Rosmarinic acid and scutellarin compatible composition and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a rosmarinic acid and scutellarin compatible composition and application thereof. The invention specifically discloses a rosmarinic acid and scutellarin composition as shown in formulas (I) and (II), and the rosmarinic acid and scutellarin composition is applied to treatment and / or prevention of acute lung injury. The screened rosmarinic acid and scutellarin composition is simple in preparation method, has a potential synergistic treatment effect on acute lung injury, has a remarkable treatment effect on lung functions, alveolar lavage fluid inflammatory factors and serum inflammatory indexes of LPS-induced mouse acute lung injury, and has a good application prospect. And compared with the toxic and side effects of dexamethasone on reducing body weight, the dexamethasone shows good safety, and meanwhile, the dexamethasone shows a remarkable inhibition effect on secretion of primary peritoneal macrophages NO, TNF-alpha and IL-6 in vitro. In conclusion, the rosmarinic acid and scutellarin composition can be in the form of a single composition or a medicinal composition, and has potential application value in treatment of acute lung injury and immune-related diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an optimal ratio composition of rosmarinic acid and scutellariae baicalin or compositions of different ratios, and different preparations containing the compatible compositions, and their use in preparing drugs for preventing and / or treating acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a disease caused by diffuse pulmonary interstitial and alveolar edema caused by damage to pulmonary capillary endothelial cells and alveolar epithelial cells during non-cardiogenic diseases such as severe infection, shock, trauma and burns, leading to acute hypoxic respiratory insufficiency or failure. Hypoxemia is difficult to correct after conventional oxygen inhalation. ALI is a common clinical critical illness, which is manifested as exudative lesions in both lungs on imaging. The presence or absence of sepsis and multiple organ dysfunction is one of the determining factors of ALI mortality.

[0003] The basic pathophysiological changes of ALI are the damage of alveolar capillary endothelial cells and alveolar epithelial cells caused by the uncontrolled acute inflammatory response after direct or indirect lung injury. The increased permeability leads to non-cardiogenic pulmonary edema and hyaline membrane formation, accompanied by pulmonary interstitial fibrosis in the late stage. In the early stage of ALI, diffuse lung injury, hyaline membrane formation, necrosis and edema of type I alveolar epithelial or endothelial cells, hyperplasia of type II alveolar epithelial cells and pulmonary interstitial fibrosis can be seen pathologically. Severe ventilation / perfusion imbalance due to alveolar edema and alveolar collapse, especially a significant increase in intrapulmonary shunt, leads to severe hypoxemia. Coagulation and fibrinolysis disorders are also involved in the course of ALI. The early procoagulant mechanism is enhanced, while the fibrinolytic process is inhibited, causing extensive thrombosis and massive deposition of fibrin. Fibrin deposition in the alveolar cavity forms a hyaline membrane covering the alveolar surface, leading to vascular blockage and damage to the microcirculatory structure, which can cause pulmonary hypertension.

[0004] ALI treatment strategies include controlling the primary disease, supportive treatment, and drug therapy. There is currently no specific treatment plan. Mechanical ventilation is the main treatment in clinical practice, but it has no effect on mortality and may cause further lung damage. Existing anti-inflammatory drugs such as glucocorticoids have a significant improvement effect in ALI preclinical experiments, but in clinical treatment, due to individual factors such as the timing and dosage of medication, the therapeutic effect is difficult to confirm, and long-term high-dose use is prone to toxic side effects. Therefore, hormone therapy is not recommended. In recent years, studies have found that natural products and their active ingredients, used alone or in combination, have shown significant therapeutic effects in ALI animal models and clinically. Therefore, the synergistic enhancement and toxicity reduction of active ingredients derived from natural products after combination may be an important direction for the future development of acute lung injury drugs.

[0005] Rosmarinic acid (RosA) is a water-soluble phenolic acid compound, and scutellarin (SCU) is a flavonoid compound. Both are widely distributed in plants such as the Lamiaceae family, and are found in relatively high concentrations in the traditional Chinese medicine Perilla leaves. Both have strong anti-inflammatory, antioxidant, and anti-apoptotic activities in vivo and in vitro.

[0006] Rosmarinic acid has various administration routes and significant pharmacological effects. It has a good alleviating effect on various in vitro and in vivo models of acute lung injury induced by modeling agents. In recent years, literature research has mainly focused on the improvement of the pharmacological activity of rosmarinic acid derivatives and their compositions. Among them, the dose of rosmarinic acid for oral administration is mostly 50-100 mg / kg, and the intraperitoneal injection dose is reduced to 5-20 mg / kg. The concentration of cell experiments is concentrated in 5-60 μM. Rosmarinic acid in this concentration range can not only inhibit the complement system, but also regulate the expression of pro-inflammatory molecules, antioxidant activity and apoptosis levels of inflammatory cells (mast cells, mononuclear macrophages and neutrophils, etc.) or lung parenchymal cells, and effectively improves acute lung injury in mice induced by modeling conditions such as lipopolysaccharides (LPS) / diesel exhaust particles / cobra venom factor / malathion / reperfusion. At the same time, rosmarinic acid and other active ingredients can exert a combined anti-inflammatory and antioxidant effect, which has been verified in the LPS-induced RAW264.7 (mouse mononuclear macrophage leukemia cell) cell model and the LPS-induced mouse acute lung injury model. The glucoside derivatives of rosmarinic acid (20, 50 mg / kg, intraperitoneal injection for 5 days) also have significant anti-inflammatory and antioxidant activities, and can alleviate acute lung injury caused by influenza virus by regulating Th1 / Th2 function.

[0007] The main administration method of baicalin is intraperitoneal injection, with the dosage concentrated in 15-50 mg / kg. It can improve the acute lung injury induced by LPS intravenous injection in rats or the lung injury induced by bilateral hind limb ischemia-reperfusion in rats by regulating mitochondrial dysfunction, oxidative stress, inflammation and cell apoptosis.

[0008] The patent does not mention the two drugs alone or in combination for the treatment of lung injury. Existing pharmacological results show that the intersection of the targets of rosmarinic acid and baicalin for the treatment of acute lung injury is concentrated in the inflammation, apoptosis, and oxidative stress pathways. Therefore, it is speculated that the two may jointly regulate the relevant pathway network, showing a synergistic effect on inflammatory lung diseases. At the same time, as active ingredients derived from natural products, rosmarinic acid and baicalin have clarified their synergistic and toxic-reducing effects, which may provide a basis for further research on the multi-component and multi-target synergistic effects of natural products.

[0009] Therefore, the researchers of the present invention combined rosmarinic acid and baicalin in different proportions to form a combination, and through in vitro experimental screening, obtained the best ratio of rosmarinic acid: baicalin = 4:1 (molar concentration ratio in cell experiments) or 3:1 (mass concentration ratio in animal experiments). In vivo and in vitro pharmacological experiments showed that the composition at this ratio has a synergistic effect on the treatment of acute lung injury. There are currently no reports on the use of rosmarinic acid and baicalin in the treatment and / or prevention of acute lung injury. This patent discloses for the first time a combination of rosmarinic acid and baicalin as shown in formula (I) and (II) for the synergistic treatment and / or prevention of acute lung injury.

[0010] Summary of the invention

[0011] In order to overcome the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a composition of rosmarinic acid and baicalin in an optimal ratio or a composition in different ratios, and the use of the pharmaceutical composition in the preparation of a drug for the prevention, treatment or auxiliary treatment of acute lung injury.

[0012] In order to achieve the technical problem of the present invention, the present invention provides the following technical solutions:

[0013] The present invention screens different ratios of compatibility combinations, preferably rosmarinic acid: baicalin = 4:1 (cell experiment molar concentration ratio) or 3:1 (animal experiment mass concentration ratio) to form a compatibility composition, and studies the application of the compatibility composition in the treatment of acute lung injury in vivo and in vitro.

[0014] The first aspect of the technical solution of the present invention is to provide a composition of rosmarinic acid and scutellariae in combination or compositions in different proportions.

[0015]

[0016] In order to achieve the purpose of the present invention, the present invention relates to different ratios of rosmarinic acid: baicalin, the molar concentration ratio is 1:32 to 32:1, preferably 16:1 to 1:1, the best ratio is 4:1 (cell experiment molar concentration ratio) or 3:1 (animal experiment mass concentration ratio), and the application of the optimal synergistic compatibility ratio in acute lung injury and other immune-related diseases is studied.

[0017] In this study, LPS was used as a modeling agent to construct a primary mouse peritoneal macrophage inflammation model, and LPS was instilled into the trachea to induce a mouse acute lung injury model. The NO inhibition rate of the primary mouse peritoneal macrophage supernatant was used as a screening index, and the HSA synergistic index of rosmarinic acid and baicalin at different ratios was calculated using the synergy software HSA model, and then the optimal ratio was selected for verification. The cell concentrations of the optimal ratio of the drug pair were rosmarinic acid (10μM), baicalin (2.5μM), and the combination (12.5μM), and the mice were intraperitoneally administered with rosmarinic acid (30mg / kg / d), baicalin (10mg / kg / d), and the combination (40mg / kg / d). The results of the study showed that the compatibility composition of the present invention has a potential synergistic inhibitory effect on the release of inflammatory mediators (NO) and inflammatory factors (TNF-ɑ, IL-6) in LPS-induced primary mouse peritoneal macrophages, and the synergistic indexes are Q=1.89, 3.13, and 1.32, respectively. The compatibility composition of the present invention has synergistic indexes of Q=1.50, 1.19, and 2.54 in improving the three indicators of quasi-static lung compliance (Cchord), dynamic lung compliance (Cydn), and inspiratory capacity (IC) of mice with acute lung injury induced by LPS, respectively; the synergistic index in inhibiting the increase of TNF-ɑ in alveolar lavage fluid BALF is Q=1.46; the synergistic index in inhibiting the increase of serum LDH is Q=1.56; compared with the significant weight loss of the clinical medication dexamethasone (DXMS) group, the composition has no toxic side effects and can significantly improve the weight loss after LPS modeling.

[0018] The second aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which contains a pharmaceutically effective dose of rosmarinic acid and baicalin in different proportions and a pharmaceutically acceptable carrier or excipient.

[0019] According to an embodiment of the present invention, the compatible composition of the present invention also includes a pharmaceutically acceptable salt thereof.

[0020] The present invention also relates to a pharmaceutical composition containing the compatible composition of the present invention as an active ingredient and a conventional pharmaceutical excipient or adjuvant. Usually, the pharmaceutical composition of the present invention contains 0.1 to 95% by weight of the compatible composition of the present invention. In a unit dosage form, the compatible composition of the present invention generally contains 0.1 to 100 mg, and a preferred unit dosage form contains 4 to 50 mg.

[0021] The pharmaceutical composition of the compatible composition of the present invention can be prepared according to methods well known in the art. When used for this purpose, if necessary, the compatible composition of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to make suitable application forms or dosage forms that can be used as human medicine or veterinary medicine.

[0022] The invention also relates to a method for preparing a pharmaceutical composition. For this purpose, if necessary, the active ingredient can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to prepare a suitable administration form or dosage form that can be used as a human medicine.

[0023] The pharmaceutical composition of the present invention can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum.

[0024] The administration route of the pharmaceutical composition of the present invention can be injection. Injection includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection. The dosage form can be a liquid dosage form or a solid dosage form. For example, the liquid dosage form can be a true solution, a colloid, a microparticle dosage form, an emulsion dosage form, or a suspension dosage form. Other dosage forms include tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, etc.

[0025] The composition of the present invention can be made into a common preparation, or a sustained-release preparation, a controlled-release preparation, a targeted preparation and various microparticle drug delivery systems.

[0026] In order to prepare a unit dosage form into tablets, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0027] In order to make the dosing unit into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders, such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.

[0028] In order to prepare the administration unit as a suppository, various carriers known in the art can be widely used, such as polyethylene glycol, lecithin, cocoa butter, higher alcohols, enzymes of higher alcohols, gelatin, semi-synthetic glycerol enzymes, and the like.

[0029] In order to prepare the dosing unit into capsules, the active ingredient is mixed with the above-mentioned various carriers, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredient can also be prepared into microcapsules, suspended in an aqueous medium to form a suspension, or can be loaded into a hard capsule or prepared into an injection for use.

[0030] For example, the composition of the present invention is made into an injection preparation, such as a solution, a suspension solution, an emulsion, or a freeze-dried powder injection. This preparation can be aqueous or non-aqueous, and can contain one and / or more pharmacodynamically acceptable carriers, diluents, adhesives, lubricants, preservatives, surfactants, or dispersants. For example, the diluent can be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitol fatty acid enzyme, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH regulators, etc. can also be added. These adjuvants are commonly used in the art.

[0031] Furthermore, if necessary, colorants, preservatives, perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations.

[0032] The dosage of the pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, etc., so the therapeutic dose of the present invention can vary widely. Generally speaking, the dosage of the compatibility composition of the present invention is well known to those skilled in the art. It can be appropriately adjusted according to the actual effective drugs contained in the final preparation of the pharmaceutical composition of the present invention to achieve the requirements of its therapeutically effective amount and complete the purpose of treating inflammatory diseases of the present invention.

[0033] Usually for patients weighing about 75 kg, the daily dose of the compatibility composition of the present invention is 0.001 mg / kg body weight to 200 mg / kg body weight, preferably 1 mg / kg body weight to 100 mg / kg body weight. The above dosage can be administered in a single dosage form or divided into several, for example two, three or four dosage forms, which is limited by the clinical experience of the doctor administering the drug and the dosage regimen.

[0034] The third aspect of the technical solution of the present invention provides the use of the combination composition described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of drugs for treating and / or preventing immune-related diseases. The immune-related diseases include acute lung injury.

[0035] The combination composition or pharmaceutical composition of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs, and is used to treat and / or prevent immune-related diseases.

[0036] Beneficial technical effects

[0037] The advantages of the present invention are: (1) the compatibility composition of the present invention has a potential synergistic effect compared with its constituent active ingredients rosmarinic acid and baicalin; (2) the compatibility composition of the present invention has significant in vivo and in vitro anti-inflammatory activity; (3) the compatibility composition of the present invention has a significant improvement effect on the lung function and inflammatory indicators of mice with LPS-induced acute lung injury; (4) the administration of the compatibility composition did not cause the toxic side effect of weight loss in mice before modeling, and significantly improved the weight loss of mice after modeling, while the clinical drug dexamethasone caused a significant weight loss in mice before modeling; (5) the raw materials of the compatibility composition are easily available, the preparation process is simple, and it is easy to standardize production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 . Effect of the combined composition on the body weight of mice with LPS-induced acute lung injury before and after modeling (n=13) DETAILED DESCRIPTION

[0039] The present invention is further described in the following examples, but the present invention is not limited to these examples, which are purely illustrative and are only used to specifically describe the present invention.

[0040] Example 1: Preparation of a compatibility composition.

[0041] The preparation steps of the compatibility composition are as follows:

[0042] Cell experiment: Prepare the dimethyl sulfoxide stock solutions of rosmarinic acid and baicalin respectively, add a certain amount of stock solution to the culture medium according to the volume of the well plate, so that the final concentration is rosmarinic acid (10 μM) and baicalin (2.5 μM), and the administration method of the combined composition (12.5 μM) is to add the rosmarinic acid and baicalin stock solutions to the culture medium at the same time, so that the final concentration is rosmarinic acid (10 μM) and baicalin (2.5 μM), and the other concentrations are the same.

[0043] Animal experiments: 30 mg of rosmarinic acid was weighed and dissolved in 10 ml of normal saline by ultrasonic wave, and the intraperitoneal injection volume was 0.1 ml / 10 g for mice; 10 mg of scutellariae was weighed and suspended in 10 ml of normal saline by ultrasonic wave, and the intraperitoneal injection volume was 0.1 ml / 10 g for mice; for the combined composition, 30 mg of rosmarinic acid and 10 mg of scutellariae were weighed, both were suspended in 10 ml of normal saline by ultrasonic wave, and the intraperitoneal injection volume was 0.1 ml / 10 g for mice.

[0044] Pharmacological experiments:

[0045] Experimental Example 1: Screening of the optimal ratio of the combination in the LPS-induced C57 mouse primary peritoneal macrophage inflammation model

[0046] Experimental methods:

[0047] C57 mice, male, 20-22g, were intraperitoneally injected with 1.5ml 3% sodium thioglycolate solution. On the fourth day after induction, primary peritoneal macrophages were removed and diluted with 1640 medium containing 10% FBS and 0.1% penicillin / streptomycin mixture. 150,000 cells per well were inoculated in 96-well plates for culture. After 2 hours of adherence, non-adherent cells were washed with PBS. After the cells were pre-protected with drugs for 1 hour, they were stimulated with 1μg / ml LPS for 24 hours, and the supernatant was taken to determine the content of inflammatory mediators (NO), and the NO production inhibition rate was calculated. The synergy index was calculated using the synergy software HSA model. HSA synergy index>10 indicates that there may be potential synergy. The compatibility combinations with synergy index>10 were screened and ranked according to the index. The drug concentrations were selected according to preliminary experiments: RosA (20 / 10 / 5 / 2.5 / 1.25 / 0.625 μM), SCU (20 / 10 / 5 / 2.5 / 1.25 / 0.625 μM), N=6.

[0048] Experimental results:

[0049] The main pathological characteristics of acute lung injury include the activation and recruitment of macrophages and the release of related inflammatory factors. By calculating the NO inhibition rate of single drugs and combinations, the drug ratio with the highest synergistic index was selected for subsequent cell animal verification experiments. The results are shown in Table 1. There are differences in the NO inhibition rate of single drugs at different concentrations and combinations of different ratios. At the selected cell concentration, rosmarinic acid has no significant inhibition on NO production, but it can sensitize the NO inhibition of baicalin in a low concentration range. As an adjuvant, it can achieve the goal of reducing the dose of the main drug and increasing the efficacy of the combination. As shown in Table 2, after calculation by synergy software, 11 combinations with HSA synergy index>10 were screened. It was found that the optimal synergistic doses of RosA and SCU in this experiment were 10 and 2.5 μM, respectively. Because of its highest synergistic index, HSA synergy index = 33.8, this compatibility ratio was used for subsequent experiments.

[0050] Table 1. NO inhibition rate (%) of different proportions of the combination of primary peritoneal macrophage inflammation model

[0051]

[0053] Table 2. Ranking of scores of compatibility combinations with HSA synergy index>10

[0054] Combination Sorting RosA (μM) SCU(μM) HSA score (>10) 1 10 2.5 33.8 2 0.625 1.25 27.67 3 20 1.25 22.1 4 1.25 1.25 20.8 5 2.5 1.25 20.3 6 5 2.5 19.1 7 10 1.25 18.3 8 5 1.25 18.2 9 20 2.5 17.7 10 20 0.625 13.5 11 2.5 2.5 10.6

[0055] Experimental Example 2: Anti-inflammatory effect of the combined composition on LPS-induced C57 mouse primary peritoneal macrophages

[0056] Experimental methods:

[0057] C57 mice, male, 20-22g, were intraperitoneally injected with 1.5ml 3% sodium thioglycolate solution. On the fourth day after induction, primary peritoneal macrophages were removed and diluted with 1640 medium containing 10% FBS, 0.1% penicillin and streptomycin mixture. 150,000 cells per well were inoculated in 96-well plates for culture. After 2 hours of adherence, non-adherent cells were washed with PBS. After the cells were pre-protected by drugs for 1 hour, they were stimulated with 1μg / ml LPS for 24 hours, and the supernatant was taken to determine the content of inflammatory mediators (NO) and inflammatory factors (TNF-ɑ, IL-6). Synergistic index Q = (inhibition rate of the combination) / (inhibition rate of rosmarinic acid group + inhibition rate of scutellaria baicalin group - inhibition rate of rosmarinic acid group * inhibition rate of scutellaria baicalin group), Q>1.15 indicates that there may be synergistic effects.

[0058] Experimental results:

[0059] The main pathological characteristics of acute lung injury include the activation and recruitment of macrophages and the release of related inflammatory factors. NO, TNF-ɑ, and IL-6 are inflammatory mediators and inflammatory factors secreted by macrophages and other cells, which in turn activate and recruit immune cells in the lungs, promote the amplification of the inflammatory cascade, and cause diffuse damage to pulmonary capillary endothelial cells and alveolar epithelial cells, thereby aggravating the symptoms of acute lung injury. The results are shown in Table 6. Compared with the blank group, the concentrations of NO, TNF-ɑ, and IL-6 in the cell culture supernatant of the model group were significantly increased, indicating that the modeling was successful. Compared with the model control group, the combination composition can significantly reduce the concentrations of NO, TNF-ɑ, and IL-6 in the cell culture supernatant. Compared with individual drugs, the combination composition may have synergistic effects on these three indicators, and the synergistic index was calculated to be Q = 1.89, 3.13, and 1.32.

[0060] Table 3. Effects of the combined composition on inflammatory indicators of primary peritoneal macrophages of C57 mice induced by LPS (Mean±Std)

[0061]

[0062]

[0063] Note: Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the model control group, * p<0.05, ** p<0.01, *** p<0.001. n=6 in each group.

[0064] Experimental Example 3: Improvement of the combination composition on LPS-induced acute lung injury in mice

[0065] Experimental methods:

[0066] KM mice, male, 18-22g, were randomly divided into groups according to body weight, and body weight was recorded every day. On days 1-4, except for the blank control group and the model group (administered with normal saline), the animals in each group were intraperitoneally injected with drug solution (0.1ml / 10g); on day 4, the mice were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital 1h after administration. Except for the blank group, which was instilled with an equal volume of PBS, the other groups were slowly instilled with 50μL of 2.4mg / mL LPS solution containing 120μg into each trachea, that is, a dose of 5mg / kg (calculated based on 24g body weight); on day 5, the last administration was given 22h after modeling, and anesthesia was performed 2h after administration, endotracheal intubation was performed, lung function was tested, and samples (alveolar lavage fluid, serum) were taken. Synergistic index Q = (inhibition rate of the combined drug) / (inhibition rate of the rosmarinic acid group + inhibition rate of the baicalin group - inhibition rate of the rosmarinic acid group * inhibition rate of the baicalin group), Q>1.15 indicates that there may be a synergistic effect.

[0067] Experimental results:

[0068] (1) Effect of the combined composition on the improvement of lung function in mice with acute lung injury induced by LPS:

[0069] The manifestations of acute lung injury include alveolar edema and alveolar collapse leading to severe ventilation / perfusion imbalance. Therefore, in the experiment, the degree of lung dysfunction was reflected by measuring quasi-static lung compliance (Cchord), dynamic lung compliance (Cydn), and inspiratory capacity (IC), thereby characterizing the efficacy of the drug. The results are shown in Table 1. Compared with the blank group, the quasi-static lung compliance, dynamic lung compliance, and inspiratory capacity of the model group decreased significantly, indicating that the model was successfully established. Compared with the model control group, the combination composition can significantly improve the quasi-static lung compliance, dynamic lung compliance, and inspiratory capacity. Compared with a single drug, the combination composition may have a synergistic effect on these three indicators, and the synergistic index was calculated to be Q=1.50, 1.19, and 2.54.

[0070] Table 4. Effect of the combined composition on the improvement of lung function in mice with acute lung injury induced by LPS (Mean±Std)

[0071]

[0072] Note: Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the model control group, * p<0.05, ** p<0.01, *** p<0.001. n=5 for each group.

[0073] (2) The effect of the combined composition on reducing inflammatory factors in BALF of mice with LPS-induced acute lung injury:

[0074] The concentration of inflammatory factors in the bronchoalveolar lavage fluid of mice can reflect the overall severity of the inflammatory response to acute lung injury and the effect of the drug. The results are shown in Table 2. Compared with the blank group, the concentration of TNF-ɑ in the BALF of the model group was significantly increased, indicating that the model was successfully established. Compared with the model control group, the combination composition can significantly reduce the concentration of TNF-ɑ in the BALF. Compared with a single drug, the combination composition may have a synergistic effect on this indicator, and the synergistic index was calculated to be Q = 1.46.

[0075] Table 5. Effect of the combined composition on reducing inflammatory factors in BALF of mice with acute lung injury induced by LPS (Mean±Std)

[0076]

[0077] Note: Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the model control group, * p<0.05, ** p<0.01, *** p<0.001. n=8 in each group.

[0078] (3) Effect of the combined composition on reducing serum lactate dehydrogenase in mice with LPS-induced acute lung injury:

[0079] Acute lung injury is often accompanied by an increase in serum lactate dehydrogenase levels, reflecting inflammatory damage to the body. The determination of serum lactate dehydrogenase levels can reflect the degree of lung injury inflammation and measure the efficacy of the drug. The results are shown in Table 3. Compared with the blank group, the concentration of serum lactate dehydrogenase in the model group was significantly increased, indicating that the model was successfully established. Compared with the model control group, the combination composition can significantly reduce the concentration of serum lactate dehydrogenase. Compared with a single drug, the combination composition may have a synergistic effect on this indicator, and the synergistic index is calculated to be Q = 1.56.

[0080] Table 6. Effect of the combined composition on reducing serum lactate dehydrogenase in mice with LPS-induced acute lung injury (Mean±Std)

[0081]

[0082] Note: Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the model control group, * p<0.05, ** p<0.01,*** p<0.001. n=10 for each group.

[0083] (4) Effect of the combined composition on the improvement of body weight before and after LPS-induced acute lung injury modeling in mice:

[0084] The weight changes of mice from the first day to the fourth day of the experiment (before modeling) can reflect the safety of the drug and thus measure the development and application prospects of the drug. Figure 1 As shown in the figure, the mice in the blank group, model group, and drug-treated group were in good condition during the experiment, and there was no significant difference in body weight among the groups. However, the dexamethasone group showed a toxic side effect of significant weight loss compared with the blank group starting from the second day, and the difference in mouse weight continued and reached the maximum before modeling on the fourth day.

[0085] After modeling on the fifth day, the results are shown in Table 4. Compared with the blank group, the model group had significantly reduced body weight, depressed mental state, and chills. Compared with the model control group, the combination can significantly increase body weight and improve the vitality of mice.

[0086] Table 7. Effect of the combined composition on the improvement of body weight in mice with acute lung injury induced by LPS (Mean±Std)

[0087]

[0088] Note: Compared with the blank control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the model control group, * p<0.05, ** p<0.01, *** p<0.001. n=13 in each group.

[0089] The above results show that the optimal ratio of the combination obtained by screening in the present invention can synergistically reduce the concentrations of NO, TNF-ɑ, and IL-6 in the culture supernatant of primary peritoneal macrophages of mice in vitro, and can synergistically improve the quasi-static lung compliance (Cchord), dynamic lung compliance (Cydn), and inspiratory capacity of the acute lung injury model of mice induced by LPS tracheal instillation in vivo, inhibit the increase of TNF-ɑ in alveolar lavage fluid and the increase of LDH in serum, indicating that the combination has a potential synergistic improvement effect on the airway inflammatory response of acute lung injury. In addition, the combination has no toxic side effect of weight loss, and can significantly improve the weight loss after LPS modeling, while DXMS causes a significant weight loss in animals in a time-dependent manner before modeling. It can be seen that the combination has a broad application and development prospect in the treatment of acute lung injury.

Claims

1. A compatibility composition, characterized in that: The pharmaceutical composition is composed of rosmarinic acid and baicalin as shown in formula (I) and (II); 2. The compatibility composition according to claim 1, characterized in that: It is composed of rosmarinic acid and baicalin in different proportions, with the molar concentration ratio being 16:1 to 1:

1.

3. The compatibility composition according to claim 2, characterized in that: It is composed of rosmarinic acid and baicalin in different proportions, with the molar concentration ratio being 4:1 to 1:

1.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition is composed of an effective dose of the combination composition according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition also contains other effective ingredients, which include one or more of antiviral drugs, antitumor drugs, anti-tuberculosis drugs, immunosuppressants, liver-protecting drugs and nutrients.

6. A pharmaceutical composition according to any one of claims 4-5, characterized in that The pharmaceutical composition is selected from tablets, capsules, pills and injections.

7. A pharmaceutical composition according to any one of claims 4-5, characterized in that The pharmaceutical composition is selected from sustained-release preparations, controlled-release preparations, and various microparticle delivery systems.

8. Use of the compatible composition according to any one of claims 1 to 3 or the pharmaceutical composition according to any one of claims 4 to 7 in the preparation of drugs for treating and / or preventing immune-related diseases.

9. The use according to claim 8, characterized in that The immune-related diseases include acute lung injury.

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