A nauclea diderrichii and alpinia oxyphylla composition for regulating macrophage polarization and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a Nauclea diderrici Kotschy and Alpiniae oxyphyllae Fructus composition for regulating macrophage polarization and application thereof. The Nauclea diderrici Kotschy and Alpiniae oxyphyllae Fructus composition is obtained by mixing Nauclea diderrici Kotschy and Alpiniae oxyphyllae Fructus, adding water, heating extraction, reducing pressure concentration and drying. The composition obtained by the application can significantly reduce pro-inflammatory factors and improve anti-inflammatory factors through bidirectional regulation of alveolar macrophage polarization, and promote lung tissue repair, thereby providing a new scheme for clinical treatment of acute lung injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a Radix Euonymi and Fructus Alpiniae oxyphyllae composition for regulating macrophage polarization and application thereof. BACKGROUND

[0002] Acute lung injury (ALI) is a common acute and critical illness in clinic, and its core pathological features are increased lung permeability, non-cardiogenic pulmonary edema, lung tissue inflammatory infiltration and intractable hypoxemia caused by lung microvascular endothelial cell and alveolar epithelial cell damage. Severe cases can progress to acute respiratory distress syndrome (ARDS), and even lead to multiple organ failure, with a mortality rate of 30% to 50%.

[0003] At present, the clinical treatment of ALI mainly includes mechanical ventilation, fluid resuscitation, anti-infection and other supportive therapies. There is a lack of specific therapeutic drugs targeting pathological mechanisms, and it is difficult to achieve precise regulation of inflammatory response and synergistic effect of tissue repair. Therefore, it has high clinical value to develop safe and efficient new therapeutic drugs.

[0004] Alveolar macrophages (AMs) are the core regulatory cells of the lung inflammatory microenvironment, and can be differentiated into classical activated type (M1 type) and alternative activated type (M2 type) after stimulation. M1 / M2 polarization imbalance is a key mechanism of ALI. M1 macrophages exacerbate lung inflammatory infiltration and tissue damage by secreting pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6); M2 macrophages promote inflammation resolution, tissue repair and phagocytosis of apoptotic cells by secreting anti-inflammatory factors such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). The overactivation of M1 macrophages or the lack of M2 macrophages is an important factor leading to uncontrolled lung inflammation. Therefore, regulating macrophage polarization to M2 type has become a key target for the treatment of ALI.

[0005] Radix Euonymi, also known as Wutan or Shanxiongdan, is the dried stem and branch of the plant Euonymus alatus of Rubiaceae. As a traditional Chinese medicinal material, it tastes cold and bitter, and belongs to the lung, large intestine, gallbladder and bladder channels. It has the effects of clearing heat and resolving toxicity, and relieving swelling and pain. It is commonly used in the treatment of upper respiratory tract infection, tonsillitis, pneumonia and other infectious diseases. The core active components of Radix Euonymi are alkaloids, terpenes, phenolic acids and flavonoids. Its anti-inflammatory and lung protection effects have been widely verified. However, in the prior art, Radix Euonymi is mostly used as a single preparation. Its efficiency in regulating macrophage polarization in a complex inflammatory microenvironment still needs to be improved, and there is a lack of related reports on its synergistic effect with other traditional Chinese medicines.

[0006] Yizhi, also known as Yizhi Ren, is a perennial herb of the genus Alpinia, one of the "four major southern medicines" in China, with a pungent and warm nature, and is returned to the spleen and kidney channels. The traditional effect is to warm the spleen to stop diarrhea, saliva, warm the kidney, and solidify the essence and shrink the urine, and has high safety. Modern pharmacological studies have shown that the active ingredients of Yizhi mainly include volatile oil (eucalyptol, etc.), flavonoids, diaryl heptanes (yizhi ketone A, yizhi alcohol) and polysaccharides, and have significant immune regulation, anti-inflammatory and antioxidant effects, but the current application is mainly concentrated in the fields of digestive system diseases and cognitive function improvement, and its application research in the treatment of ALI is less. SUMMARY

[0007] In view of the technical defects in the background art, the present application provides a combination of Radix Meliae and Yizhi for regulating macrophage polarization and its application. The combination of Radix Meliae and Yizhi can precisely regulate the polarization direction of alveolar macrophages, and effectively relieve lung tissue inflammation and pathological damage. Compared with the existing single drug or conventional treatment method, the combination of Radix Meliae and Yizhi has better treatment effect on ALI, and provides a more efficient treatment scheme.

[0008] The technical scheme adopted by the present application is as follows:

[0009] A combination of Radix Meliae and Yizhi for regulating macrophage polarization, which is composed of Radix Meliae and Yizhi, and the mass ratio of Radix Meliae to Yizhi is (2-1):(1-2).

[0010] The present application also provides a preparation method of the combination of Radix Meliae and Yizhi. Radix Meliae and Yizhi medicinal materials are mixed, crushed into coarse powder, sieved, heated and extracted with 10-15 times the amount of water, concentrated under reduced pressure, dried, and the combination of Radix Meliae and Yizhi is obtained.

[0011] The present application also provides the application of the combination of Radix Meliae and Yizhi in preparing a drug for treating acute lung injury.

[0012] In an alternative embodiment, the drug comprises the combination of Radix Meliae and Yizhi and a pharmaceutically acceptable excipient, and is prepared into a pharmaceutically acceptable dosage form.

[0013] In an alternative embodiment, the pharmaceutically acceptable excipient comprises a filler, a diluent, a binder, a disintegrant, an emulsifying agent and a drug carrier with no toxic side effects.

[0014] In an alternative embodiment, the dosage form comprises tablets, drops, injection preparations and capsule preparations.

[0015] Compared with the prior art, the present application has the following advantages:

[0016] 1. The present invention can effectively solve the problem of insufficient specificity of existing drugs for ALI treatment. By combining Nauclea officinalis and Alpinia oxyphylla, the polarization balance of macrophages can be precisely regulated. It not only inhibits the activation of pro-inflammatory macrophages to relieve inflammation, but also promotes the differentiation of anti-inflammatory and reparative macrophages to promote lung tissue repair, synchronously achieving inflammation regulation and tissue repair.

[0017] 2. The present invention breaks through the limitation of the single application of Nauclea officinalis. Its combination with Alpinia oxyphylla can strengthen the regulatory efficacy on macrophage polarization, neutralize the cold nature of Nauclea officinalis, reduce its potential damage to the body, and broaden the applicable population. The raw materials are all safe and controllable natural medicinal ingredients with good druggability, providing an excellent solution for ALI treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a graph of the levels of inflammatory factors in the BALF of mice after treatment with different drugs;

[0019] Figure 2 It is a flow cytometry graph of the percentages of M1 and M2 macrophages in the BALF of mice after treatment with different drugs;

[0020] Figure 3 It is the percentage change in the body weight of mice on the eighth day of the experiment after treatment with different drugs;

[0021] Figure 4 It is a pathological section graph of the lung tissue of mice stained with H&E after treatment with different drugs;

[0022] Figure 5 It is a statistical graph of the wet / dry ratio of the lung tissue of mice after treatment with different drugs. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] Next, the technical solutions in the embodiments of the present application will be further clearly and completely described in conjunction with the drawings in the embodiments of the present application. It should be noted that the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present application.

[0024] In order to make the object, technical solution and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings in the specification: In order to better understand the above objects, features and advantages of the present invention, the advantages of the present invention will be further illustrated by comparing with specific embodiments in conjunction with the drawings and specific implementation manners.

[0025] I. Preparation of the Nauclea officinalis - Alpinia oxyphylla composition for regulating macrophage polarization Example 1

[0026] Take the herbs *Gentiana scabra* and *Alpinia oxyphylla* separately, mix them in a 1:1 ratio, pulverize them into coarse powder, pass them through a 20-mesh sieve, add 10 times the amount of water, heat and extract, concentrate under reduced pressure, and dry to obtain the *Gentiana scabra* and *Alpinia oxyphylla* composition. Example 2

[0027] Take the herbs *Gentiana scabra* and *Alpinia oxyphylla* separately, mix them in a 2:1 ratio, grind them into coarse powder, pass them through a 20-mesh sieve, add 15 times the amount of water, heat to extract, concentrate under reduced pressure, and dry to obtain the *Gentiana scabra* and *Alpinia oxyphylla* composition. Example 3

[0028] Take the herbs *Gentiana scabra* and *Alpinia oxyphylla* separately, mix them in a 1:2 ratio, grind them into coarse powder, pass them through a 20-mesh sieve, add 13 times the amount of water, heat to extract, concentrate under reduced pressure, and dry to obtain the *Gentiana scabra* and *Alpinia oxyphylla* composition. Comparative Example 1

[0029] Take the herbs *Gentiana scabra* and *Alpinia oxyphylla* separately, mix them in a 1:3 ratio, pulverize them into coarse powder, pass them through a 20-mesh sieve, add 10 times the amount of water, heat and extract, concentrate under reduced pressure, and dry to obtain the *Gentiana scabra* and *Alpinia oxyphylla* composition. Comparative Example 2

[0030] Take the herbs *Gentiana scabra* and *Alpinia oxyphylla* separately, mix them in a 3:1 ratio, grind them into coarse powder, pass them through a 20-mesh sieve, add 10 times the amount of water, heat to extract, concentrate under reduced pressure, and dry to obtain the *Gentiana scabra* and *Alpinia oxyphylla* composition.

[0031] II. Evaluation of the efficacy of different drugs in acute lung injury

[0032] 169 male C57BL / 6 mice of uniform weight were randomly divided into 13 groups to test the efficacy of the Danmu Yizhi composition in treating acute lung injury. The experimental groups are as follows:

[0033] Control group: No treatment was given;

[0034] Model group: Acute lung injury was induced in mice by injecting them with lipopolysaccharide (LPS) solution;

[0035] Dexamethasone group: Take an appropriate amount of dexamethasone (DXM) injection and dilute it with physiological saline to the required concentration for the experiment; after LPS-induced modeling, administer DXM (2mg / kg) as a positive control.

[0036] Gallnut extract group: Gallnut medicinal material was pulverized into coarse powder, passed through a 20-mesh sieve, and extracted with 10 times the amount of water by heating. The extract was concentrated under reduced pressure and dried to obtain gallnut extract. After LPS-induced modeling, gallnut extract (500 mg / kg) was administered for treatment.

[0037] Alpinia oxyphylla extract group: Alpinia oxyphylla was pulverized into coarse powder, passed through a 20-mesh sieve, and extracted with 10 times the amount of water by heating. The extract was then concentrated under reduced pressure and dried to obtain Alpinia oxyphylla extract. After LPS induction, Alpinia oxyphylla extract (500 mg / kg) was administered for treatment.

[0038] The *Gastrodia elata* group: an appropriate amount of the *Gastrodia elata* composition obtained in Example 1 was taken and prepared with physiological saline to the required concentration for the experiment; after LPS-induced modeling, the *Gastrodia elata* composition (500 mg / kg) was administered for treatment.

[0039] The 2:1 group of *Alpinia buergeriana*: Take an appropriate amount of the *Alpinia buergeriana* composition obtained in Example 2 and prepare it with physiological saline to the required concentration for the experiment; after LPS-induced modeling, administer the *Alpinia buergeriana* composition (500 mg / kg) for treatment;

[0040] The 1:2 group of *Alpinia buergeriana*: Take an appropriate amount of the *Alpinia buergeriana* composition obtained in Example 3 and prepare it with physiological saline to the required concentration for the experiment; after LPS-induced modeling, administer the *Alpinia buergeriana* composition (500 mg / kg) for treatment;

[0041] The 1:3 group of *Gynostemma pentaphyllum*: Take an appropriate amount of the *Gynostemma pentaphyllum* composition obtained in Comparative Example 1 and prepare it with physiological saline to the required concentration for the experiment; after LPS-induced modeling, administer the *Gynostemma pentaphyllum* composition (500 mg / kg) for treatment.

[0042] The 3:1 group of *Gynostemma pentaphyllum*: Take an appropriate amount of the *Gynostemma pentaphyllum* composition obtained in Comparative Example 2 and prepare it with physiological saline to the required concentration for the experiment; after LPS-induced modeling, administer the *Gynostemma pentaphyllum* composition (500 mg / kg) for treatment;

[0043] The *Scutellaria baicalensis* and *Gnaphalium affine* group: *Gnaphalium affine* and *Scutellaria baicalensis* were taken separately, mixed in a 1:1 ratio, pulverized into coarse powder, passed through a 20-mesh sieve, extracted with 10 times the amount of water by heating, concentrated under reduced pressure, and dried to obtain *Gnaphalium affine* and *Scutellaria baicalensis* extract; after LPS induction, *Gnaphalium affine* and *Scutellaria baicalensis* extract (500 mg / kg) was administered for treatment.

[0044] The *Gentiana scabra* and *Gardenia jasminoides* group: *Gentiana scabra* and *Gardenia jasminoides* were taken separately, mixed in a 1:1 ratio, pulverized into coarse powder, passed through a 20-mesh sieve, extracted with 10 times the amount of water by heating, concentrated under reduced pressure, and dried to obtain *Gentiana scabra* and *Gardenia jasminoides* extract; after LPS induction, *Gentiana scabra* and *Gardenia jasminoides* extract (500 mg / kg) was administered for treatment.

[0045] The *Callicarpa nudiflora* group: *Callicarpa nudiflora* and *Callicarpa nudiflora* were mixed at a ratio of 1:2, pulverized into coarse powder, passed through a 20-mesh sieve, extracted with 10 times the amount of water by heating, concentrated under reduced pressure, and dried to obtain *Callicarpa nudiflora* extract; after LPS induction, *Callicarpa nudiflora* extract (500 mg / kg) was administered for treatment.

[0046] The experiment is as follows:

[0047] 1. Determination of the concentrations of IL-1β, TNF-α, IL-6, and IL-10 in mouse BALF

[0048] Twenty-four hours after modeling (12 hours of fasting), mice in the control group, model group, and experimental groups treated with different drugs were anesthetized, and bronchoalveolar lavage fluid (BALF) was collected by endotracheal intubation. The BALF was centrifuged at 500 rpm for 10 minutes at 4°C, and the supernatant was collected and transferred to sterile tubes.

[0049] The collected BALF supernatant was centrifuged again, and the concentrations of IL-1β, TNF-α, IL-6 and IL-10 were determined according to the specific experimental steps in the ELISA kit instructions.

[0050] The effects of Alpinia oxyphylla, Alpinia scabra alone, the Alpinia oxyphylla and Alpinia scabra compositions obtained in Examples 1-3 and Comparative Examples 1-2, and Alpinia oxyphylla in combination with other drugs on the expression levels of inflammatory factors in ALI model mice are shown in Table 1:

[0051] Table 1. Expression levels of inflammatory factors in mice after treatment with different drugs (pg / mL)

[0052]

[0053] As shown in Table 1, the *Gnaphalium affine* and *Alpinia oxyphylla* compositions obtained in Examples 1-3 showed significantly better effects than Alpinia oxyphylla monotherapy, *Gnaphalium affine* monotherapy, and the comparative group in inhibiting the expression of pro-inflammatory factors (IL-1β, TNF-α, IL-6) and increasing the expression of anti-inflammatory factors (IL-10) after treatment. This indicates that *Gnaphalium affine* and *Alpinia oxyphylla* have the best synergistic effect at a ratio of (2-1):(1-2). After treatment with the *Gnaphalium affine* and *Alpinia oxyphylla* compositions obtained in Comparative Examples 1-2, the reduction of pro-inflammatory factors and the increase of anti-inflammatory factors were improved compared with monotherapy, but the effect was not significant. Therefore, the optimal ratio for achieving the synergistic effect of *Gnaphalium affine* and *Alpinia oxyphylla* is to combine them at a ratio of (2-1):(1-2).

[0054] Compared with the model group, the reduction rates of pro-inflammatory factors IL-1β, TNF-α, and IL-6 and the increase rate of anti-inflammatory factor IL-10 in mice treated with dexamethasone, *Gynostemma pentaphyllum* alone, *Alpinia oxyphylla* alone, and *Gynostemma pentaphyllum* in combination with different drugs are calculated as follows:

[0055]

[0056]

[0057] The calculation results are shown in Table 2:

[0058] Table 2. Regulation rates of IL-1β, TNF-α, IL-6, and IL-10 in mouse BALF

[0059]

[0060] As shown in Table 2, the single drug *Gnaphalium affine* showed better regulatory effects on IL-1β and IL-10 than the positive control dexamethasone, but its inhibitory effects on TNF-α and IL-6 were relatively limited. The single drug *Alpinia oxyphylla* showed poor overall anti-inflammatory effects and had no significant effect on reducing IL-6. Among these, the combination of *Gnaphalium affine* and *Alpinia oxyphylla* exhibited a strong synergistic anti-inflammatory effect; its inhibitory effects on IL-1β, TNF-α, and IL-6, as well as its upregulation effect on IL-10, were significantly better than those of *Gnaphalium affine* and *Alpinia oxyphylla* alone. p <0.05); while the inhibitory effects of *Gentiana scabra* combined with *Scutellaria baicalensis*, *Gardenia jasminoides*, and *Callicarpa nudiflora* on IL-1β, TNF-α, and IL-6, and the upregulation effect on IL-10, were not significantly different from those of *Gentiana scabra* alone compared with those of *Gentiana scabra* alone. p (>0.05), showing additive or weak synergistic effects. The results indicate that the *Alpinia buergeriana* and *Alpinia oxyphylla* composition can exert a significant synergistic anti-inflammatory effect by enhancing the inhibition of pro-inflammatory factors and the upregulation of anti-inflammatory factors.

[0061] Figure 1 Figures a, b, c, and d show the levels of inflammatory factors in the BALF of mice in the control group, model group, dexamethasone group, *Evodia rutaecarpa* extract group, *Alpinia oxyphylla* extract group, and *Evodia rutaecarpa* and *Alpinia oxyphylla* groups, respectively. Figures a, b, c, and d show the levels of inflammatory factors IL-1β, TNF-α, IL-6, and IL-10, respectively (compared to the control group). p <0.0001; compared with the model group, ns>0.05, △△△ p <0.001, △△△△ p <0.0001, n=6). For example... Figure 1 As shown in the results compared to the control group, the ALI model group promoted the expression levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6, thereby significantly increasing their levels and inhibiting the expression level of the anti-inflammatory factor IL-10, showing a significant difference from the control group, indicating that the model construction was effective. Compared with the model group, the Alpinia oxyphylla extract reduced the levels of IL-1β and TNF-α in BALF and increased the level of IL-10, but the effect was poor, and it had no significant effect on reducing the level of IL-6. p >0.05); Dexamethasone, *Gnaphalium affine* extract, and the combination of *Gnaphalium affine* and *Alpinia oxyphylla* all reduced the levels of IL-1β, TNF-α, and IL-6 in BALF, while significantly increasing the level of IL-10, with the combination of *Gnaphalium affine* and *Alpinia oxyphylla* showing better effects. The results indicate that the combination of *Gnaphalium affine* and *Alpinia oxyphylla* can alleviate LPS-induced ALI in mice by regulating the production and secretion of pulmonary inflammatory factors, and its regulation of inflammatory factors is superior to that of *Alpinia oxyphylla* extract alone and *Gnaphalium affine* extract alone, demonstrating a synergistic effect when used in combination with *Alpinia oxyphylla*.

[0062] 2. Detection of M1 and M2 alveolar macrophage levels in mouse BALF

[0063] Twenty-four hours after modeling (12 hours of fasting), mice in the control group, model group, dexamethasone group, Alpinia oxyphylla extract group, Gynostemma pentaphyllum extract group, and Gynostemma pentaphyllum Alpinia oxyphylla group were anesthetized, and basilar alveolar lavage fluid (BALF) was collected via endotracheal intubation. The cells were centrifuged at 1500 rpm for 10 minutes at 4°C. The cell pellet was resuspended in 2 mL of DMEM complete medium (20% fetal bovine serum, 1% penicillin-dextrose antibody), and the suspension was evenly dispensed into sterile six-well plates and incubated in a cell culture incubator for adhesion. After 2 hours, the culture medium and any floating, non-adhered red blood cells were removed, and the adhered alveolar macrophages were collected. 100 µL of cell staining buffer was added, and the cells were resuspended. 0.7 μg of CD16 / 32 monoclonal antibody was added to each tube to block the Fc receptor on the surface of the alveolar macrophages, and the cells were incubated at room temperature for 10 minutes.

[0064] Divide the EP tubes into single-stain tubes, FMO control tubes, blank tubes, and sample tubes, and add the corresponding solutions. Add 5µL of flow cytometry antibody F4 / 80 (FITC), CD11c (PE-Cy7), and CD86 (PE) to the corresponding tubes respectively, and incubate at 4℃ in the dark for 30 min.

[0065] Prepare the fixation and permeabilization buffer in advance according to the instructions. First, wash the cells with cell staining buffer, centrifuge and collect the precipitate. Resuspend the precipitate in 200µL of fresh cell staining buffer, add 200µL of fixation buffer, mix well by pipetting, and incubate at room temperature in the dark for 30-60 minutes. After cell membrane fixation, add 1mL of 1× permeabilization agent, centrifuge and collect the cell pellet.

[0066] Add 100 µL of fresh 1× cell permeabilizing agent to the cell pellet, mix well by pipetting, add 5 µL of flow cytometry antibody CD206 (APC) to the corresponding single staining tube and sample tube, mix well by pipetting, incubate in an ice box for 30 min, then add 1.8 mL of 1× cell permeabilizing agent, mix well by pipetting, centrifuge, and retain the cell pellet; resuspend the cells in 200 µL of PBS, adjust the instrument parameters, and perform analysis.

[0067] Figure 2 The flow cytometry plot shows the percentage of M1 and M2 alveolar macrophages in mouse BALF, where CD86 is a marker of M1 pro-inflammatory macrophages and CD206 is a marker of M2 anti-inflammatory macrophages. Figure 2As shown, compared with the control group, the expression levels of CD86 and CD206 in the BALF of mice in the ALI model group were increased, and the number of M1 macrophages significantly increased to 8.62%, indicating that the polarization of M1 macrophages was significantly aggravated under ALI. Compared with the model group, dexamethasone, Alpinia oxyphylla extract, Evodia rutaecarpa extract, and the combination of Alpinia oxyphylla and Evodia rutaecarpa significantly reduced CD86 expression and increased CD206 expression. Among them, the number of M2 macrophages increased to 42.78% in the dexamethasone group, 52.25% and 59.81% respectively in Alpinia oxyphylla and Evodia rutaecarpa extract alone, and 73.38% in the combination of Alpinia oxyphylla and Evodia rutaecarpa, indicating that the Alpinia oxyphylla group can enhance the polarization of M2 macrophages. The results show that the combination of Alpinia oxyphylla and Evodia rutaecarpa can reduce the number of M1 macrophages, increase the number of M2 macrophages, and has a synergistic effect on the regulation of alveolar macrophage polarization.

[0068] 3. Measurement of mouse body weight change

[0069] Mice in the control group, model group, dexamethasone group, Alpinia oxyphylla extract group, Cinnamomum cassia extract group, and Cinnamomum cassia Alpinia oxyphylla group had their basal body weight recorded before the experiment. During the experiment, the weight of mice in each group was measured and recorded at fixed times daily, and the percentage change in body weight was calculated. Monitoring continued until day eight, and the percentage decrease in body weight was statistically analyzed on day eight. The calculation formula is as follows:

[0070]

[0071] Figure 3 The graph shows the changes in mouse body weight. Graph a shows the percentage change in mouse body weight from day 0 to day 8, and graph b shows the percentage decrease in mouse body weight on day 8 (compared to the control group, *). p <0.05; compared with the model group, △ p <0.05, △△△ p <0.001, △△△△ p <0.0001, n=13); according to Figure 3 The changes in body weight of mice during the experiment showed that, compared with the control group, the percentage change in body weight in the model group was significantly lower in the later stage of the experiment (D7-D8), and the percentage decrease in body weight on day 8 was significantly higher than that in the control group (*). p <0.05 indicates that ALI causes rapid weight loss in mice; while the dexamethasone group, Alpinia oxyphylla extract group, Evodia rutaecarpa extract group, and Evodia rutaecarpa extract group showed smaller weight loss, and the Evodia rutaecarpa extract group was significantly more effective than other drugs (△△△△). p <0.0001).

[0072] 4. Pathological examination of mouse lung tissue sections

[0073] Twenty-four hours after modeling (12 hours of fasting), mice in the control group, model group, dexamethasone group, Alpinia oxyphylla extract group, Evodia rutaecarpa extract group, and Evodia rutaecarpa extract group were anesthetized and sacrificed. The thoracic cavity was opened to expose the heart and lungs, and the intact lung tissue was quickly separated. The left lung tissue of each group of mice was taken and placed in a 10 mL sterile centrifuge tube, which was quickly filled with tissue fixative and left at room temperature overnight. After the lung tissue was fixed, paraffin-embedded sections with a thickness of 4 μm were prepared.

[0074] Paraffin sections were thoroughly dewaxed, stained with hematoxylin and eosin (H&E), and pathological section images were observed and collected for H&E staining pathological analysis.

[0075] Figure 4 H&E-stained pathological sections of lung tissue from mice in the control group, model group, dexamethasone group, Alpinia oxyphylla extract group, Evodia rutaecarpa extract group, and Evodia rutaecarpa and Alpinia oxyphylla groups, such as... Figure 4 As shown, the lung tissue structure of the control group mice was intact, with less inflammatory cell infiltration and no inflammatory damage. Pathological sections of the LPS-induced ALI model mice showed significant alveolar and vascular inflammation, manifested as alveolar wall thickening, extensive neutrophil infiltration, tissue fluid exudation, and alveolar hemorrhage. After treatment with dexamethasone and *Alpinia oxyphylla* extract, inflammatory cell infiltration was reduced compared to the model group, improving lung tissue damage. After treatment with *Alpinia oxyphylla* extract, inflammatory cell infiltration remained significant, and the improvement in lung tissue damage was relatively limited. After treatment with *Alpinia oxyphylla* extract, inflammatory cell infiltration was significantly reduced, and the alveolar cavity structure was close to that of the control group, indicating that *Alpinia oxyphylla* had the most prominent effect in improving lung tissue damage.

[0076] 5. Measurement of wet / dry ratio (W / D) of mouse lung tissue

[0077] Twenty-four hours after modeling (12 hours of fasting), mice in the control group, model group, dexamethasone group, Alpinia oxyphylla extract group, Evodia rutaecarpa extract group, and Evodia rutaecarpa extract group were anesthetized and sacrificed. The thoracic cavity was opened to expose the heart and lungs, and the intact lung tissue was quickly separated. Fresh right upper lobe lung tissue from each group of mice was taken onto clean weighing paper, the surface moisture was absorbed with absorbent paper and the wet weight (W) was recorded, and the tissue was placed in an 80℃ oven to dry for 48 hours until constant weight was achieved. The dry weight (D) was recorded, and the W / D ratio was calculated to assess the edema of the lung tissue.

[0078] Figure 5 A wet / dry ratio diagram of mouse lung tissue (compared to the control group, *). p <0.05; compared with the model group, △△△ p <0.001, △△△△ p <0.0001, n=13), such as Figure 5As shown, the lung tissue W / D ratio of the model group mice was significantly higher than that of the control group, indicating that ALI causes pulmonary edema. After treatment with dexamethasone, Alpinia oxyphylla extract, Evodia rutaecarpa extract, and a combination of Evodia oxyphylla and Alpinia oxyphylla, the lung tissue W / D ratio of the mice was significantly lower than that of the model group, indicating that these drugs can effectively reduce pulmonary edema. The improvement effect of dexamethasone was close to that of the control group, while the combination of Evodia oxyphylla and Alpinia oxyphylla showed better improvement than Alpinia oxyphylla and Evodia rutaecarpa extract alone. This suggests that the combination of Evodia oxyphylla and Alpinia oxyphylla can effectively alleviate the symptoms of pulmonary edema.

[0079] The results showed that the combination of *Gnaphalium affine* and *Alpinia oxyphylla* can treat acute lung injury by regulating the production and secretion of inflammatory factors and modulating macrophage polarization. It can effectively alleviate typical symptoms of acute lung injury such as weight loss, pulmonary edema, alveolar wall thickening, massive neutrophil infiltration, tissue fluid exudation and alveolar hemorrhage. Moreover, the effect is more significant than that of *Alpinia oxyphylla* and *Gnaphalium affine* used alone, providing a new and effective solution for the intervention and treatment of acute lung injury.

[0080] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0081] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A composition of *Alpinia oxyphylla* for treating acute lung injury, characterized in that, The raw materials consist of *Gnaphalium affine* and *Alpinia oxyphylla*, with a mass ratio of (2-1):(1-2).

2. The method for preparing the *Gynostemma pentaphyllum* composition according to claim 1, characterized in that, Mix the herbs *Gentiana scabra* and *Alpinia oxyphylla*, grind them into coarse powder, sieve them, add 10-15 times the amount of water, heat to extract, concentrate under reduced pressure, and dry to obtain the *Gentiana scabra* and *Alpinia oxyphylla* composition.

3. The use of the *Alpinia buergeriana* composition as described in any one of claims 1-2 in the preparation of a medicament for treating acute lung injury.

4. The application according to claim 3, characterized in that, The drug is prepared into a pharmaceutically acceptable dosage form from a composition of Alpinia buergeriana and pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients include fillers, diluents, binders, disintegrants, and emulsifiers.

6. The application according to claim 4, characterized in that, The dosage forms include tablets, drops, injections, and capsules.

Images