the body weight of both male and female mice decreased significantly on the third day (p<0.01); compared with the LPS group, after 3 days of therapeutic administration of the Chinese medicine composition provided by the present invention, the body weight of mice in the Chinese medicine composition-M group provided by the present invention and the Chinese medicine composition-H group provided by the present invention was significantly adjusted, among which the adjustment of the Chinese medicine composition-H group provided by the present invention was statistically significant (p<0.05). The results are shown in Tables 8 and Figure 4 .

[0155] Table 8 Effect of the Chinese medicine composition provided by the present invention on the body weight of mice with acute pneumonia - therapeutic administration

[0156]

[0157]

[0158] Note: Compared with the Sham group, ## p<0.01, ### p<0.001, compared with LPS group, * p<0.05.

[0159] 2.2 Effect of the Chinese medicine composition provided by the present invention on the lung index of mice with acute pneumonia model

[0160] In the preventive administration experiment, compared with the Sham group, the lung index of male and female mice in the LPS group was extremely significantly increased, with extremely significant statistical significance (p<0.001); compared with the LPS group, the lung index of male and female mice in the DEX group, the Chinese medicine composition-M provided by the present invention, and the Chinese medicine composition-H group provided by the present invention was extremely significantly reduced (p<0.01 or p<0.001); the lung index of female mice in the preventive administration group of the Chinese medicine composition-L provided by the present invention was also extremely significantly reduced compared with the LPS group (p<0.001) (the results are shown in Tables 9 and Figure 5 ). In the therapeutic administration experiment, compared with the Sham group, the lung indexes of male and female mice in the LPS group were extremely significantly increased, with extremely significant statistical significance (p<0.001); compared with the LPS group, the lung indexes of male mice in the DEX group, the Chinese medicine composition-M provided by the present invention, and the Chinese medicine composition-H group provided by the present invention were significantly reduced (p<0.05 or p<0.01 or p<0.001), and the lung indexes of female mice in the DEX group and the Chinese medicine composition-H group provided by the present invention were significantly reduced (p<0.05) (the results are shown in Tables 9 and Figure 6 ).

[0161] Table 9 Effect of the Chinese medicine composition provided by the present invention on the lung index of mice with acute pneumonia model

[0162]

[0163]

[0164] Note: Compared with the Sham group, ### p<0.001, compared with LPS group, * p<0.05, ** p<0.01, *** p<0.001

[0165] 2.3 Effect of the Chinese medicine composition provided by the present invention on the content of inflammatory factors in mice with acute pneumonia model

[0166] (1) Effect on serum IL-6 content: Under the preventive administration mode, compared with the Sham group, the serum IL-6 content of male and female mice in the LPS group was extremely significantly increased (p<0.001). Compared with the LPS group, only the DEX and the Chinese medicine composition-H group provided by the present invention had significantly reduced serum IL-6 content in male mice (p<0.05 or p<0.001), while the serum IL-6 content in female mice in the three dose groups of DEX and the Chinese medicine composition provided by the present invention was significantly reduced (p<0.05, p<0.01 or p<0.001). In the therapeutic administration mode, compared with the Sham group, the serum IL-6 levels of male and female mice in the LPS group were also significantly increased (p<0.001); compared with the LPS group, the serum IL-6 levels of DEX and the three dose groups of the Chinese medicine composition provided by the present invention were significantly reduced in both male and female mice (p<0.05, p<0.01 or p<0.001) (the results are shown in Tables 10 and Figure 7 , Fig.10 ).

[0167] (2) Effect on serum IL-1β content: Under the preventive administration mode, compared with the Sham group, the serum IL-1β content of male and female mice in the LPS group was extremely significantly increased (p<0.001); compared with the LPS group, the serum IL-1β content of the Chinese medicine composition-H group provided by the present invention in male mice was extremely significantly decreased (p<0.001); the serum IL-1β content of female mice in the DEX and Chinese medicine composition-H groups provided by the present invention was significantly decreased (p<0.05 or p<0.001). In the therapeutic administration mode, compared with the Sham group, the serum IL-1β levels of male and female mice in the LPS group were significantly increased (p<0.05 or p<0.001); compared with the LPS group, the serum IL-1β levels of male mice in the DEX and Chinese medicine composition-H groups provided by the present invention were significantly reduced (p<0.05), while the serum IL-1β levels of female mice in the three dose groups of DEX and the Chinese medicine composition provided by the present invention were significantly reduced (p<0.05, p<0.01) (the results are shown in Tables 10 and Figure 8 , Fig.11 ).

[0168] (3) Effect on serum TNF-α content: Under the preventive administration mode, compared with the Sham group, the serum TNF-α content of male and female mice in the LPS group was extremely significantly increased (p<0.05 or p<0.001); compared with the LPS group, the serum TNF-α content of male mice in the three dose groups of DEX and the Chinese medicine composition provided by the present invention was extremely significantly reduced (p<0.01 or p<0.001). Under the therapeutic administration mode, the serum TNF-α content of female mice in the LPS group was significantly higher than that in the Sham group (p<0.001), while the serum TNF-α content of the DEX, the Chinese medicine composition-L provided by the present invention, and the Chinese medicine composition-H provided by the present invention were significantly lower than those in the LPS group (p<0.05 or p<0.01) (the results are shown in Tables 10 and Fig. 9 , Fig.12 ). Table 10 Effect of the Chinese medicine composition provided by the present invention on the levels of serum inflammatory factors IL-6, IL-1β and TNF-α in mice with acute pneumonia model

[0169]

[0170] Note: Compared with the Sham group, # p<0.05, ### p<0.001, compared with LPS group, * p<0.05, ** p<0.01, *** p<0.001

[0171] 2.4 Effect of the Chinese medicine composition provided by the present invention on serum IgM content in mice with acute pneumonia

[0172] For female mice, compared with the Sham group, the IgM levels in the serum of both male and female mice in the LPS group were significantly increased (p<0.001). Compared with the LPS group, the IgM levels in the serum of the DEX and the Chinese medicine composition-H groups provided by the present invention were significantly reduced (p<0.01) regardless of preventive or therapeutic administration (results are shown in Tables 11, Fig.13 , Fig.14 ).

[0173] Table 11 Effect of the Chinese medicine composition provided by the present invention on serum IgM content in mice with acute pneumonia model

[0174]

[0175]

[0176] Note: Compared with the Sham group, ### p<0.001, compared with LPS group, *p<0.05, ** p<0.01, *** p<0.001

[0177] 2.5 Effect of the Chinese medicine composition provided by the present invention on peripheral blood T cell subsets in mice with acute pneumonia

[0178] The results are shown in Table 12. In the preventive administration mode, the peripheral blood CD 3+ , CD 4+ The percentage of T cells decreased significantly (p<0.01), 8+ The percentage of T cells increased significantly (p<0.01). Compared with the LPS group, the peripheral blood CD4+ T cells of male and female mice in each drug-treated group 3+ The percentage of T cells increased significantly (p<0.05 or p<0.01 or p<0.001); the peripheral blood CD4+ T cells of male mice in the Chinese medicine composition provided by the present invention-H group 4+ The percentage of T cells increased significantly (p<0.05), and the peripheral blood CD4+ T cells of female mice in all drug-treated groups increased significantly (p<0.05). 4+ The percentage of T cells was significantly increased (p<0.01 or p<0.001); 8+ As for T cells, except for the male mice treated with the Chinese medicine composition-H provided by the present invention, which showed no significant decrease, the peripheral blood CD 8+ The percentage of T cells in the LPS group was significantly decreased compared with that in the Sham group (p<0.05 or p<0.01). 3+ , CD 4+ The percentage of T cells was significantly decreased (p<0.01 or p<0.001), and the CD 8+ The percentage of T cells increased significantly (p<0.01 or p<0.001), which was basically consistent with the effect of preventive administration on T cell subsets. Compared with the LPS group, the peripheral blood CD 3+ The percentage of T cells increased significantly, while CD 8+ The percentage of T cells was significantly decreased (p<0.05 or p<0.01 or p<0.001), and the 4+ In terms of T cell percentage, except for the Chinese medicine composition-L group provided by the present invention, which showed an increasing trend compared with the LPS group, the CD4 T cells in the other drug-treated groups, regardless of whether they were female or male mice, 4+ The percentage of T cells was significantly higher than that in the LPS group (p<0.05 or p<0.01 or p<0.001).

[0179] Table 12 Effects of the Chinese medicine composition provided by the present invention on peripheral blood T cell subsets in mice with acute pneumonia model

[0180]

[0181]

[0182] Note: Compared with the Sham group, # p<0.05, ## p<0.01, ### p<0.001, compared with LPS group, * p<0.05, ** p<0.01, *** p<0.001

[0183] 2.6 Effect of the Chinese medicine composition provided by the present invention on the pathological morphology of lung tissue in mice with acute pneumonia model

[0184] In the preventive administration, the alveolar structure of the mice in the Sham group was normal, the alveoli were intact, the pulmonary artery lumen was large, the wall thickness was uniform and there was no thickening, the number of eosinophils in the alveolar space and bronchial goblet cell metaplasia was very small, there was no edema around the blood vessels, and the pathological score was recorded as 0.00. The lung structure of the mice in the LPS group was damaged to varying degrees, the alveolar structure was disordered, the alveolar wall was thinned or broken, the alveolar space was thickened, the lumen was narrowed, eosinophil infiltration was visible in the alveolar space, and there was bronchial goblet cell metaplasia, obvious edema appeared around the blood vessels, and the pathological score was significantly higher than that of the Sham group (p<0.001); after the intervention treatment with DEX and the Chinese medicine composition provided by the present invention, it can be seen that the lung structure has recovered, the eosinophil infiltration in the alveolar space and the bronchial goblet cell metaplasia have decreased, the edema around the blood vessels has weakened, and the pathological score is significantly lower than that of the LPS group (p<0.05). The representative pathological pictures and scoring pictures are shown in Figure 15-16 .

[0185] During therapeutic administration, the mice in the Sham group had normal alveolar structure, intact alveoli, larger pulmonary artery lumen, uniform wall thickness without thickening, fewer eosinophils in the alveolar space and bronchial goblet cell metaplasia, and no edema around the blood vessels. The pathological score was recorded as 0.00. The lung structure of mice in the LPS group was damaged to varying degrees, with disordered alveolar structure, thinning or rupture of alveolar wall, thickening of alveolar space, stenosis of lumen, eosinophil infiltration in alveolar space, bronchial goblet cell metaplasia, obvious edema around blood vessels, and significantly higher pathological scores than those in the Sham group (p<0.001). After intervention treatment with DEX and the Chinese medicine composition provided by the present invention, it was found that the lung structure was restored, eosinophil infiltration in alveolar space and bronchial goblet cell metaplasia were reduced, and edema around blood vessels was weakened. The pathological score of the DEX group was significantly lower than that of the LPS group (p<0.01), and the pathological score of the Chinese medicine composition group provided by the present invention was significantly lower than that of the LPS group (p<0.001). The representative pathological figures and scoring figures are shown in Figure 17-Figure 18 .

[0186] In the preventive and therapeutic drug intervention, there was no significant difference in the weight change of mice; the lung index of mice in each group of the Chinese medicine composition provided by the present invention was improved to varying degrees compared with the LPS group, and the pharmacodynamic relationship was dose-dependent; in the detection of serum inflammatory factors and immunoglobulins, the medium and high dose groups of the Chinese medicine composition provided by the present invention had a significant antagonistic effect on the increase of inflammatory factors IL-6, IL-1β, TNF-α and IgM caused by LPS, and had a significant callback effect on T cell subset disorders. Pathological morphology found that the Chinese medicine composition provided by the present invention can significantly improve the inflammatory changes and pathological scores of mouse lung tissue caused by LPS.

[0187] Example 2 In vitro and in vivo efficacy against influenza virus H3N2 strains

[0188] 1. Test substances and their management

[0189] Drug: The Chinese medicine composition provided by the present invention (prepared in Preparation Example 1)

[0190] Content: 3.98g crude drug / g sample

[0191] Storage of test substance: Protect from light, seal and dry, store at 15-25℃.

[0192] Note: The client is responsible for the content, stability, uniformity and consistency of the samples provided by the client.

[0193] Preparation method and environmental requirements: For in vitro tests, prepare the stock solution with PBS, divide it into small portions, and store it at 4°C for later use. When preparing the solution, dilute it with the cell working solution to the required concentration for testing. For in vivo tests, prepare it with physiological saline to the required concentration.

[0194] 2. Cells and viruses

[0195] Cells and sources: MDCK cells, maintained by the Virus Laboratory of the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health.

[0196] Virus source and quality attributes: Influenza virus H3N2 strain, preserved by the Virus Laboratory of the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health.

[0197] Storage conditions: Store in -80℃ ultra-low temperature refrigerator.

[0198] Culture method: Virus isolation and culture were performed in MDCK cell carriers using DMEM medium containing 2% fetal bovine serum.

[0199] 3. Experimental Animals

[0200] Animal strain: BALB / c mice

[0201] Number of animals: 60.

[0202] Age: 5-6 weeks

[0203] Gender: Female

[0204] Source: Zhuhai Baishitong Biotechnology Co., Ltd.

[0205] Grade: SPF grade

[0206] Laboratory Animal Production License Number: SYXK(Guangdong)2020-0051; Laboratory Animal Quality Certificate Number: No.44822700036463. Animal Ethics Approval Number: 20230474.

[0207] Reason for the use of number of experimental animals: The animals were BALB / c mice. The experiment set up 6 groups, with 10 mice in each group. The number of animals used in this experiment can meet the requirements of statistical tests.

[0208] 4. Experimental reagents and instruments

[0209] 4.1 Main experimental reagents

[0210] Table 13

[0211]

[0212] Table 13

[0213]

[0214]

[0215] 6.2 Main experimental instruments

[0216] Table 14

[0217]

[0218] 5. Test methods

[0219] 5.1 Overview of test methods

[0220] 5.1.1 In vitro antiviral test

[0221] The experiment was conducted in a BSL-2 laboratory. A cell control, a virus control (negative control), and a drug group were set up. The cell density was 2×10 5 The MDCK cell suspension of 10 cells / mL was plated in a sterile 96-well culture plate, 100 μL was added to each well, and cultured at 37°C and 5% CO2 for 24 h; the culture supernatant was discarded, and 100 TCID 50Virus solution, 100 μL / well, conventional culture adsorption for 2 hours; discard the culture supernatant, add 100 μL of different concentrations of drugs to each well, 100 μL / well, 3 replicates for each concentration, and conventional culture incubation for 48 hours. The cell lesion (CPE) was observed under an optical microscope, and the degree of cell lesions was recorded according to the following 6-level standards: "-" means no cell lesions; "±" means less than 10% cell lesions; "+" means about 25% cell lesions; "++" means about 50% cell lesions; "+++" means about 75% cell lesions; "++++" means more than 75% cell lesions. GraphPad Prism 5.0 software was used to calculate the half inhibitory concentration (IC 50 ).

[0222] 5.1.2 In vivo studies

[0223] Antiviral test: BALB / c mice were divided into a normal group, a virus infection group, a 10 g / kg, 5 g / kg and 2.5 g / kg dose group of the Chinese medicine composition provided by the present invention, and a positive control group (OSE-30 mg / kg), with 10 mice in each group. Except for the normal group, mice in other groups were intranasally infected with PBS for 10 4 PFU of H3N2 virus. 2h after infection, mice in the drug group were gavaged with the Chinese medicine composition provided by the present invention once a day for 5 consecutive days. Body weight changes were recorded daily after infection, lungs were dissected and taken on the 5th day after infection, and the weight of lung tissue was recorded. The lung tissue of animals in the infection group was homogenized to detect the virus titer and the expression of inflammatory factors; lung tissue was taken for pathological study.

[0224] The specific steps are as follows:

[0225] (1) Detection of virus titer in mouse lung homogenate

[0226] After removing the lung tissue of the mouse, place it in a culture dish, cut it into pieces, transfer it to a homogenizer tube, dilute it with physiological saline 1:10 (w / v), and homogenize it at 8000rpm for 10 minutes. All the above operations are performed in an ice bath. Transfer the homogenate to a 1.5mL EP tube and centrifuge it at 10000rpm for 10 minutes at 4℃. Pipette the supernatant, aliquot it, and store it at -80℃ for later use.

[0227] MDCK cells with good growth status were seeded in 96-well plates, and 1×10 4 cells / well, and continue to culture for 24 h. After the cells adhere to the wall and grow into a complete monolayer, the supernatant is discarded and the cells are washed twice with PBS. The frozen lung homogenate supernatant is thawed and diluted 10 times to 10 -1 -10 -5Five concentrations, add the diluted lung homogenate supernatant of each group of mice to the 96-well plate, add 100 μL of lung supernatant to each well, set up blank control wells at the same time, add cell culture medium to the blank control wells, make 4 replicates for each concentration in parallel, and culture in an incubator. Continuously observe for 48 hours, observe the cytopathic effect (CPE) every day, record the number of lesion wells for each gradient concentration, and calculate its TCID for MDCK cells 50 value.

[0228] (2) Observation of mouse lung tissue pathology

[0229] Fixation and embedding: Fixation (with 4% paraformaldehyde) → Wash and trim the block, trim the tissue to a smooth section → Dehydrate with 50%, 70%, 80%, 95%, 100% ethanol solution → Immerse in xylene I and II for transparency → Immerse in 60℃ wax I and II for permeation → Embed in paraffin

[0230] Section staining: Section (thickness is about 4um) → paraffin sections are immersed in transparent agent I, transparent agent II, and transparent agent III for dewaxing → immersed in 100%, 90%, 80%, 70% and other alcohol solutions for 5 minutes each → wash with water → hematoxylin staining for 3 minutes → wash with water → 0.5% hydrochloric acid ethanol differentiation for a few seconds → wash with tap water for 15 minutes to reverse blue → eosin staining for 2 minutes → immersed in 95% ethanol I, 95% ethanol II, 100% ethanol I, 100% ethanol II, transparent agent I, transparent agent II, and transparent agent III respectively for dehydration and transparency → seal with neutral gum → observe and take pictures under a microscope.

[0231] (3) RT-qPCR detection of inflammatory factors in mouse lung tissue

[0232] 1) RNA extraction

[0233] Take 100mg of mouse lung tissue and place it in a 1.5mL EP tube. Add 1mL Trizol Reagent to the EP tube, insert it into ice, quickly cut it into pieces, and place it at room temperature for 5min. Add 200μL of chloroform, cover tightly, shake vigorously for 1min, and let it stand at room temperature for 5min. Centrifuge at 4℃, 10000rpm for 10min. The suspension is divided into three layers. The upper layer is a clear aqueous liquid. Carefully aspirate the upper aqueous phase and transfer it to another new 1.5mL EP tube, about 500μL, add an equal volume of isopropanol, shake and mix, and let it stand at room temperature for 10min. Centrifuge at 4℃, 10000rpm for 10min. After centrifugation, a milky white translucent gelatinous precipitate can be seen on the bottom side wall of the EP tube, which is the total RNA. Pour off the supernatant, add 500μL of 75% ethanol to the precipitate, repeatedly invert and wash, and centrifuge at 4℃, 10000rpm for 10min. Pour off the supernatant, add 500 μL of 75% ethanol, wash repeatedly by inversion, centrifuge at 4°C, 10,000 rpm for 5 min. After drying the ethanol, let it stand at room temperature for 5 min, add RNase-free dd H2O to dissolve the RNA, and reverse transcribe it into cDNA.

[0234] 2) Reverse transcription reaction

[0235] Before reverse transcription of RNA, a genomic DNA removal reaction is performed. The reaction system is as follows:

[0236] Table 15

[0237]

[0238]

[0239] Reaction conditions: 42℃2min

[0240] Store at 4℃

[0241] mRNA is reverse transcribed into cDNA; the reverse transcription reaction system is configured as follows:

[0242] Table 16

[0243]

[0244] The reverse transcription reaction conditions are as follows:

[0245] Table 17

[0246]

[0247] 3) Real-time fluorescence quantitative PCR detection

[0248] The reaction system is as follows:

[0249] Table 18

[0250]

[0251] The PCR reaction conditions are as follows:

[0252] Table 19

[0253]

[0254] 5.2 Data Statistical Processing Methods

[0255] SPSS 23.0 software was used for statistical analysis. The data were expressed as ANOVA. If there was a statistically significant difference between the groups (P<0.05), the LSD test was performed on the mean values ​​between the groups. The count data were tested using the X2 test.

[0256] Effect Example 4 In vitro antiviral test results

[0257] The results of the antiviral test are shown in Table 20 and Fig.19 As shown, the antiviral test results show that the IC value of the Chinese medicine composition provided by the present invention against influenza virus H3N2 is 50 It is 1331μg / mL.

[0258] Table 20 In vitro antiviral efficacy results

[0259]

[0260] Note: Compared with the virus group: *** P < 0.001, ** P<0.01, * P<0.05.

[0261] Effect Example 5 In vivo test results

[0262] (1) The effect of the Chinese medicine composition provided by the present invention on the body weight of mice infected with influenza virus

[0263] The weight results showed that after influenza virus infection, the weight of mice showed a decreasing trend to varying degrees. After intervention administration, the high-dose group (10 g / kg) of the Chinese medicine composition provided by the present invention had a certain improvement effect on the weight loss of infected mice. The results are as follows Fig. 20 shown.

[0264] (2) The results of the test on the changes of lung index of mice infected with influenza virus by the Chinese medicine composition provided by the present invention

[0265] The results of lung index change test showed that after influenza virus infection, the lung index of the model group was significantly increased. After intervention administration, the high-dose group (10 g / kg) of the Chinese medicine composition provided by the present invention had a significant inhibitory effect on the increase of lung index induced by virus infection. The results are shown in Tables 21 and Fig.21 shown.

[0266] Table 21 Effect of the Chinese medicine composition provided by the present invention on changes in lung index of mice infected with influenza virus

[0267]

[0268] Note: Compared with the virus group: *** P < 0.001, ** P<0.01, * P<0.05.

[0269] (3) The results of the test on the virus titer of the lung tissue of mice infected with influenza virus by the Chinese medicine composition provided by the present invention

[0270] The results of virus titer detection showed that after influenza virus infection, the virus titer of the lung tissue of the infected mice in the model group was significantly increased. After the intervention administration of the Chinese medicine composition provided by the present invention, the 10g / kg administration group had a significant inhibitory effect on the increase in the virus titer of the lungs of the infected mice. The results are shown in Tables 22 and Fig. 22 shown.

[0271] Table 22 Effect of the Chinese medicine composition provided by the present invention on the change of virus titer in the lungs of mice infected with influenza virus

[0272]

[0273]

[0274] Note: Compared with the virus group: *** P < 0.001, ** P<0.01, * P<0.05.

[0275] (4) Study on the effect of the Chinese medicine composition provided by the present invention on lung pathology in mice infected with influenza virus

[0276] The test results are shown in Table 23. Fig.23 and Fig.24As shown, compared with the blank group, the model group animals had interstitial pneumonia in the lung tissue, edema fluid, hemorrhage, lymphocyte exudation, hyaline membrane formation, interstitial lymphocyte infiltration, adjacent bronchiolar mucosa, bronchial mucosal degeneration / necrosis, shedding and other changes. After the intervention administration of the Chinese medicine composition provided by the present invention, the 10g / kg administration group was compared with the model group, and the lung tissue damage of the mice was significantly improved, especially interstitial pneumonia, bronchiolar mucosal epithelial degeneration / necrosis, shedding had a better improvement effect.

[0277] Table 23 The results of pathological scoring of lung tissue of mice infected with influenza virus by the Chinese medicine composition provided by the present invention

[0278]

[0279] Note: Compared with the virus group: *** P < 0.001, ** P<0.01, * P<0.05.

[0280] (5) Detection results of the Chinese medicine composition provided by the present invention on the overexpression of inflammatory factors induced by influenza virus-infected mice

[0281] Effect on the expression of inflammatory factors: After influenza virus infection of mice, compared with the normal group, the expression of IL-6, IL-8, IP-10, TNF-α, MCP-1 and IL-1β in the virus model group was significantly increased; compared with the virus model group, the Chinese medicine composition provided by the present invention can reduce the overexpression of inflammatory factors to a certain extent after intervention, among which the Chinese medicine composition provided by the present invention-10g / kg administration group has a significant inhibitory effect on the overexpression of IL-8, and the Chinese medicine composition provided by the present invention-5g / kg administration group has a significant inhibitory effect on the overexpression of MCP-1. The results are shown in Tables 24, 25 and Fig.25 shown.

[0282] Table 24 Expression of IL-6, IL-8, MCP-1 and IP-10 mRNA in lung homogenates of infected mice

[0283]

[0284] Note: Compared with the Virus group, * P<0.05, ** P<0.01, *** P<0.001.

[0285] Table 25 Expression of TNF-α and IL-1β mRNA in lung homogenate of infected mice

[0286]

[0287] Note: Compared with the Virus group, * P<0.05, ** P<0.01, *** P<0.001.

[0288] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: In parts by weight, it includes the following components:

2. The Chinese medicine composition according to claim 1, characterized in that In parts by weight, it includes the following components:

3. Use of the Chinese medicine composition as claimed in claim 1 or 2 in the preparation of drugs for improving, preventing and / or treating anti-inflammatory and / or antiviral effects.

4. The use according to claim 3, characterized in that The anti-inflammatory treatment includes any of the following: (I) inhibit ear swelling; (II), reduce lung index; (III), reducing TNF-α content; (IV), reducing IL-6 and IL-1β levels; (V) Reduce IgM content; (VI), increased percentage of CD3+ cell population; (VII), increase the percentage of CD4+T cell population; (VIII), reduce the percentage of CD8+T cell population; (IX) Reducing the degree of inflammatory cell infiltration, perivascular edema and / or alveolar structural destruction in lung tissue; (X), inhibiting the decrease in body weight of mice induced by LPS; and / or (XI) Inhibits the increase of lung index caused by LPS.

5. The use according to claim 3 or 4, characterized in that: The anti-virus includes any of the following: (I) inhibiting viral proliferation; (II), inhibiting weight loss; (III) inhibiting the increase of lung index caused by viral infection; (IV) inhibiting the upregulation of lung virus titers caused by viral infection; (V), improving lung tissue damage; and / or (VI) Reduce the overexpression of inflammatory factors.

6. The use according to claim 5, characterized in that The lung tissue damage includes interstitial pneumonia, edema fluid visible in the alveolar cavity, bleeding, lymphocyte infiltration, formation of hyaline membrane, interstitial lymphocyte infiltration, degeneration / necrosis and shedding of adjacent bronchiolar mucosa or bronchial mucosa.

7. The use according to claim 5, characterized in that The inflammatory factors include one or more of IL-6, IL-8, IP-10, TNF-α, MCP-1 or IL-1β.

8. The use according to any one of claims 3 to 7, characterized in that The antiviral effect includes anti-influenza A(H3N2) virus, and the anti-inflammatory effect includes anti-acute pneumonia.

9. A pharmaceutical preparation, characterized in that The invention comprises the Chinese medicine composition according to claim 1 or 2 and a pharmaceutically acceptable adjuvant / excipient.

10. The pharmaceutical preparation according to claim 9, characterized in that It includes pharmaceutically acceptable preparations such as tablets, capsules, injections, oral solutions, and ointments.

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