An effective component group of fructus aurantii for treating acute lung injury and a preparation method and application thereof

Abstract

The application discloses an effective component group of Quji-Shell for treating acute lung injury, which comprises naringin, naringin, naringenin 7-O-glucoside, hesperidin and neohesperidin; a preparation method comprises the following steps: (1) crushing Quji-Shell, and soaking and extracting by using ethanol; (2) adding water, and extracting by using ethyl acetate, and performing concentration under reduced pressure; (3) separating and eluting, and combining elution fractions according to chemical component types; and (4) combining chemical component groups Fr.1-4, and obtaining the effective component group of Quji-Shell. The effective component group of Quji-Shell has the advantages of clear chemical composition, high content of anti-inflammatory active components, oral preparation or inhalation preparation which can be developed in clinic, and clear pharmacology and toxicology, and has better medicine property.

Description

Technical Field

[0001] This invention relates to the field of drug extraction technology, and more specifically to a group of effective components of Citrus aurantium for treating acute lung injury, its preparation method and application. Background Technology

[0002] Acute lung injury (ALI) is a common acute and critical illness in clinical practice. It is an acute, progressive hypoxic respiratory failure caused by various intrapulmonary and extrapulmonary pathogenic factors other than cardiac causes. Clinical manifestations include refractory hypoxemia, severe edema in the alveoli and lung parenchyma, and progressively worsening respiratory distress. If the condition is not controlled in time, it is likely to further develop into acute respiratory distress syndrome (ARDS), and then into multiple organ failure. It is a common critical illness in clinical practice with a mortality rate as high as 30%-50%.

[0003] The causes of acute lung injury (ALI) are diverse and can be classified into two categories: intrapulmonary factors and extrapulmonary factors. Intrapulmonary factors include: (1) chemical factors, such as inhalation of smoke, nanoparticles, and oxygen poisoning; (2) physical factors, such as pulmonary contusion and radiation damage; and (3) biological factors, such as severe pneumonia. Extrapulmonary factors include severe shock, infectious poisoning, severe non-thoracic trauma, severe burns, massive blood transfusions, acute pancreatitis, and drug poisoning. In the early stages of ALI, alveolar damage, massive inflammatory cell infiltration, especially neutrophil aggregation, and pulmonary interstitial edema are observed. The pathogenesis of ALI is not yet clear. Most scholars believe that ALI is essentially an excessive and uncontrolled cascade of inflammatory reactions in the lungs under the influence of a large number of inflammatory cells and inflammatory factors.

[0004] Viral and bacterial infections are also major causes of ALI. The cytokine storm following COVID-19 infection is a leading cause of death in severely ill patients with ALI / ARDS. Clinically, clearing inflammation is the primary treatment for infectious ALI / ARDS, with medications mainly consisting of corticosteroids. While corticosteroids can suppress lung inflammation, they also inhibit the immune response and the body's ability to clear the virus. Therefore, developing drugs to treat inflammation in ALI / ARDS is urgently needed.

[0005] The applicant's preliminary research showed that the aqueous extract of Citrus aurantium can effectively treat acute pneumonia (Chinese Patent 201710813853.0), but the components of the aqueous extract of Citrus aurantium are relatively complex, which is not conducive to the development of new drugs.

[0006] Therefore, how to provide a group of effective components of Citrus aurantium that can effectively treat acute lung injury is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a group of effective components of Citrus aurantium for treating acute lung injury, a method for preparing the same, and its application, so as to overcome the shortcomings of the prior art.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A group of effective components of Citrus aurantium for treating acute lung injury, characterized in that it includes: naringin, naringin, naringin 7-O-glucoside, hesperidin and neohesperidin.

[0010] The preferred composition is: 0.109 parts of naringin, 0.402 parts of naringin, 0.044 parts of naringin 7-O-glucoside, 0.057 parts of hesperidin and 0.357 parts of neohesperidin.

[0011] The beneficial effects of this invention are as follows:

[0012] The applicant extracted *Citrus aurantium* (Quercus variabilis) with 50%-100% ethanol. The total extract was then extracted with petroleum ether, ethyl acetate, and n-butanol to obtain petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and aqueous fraction of *Citrus aurantium*. The applicant separated the ethyl acetate fraction of *Citrus aurantium* using ODS-C18 medium-pressure preparative chromatography, eluting with a methanol-water gradient at different ratios. 30% methanol eluent fractions 1, 2, 3, 4, 5, 6, and 7 were collected. Fractions 1-4 were combined to obtain the effective component group of *Citrus aurantium*. The composition of this effective component group is basically clear, containing five active ingredients: naringin, naringin, naringenin 7-O-glucoside, hesperidin, and neohesperidin. In vitro and in vivo experimental results show that this effective component group has a good therapeutic effect on acute lung injury. The components are basically identified, facilitating pharmacological research and drug development.

[0013] Furthermore, the above-mentioned effective component group of Quzhou Zhi Ke was obtained by extracting, separating chemical components, identifying activity, and combining active components from Quzhou Zhi Ke, a traditional Chinese medicine decoction piece.

[0014] A method for preparing an effective component group of Citrus aurantium for treating acute lung injury specifically includes the following steps:

[0015] (1) The husk of Quzhou Citrus aurantium was crushed into granules, then soaked in ethanol and extracted by reflux to obtain the total extract of Quzhou Citrus aurantium.

[0016] (2) The total extract of Quzhou Citrus aurantium was suspended in water to obtain a suspension. Then, the suspension was extracted with ethyl acetate to obtain an extract. Finally, the extract was concentrated under reduced pressure to obtain the ethyl acetate fraction of Quzhou Citrus aurantium.

[0017] (3) The ethyl acetate fraction of Citrus aurantium was separated by ODS-C18 medium-pressure preparative chromatography, and eluted with different ratios of methanol-water gradient. Under HPLC-QTOF-MS tracking, the eluted fractions were combined according to the chemical composition type to obtain 13 chemical composition groups.

[0018] (4) Combine the chemical components Fr.1-4 to obtain the effective components of Citrus aurantium for treating acute lung injury.

[0019] Furthermore, in step (1) above, the volume concentration of ethanol is 50%-100%; the soaking time is 12h; and the reflux extraction is performed 3 times, 1h each time.

[0020] Furthermore, in step (2) above, the volume ratio of the total extract of Citrus aurantium to water is 1:10; the volume ratio of the suspension to ethyl acetate is 1:1; and the extraction is performed 3 times.

[0021] The present invention also claims protection for the use of the above-mentioned group of effective components of Citrus aurantium in the preparation of a medicine.

[0022] Furthermore, the aforementioned drugs are for the treatment of acute lung injury; the routes of administration are oral or inhalation.

[0023] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The chemical composition of the aqueous extract of Citrus aurantium in the applicant's previous research (Chinese Patent 201710813853.0) is relatively complex, with a low content of anti-inflammatory active ingredients. Clinically, it can only be developed into oral preparations, and its pharmacology and toxicology are relatively complex. In contrast, the chemical composition of the effective components of Citrus aurantium in this invention is basically clear, with a high content of anti-inflammatory active ingredients. Clinically, it can be developed into oral or inhaled preparations, and its pharmacology and toxicology are basically clear, giving it better drug-like properties.

[0025] 2. Extensive experimental data confirm that the anti-inflammatory activity of the effective components of Citrus aurantium in this invention is significantly higher than that of the aqueous extract and the total ethyl acetate extract of Citrus aurantium.

[0026] 3. The effective components of the Citrus aurantium in this invention are relatively well-defined, mainly consisting of 5 effective components. When treating acute lung injury, it can be developed into an inhaled preparation, which will have a better therapeutic effect. Attached Figure Description

[0027] Figure 1 This is the chromatogram of the effective components of Citrus aurantium in Example 1;

[0028] Figure 2 The figure shows the effect of the effective components of Citrus aurantium in Example 1 on the inflammatory factor IL-6 in the bronchoalveolar lavage fluid of mice with acute lung injury after treatment.

[0029] Figure 3 The figure shows the effect of the effective components of Citrus aurantium in Example 1 on the inflammatory factor IL-1β in the bronchoalveolar lavage fluid of mice with acute lung injury after treatment.

[0030] Figure 4 The figure shows the effect of the effective components of Citrus aurantium in Example 1 on the inflammatory factor TNF-α in the bronchoalveolar lavage fluid of mice with acute lung injury after treatment.

[0031] Figure 5 The figure shows the effect of the effective components of Citrus aurantium in Example 1 on the inflammatory factor IFN-γ in the bronchoalveolar lavage fluid of mice with acute lung injury after treatment.

[0032] Figure 6 This is a graph showing the effect of the effective components of Citrus aurantium in Example 1 on the wet-to-dry weight ratio of the lungs of mice with acute lung injury after treatment.

[0033] Figure 7 The figure shows the effect of the blank control group on the pathological damage of the lungs in mice with acute lung injury.

[0034] Figure 8 Figure showing the effect of LPS on pathological damage to the lungs of mice with acute lung injury;

[0035] Figure 9 Figure 1: Effect of LPS + 100mg / kg effective components of Citrus aurantium on pathological damage of lungs in mice with acute lung injury.

[0036] Figure 10 The effect of LPS+20mg / kg effective component group of Example 1 on the pathological damage of lungs in mice with acute lung injury. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] The preparation method of the effective components of Citrus aurantium for treating acute lung injury specifically includes the following steps:

[0040] (1) 100g of Quzhou Citrus aurantium was crushed into granules, then soaked in 60% ethanol for 12 hours, and then refluxed for 3 times, 1 hour each time. The extracts were combined and concentrated under reduced pressure to obtain the total extract of Quzhou Citrus aurantium.

[0041] (2) The total extract of Quzhi Ke was suspended in 10 times the volume of water to obtain a suspension. Then the suspension was extracted three times with ethyl acetate at a volume ratio of 1:1 to obtain an extract. Finally, the extract was concentrated under reduced pressure to obtain the ethyl acetate fraction of Quzhi Ke.

[0042] (3) The ethyl acetate fraction of Citrus aurantium was separated by ODS-C18 medium-pressure preparative chromatography, and eluted with different ratios of methanol-water gradient. Under HPLC-QTOF-MS tracking, the eluted fractions were combined according to the chemical composition type to obtain 7 chemical composition groups.

[0043] (4) Combining chemical component groups Fr.1, 2, 3 and 4 yields the effective component group of Citrus aurantium for treating acute lung injury, with a yield of 1.124% (based on the weight of the medicinal material).

[0044] Example 2

[0045] The preparation method of the effective components of Citrus aurantium for treating acute lung injury specifically includes the following steps:

[0046] (1) 100g of Quzhou Citrus aurantium was crushed into granules, then soaked in 50% ethanol for 12 hours, and then refluxed for 3 times, 1 hour each time. The extracts were combined and concentrated under reduced pressure to obtain the total extract of Quzhou Citrus aurantium.

[0047] (2) The total extract of Quzhi Ke was suspended in 10 times the volume of water to obtain a suspension. Then the suspension was extracted three times with ethyl acetate at a volume ratio of 1:1 to obtain an extract. Finally, the extract was concentrated under reduced pressure to obtain the ethyl acetate fraction of Quzhi Ke.

[0048] (3) The ethyl acetate fraction of Citrus aurantium was separated by ODS-C18 medium-pressure preparative chromatography, and eluted with different ratios of methanol-water gradient. Under HPLC-QTOF-MS tracking, the eluted fractions were combined according to the chemical composition type to obtain 7 chemical composition groups.

[0049] (4) Combine the chemical components Fr.1-4 to obtain the effective components of Citrus aurantium for treating acute lung injury.

[0050] Example 3

[0051] The preparation method of the effective components of Citrus aurantium for treating acute lung injury specifically includes the following steps:

[0052] (1) 100g of Quzhou Citrus aurantium was crushed into granules, then soaked in 90% ethanol for 12 hours, and then refluxed for 3 times, 1 hour each time. The extracts were combined and concentrated under reduced pressure to obtain the total extract of Quzhou Citrus aurantium.

[0053] (2) The total extract of Quzhi Ke was suspended in 10 times the volume of water to obtain a suspension. Then the suspension was extracted three times with ethyl acetate at a volume ratio of 1:1 to obtain an extract. Finally, the extract was concentrated under reduced pressure to obtain the ethyl acetate fraction of Quzhi Ke.

[0054] (3) The ethyl acetate fraction of Citrus aurantium was separated by ODS-C18 medium-pressure preparative chromatography, and eluted with different ratios of methanol-water gradient. Under HPLC-QTOF-MS tracking, the eluted fractions were combined according to the chemical composition type to obtain 7 chemical composition groups.

[0055] (4) Combine the chemical components Fr.1-4 to obtain the effective components of Citrus aurantium for treating acute lung injury.

[0056] Performance testing

[0057] 1. Identification of the effective components of Citrus aurantium

[0058] The effective components of Citrus aurantium obtained in Example 1 were analyzed by HPLC-QTOF-MS. Using precise molecular weight, molecular formula, and secondary mass spectrometry fragment data from high-resolution mass spectrometry, combined with comparisons of standard samples and literature fragments, the structures of the main compounds were identified. The results are as follows: Figure 1 As shown.

[0059] Figure 1 This is a chromatogram of the effective components of Citrus aurantium in Example 1. Figure 1 It can be seen that the composition of the effective components of Quzhou Citrus aurantium in Example 1 of the present invention is basically clear, containing 5 active ingredients. Specifically, 1g of the effective components of Quzhou Citrus aurantium contains 0.109g of naringin, 0.402g of naringin, 0.044g of naringenin 7-O-glucoside, 0.057g of hesperidin, and 0.357g of neohesperidin.

[0060] 2. Comparison of the anti-inflammatory activities of the effective components of Citrus aurantium and its aqueous extract.

[0061] RAW264.7 cells in logarithmic growth phase were digested with 0.25% trypsin and then seeded into 24-well plates (2 × 10⁶ cells per well). 4 Cells were cultured at 37°C and 5% CO2 for 24 hours (cell confluence approximately 70%). After induction with 1 μg / mL LPS for 2 hours, the effective component group of Citrus aurantium (25 mg / mL, based on raw medicinal material) and the aqueous extract of Citrus aurantium (25 mg / mL, based on raw medicinal material, preparation method described in Chinese Patent 201710813853.0) from Example 1 were added. A model control group and a blank control group were also set up. After 24 hours of LPS induction, the cell supernatant was collected. The levels of IL-6 and IL-1β in the cell supernatant were detected by ELISA to compare the anti-inflammatory activity of the effective component group of Citrus aurantium (Example 1) and the aqueous extract of Citrus aurantium.

[0062] The results are shown in Table 1.

[0063] Table 1. Comparison of anti-inflammatory activities of effective components of Citrus aurantium and its aqueous extract.

[0064] Group IL-6 (pg / mL) IL-1β (pg / mL) Blank control group 21.54±3.02 8.25±1.38 LPS 4826.55±345.32## 4012.32±363.56## LPS + Qu Zhi Ke effective ingredient group 284.64±87.55** 183.54±79.55** LPS + Quzhou Citrus aurantium aqueous extract 2343.76±128.88** 2464.97±214.76**

[0065] Note: Compared with the blank control group, ##p<0.01; compared with the LPS model control group, **p<0.01.

[0066] As shown in Table 1, the levels of IL-6 and IL-1β inflammatory factors in the cell supernatant were significantly increased after LPS induction (p<0.01 compared with the blank control group). The levels of IL-6 and IL-1β were significantly decreased after the addition of the effective components of *Citrus aurantium* from Example 1 and the aqueous extract of *Citrus aurantium* from Example 1 (p<0.01 compared with the model control group), while the anti-inflammatory activity of the effective components of *Citrus aurantium* from Example 1 was significantly higher than that of the aqueous extract of *Citrus aurantium* from Example 1.

[0067] Conclusion: The anti-inflammatory activity of the effective components of Citrus aurantium in this invention is significantly higher than that of the aqueous extract of Citrus aurantium disclosed in the applicant's previous patent application (201710813853.0). Moreover, under the same crude drug dosage, the anti-inflammatory activity of the effective components of Citrus aurantium in this invention is more than 8 times that of the aqueous extract of Citrus aurantium.

[0068] 3. Comparison of the anti-inflammatory activity of the effective components of Citrus aurantium and the polar fraction of ethyl acetate from Citrus aurantium.

[0069] RAW264.7 cells in logarithmic growth phase were digested with 0.25% trypsin and then seeded into 24-well plates (2 × 10⁶ cells per well). 4 Cells were cultured at 37°C and 5% CO2 for 24 hours (cell confluence approximately 70%). After induction with 1 μg / mL LPS for 2 hours, the effective components of Citrus aurantium from Example 1 (25 mg / mL, based on raw medicinal material) and the polar fraction of ethyl acetate from Citrus aurantium from Example 1 (25 mg / mL, based on raw medicinal material) were added. A model control group and a blank control group were also included. Cell supernatant was collected after 24 hours of LPS induction. The levels of IL-6 and IL-1β in the cell supernatant were detected using ELISA to compare the anti-inflammatory activities of the effective components of Citrus aurantium from Example 1 and the polar fraction of ethyl acetate from Citrus aurantium from Example 1.

[0070] The results are shown in Table 2.

[0071] Table 2 Comparison of the anti-inflammatory activity of the effective components of Citrus aurantium and the polar fraction of ethyl acetate from Citrus aurantium.

[0072] Group IL-6 (pg / mL) IL-1β (pg / mL) Blank control group 22.62±3.14 10.34±2.65 LPS 4385.68±286.74## 3812.49±321.50## LPS + Qu Zhi Ke effective ingredient group 286.46±78.58** 234.60±125.43** LPS + Ethyl Acetate from Citrus aurantium (polar fraction) 924.75±148.91** 832.02±218.21**

[0073] Note: Compared with the blank control group, ##p<0.01; compared with the LPS model control group, **p<0.01.

[0074] Table 2 shows that the levels of IL-6 and IL-1β inflammatory factors in the cell supernatant were significantly increased after LPS induction (p<0.01 compared with the blank control group). The levels of IL-6 and IL-1β were significantly decreased after the addition of the effective components of Citrus aurantium from Example 1 and the polar fraction of ethyl acetate from Citrus aurantium from Example 1 (p<0.01 compared with the model control group), and the anti-inflammatory activity of the effective components of Citrus aurantium from Example 1 was superior to that of the polar fraction of ethyl acetate from Citrus aurantium from Example 1.

[0075] Conclusion: Both the effective components of Citrus aurantium and the polar fraction of ethyl acetate from Citrus aurantium exhibit good anti-inflammatory activity in this invention, with the effective components showing significantly stronger anti-inflammatory activity than the ethyl acetate fraction. Furthermore, the composition of the effective components of Citrus aurantium is well-defined, while the composition of the polar fraction of ethyl acetate is more complex, leading to a much more complex toxicity at the same crude drug dosage. Therefore, the effective components of Citrus aurantium possess stronger medicinal properties and are more easily developed into anti-inflammatory drugs.

[0076] 4. Comparison of the anti-inflammatory activities of the effective components of Quzhou bitter orange peel with those of naringin, hesperidin, and neohesperidin.

[0077] Drug concentration selection: ① Example 1: effective component group of Quzhou citrus aurantium 10 μg / mL (including 1.09 μg / mL of rutin, 4.02 μg / mL of naringin, 0.57 μg / mL of hesperidin, and 3.57 μg / mL of neohesperidin); ② Rutin naringin 10 μg / mL; ③ Naringin 10 μg / mL; ④ Hesperidin 10 μg / mL; ⑤ Neohesperidin 10 μg / mL.

[0078] RAW264.7 cells in logarithmic growth phase were digested with 0.25% trypsin and seeded into 24-well plates (2 × 10⁴ cells per well). The cells were then cultured at 37°C in a 5% CO₂ incubator for 24 h (cell confluence approximately 70%). After induction with 1 μg / mL LPS for 2 h, the effective components of *Citrus aurantium* from Example 1, along with naringin, hesperidin, and neohesperidin, were added. A model control group and a blank control group were also included. After 24 h of LPS induction, the cell supernatant was collected. The levels of IL-6 and IL-1β in the cell supernatant were detected using ELISA. The anti-inflammatory activity of the effective components of *Citrus aurantium* was compared with that of individual components of naringin, hesperidin, and neohesperidin. The results are shown in Table 3.

[0079] Table 3. Comparison of the anti-inflammatory activities of the effective components of Citrus aurantium with those of naringin, hesperidin, and neohesperidin.

[0080] Group IL-6 (pg / mL) IL-1β (pg / mL) Blank control group 36.52±4.98 22.70±3.71 LPS 3817.34±317.92## 3153.65±291.57## LPS + Citrus aurantium active ingredient group (10μg / mL) 238.36±49.02** 198.55±64.98** LPS + Rutin (10 μg / mL) 1364.81±139.61** 1634.21±210.03** LPS + naringin (10 μg / mL) 2829.34±318.99* 2352.44±274.34* LPS + hesperidin (10 μg / mL) 2463.03±148.88** 1855.88±316.43* LPS + neohesperidin (10 μg / mL) 1861.75±392.59** 1773.65±148.93**

[0081] Note: Compared with the blank control group, ##p<0.01; compared with the LPS model control group, *p<0.05, **p<0.01.

[0082] Table 3 shows that the levels of IL-6 and IL-1β inflammatory factors in the cell supernatant were significantly increased after LPS induction (p<0.01 compared with the blank control group). The levels of IL-6 and IL-1β were significantly decreased after the addition of the effective components of *Citrus aurantium* from Example 1 and rutin, naringin, hesperidin, and neohesperidin (p<0.05 and p<0.01 respectively compared with the model control group). Furthermore, the anti-inflammatory activity of the effective components of *Citrus aurantium* from Example 1 was significantly higher than that of any single component of rutin, naringin, hesperidin, or neohesperidin.

[0083] Conclusion: The effective components of Citrus aurantium in this invention, along with the single components of naringin, hesperidin, and neohesperidin, all exhibit anti-inflammatory activity. Moreover, the anti-inflammatory activity of the effective components of Citrus aurantium in this invention is significantly stronger than that of the single components of naringin, hesperidin, and neohesperidin, and the anti-inflammatory activity is more than 5 times that of each of the single components.

[0084] 5. The effect of effective components of Citrus aurantium in treating acute lung injury in mice.

[0085] (1) Experimental animals: SPF-grade male BALB / c mice were housed in an SPF-grade barrier system with free access to food and water at a temperature of 20-25℃ and humidity of 50-60%, following a 12h day / 12h night routine. Husbandry and management met the requirements of the international AAALAC accreditation. The experimental design was approved by the Ethics Committee of Zhejiang Academy of Medical Sciences. All experiments were conducted in accordance with relevant guidelines and regulations.

[0086] (2) Preparation of inhalation formulation of effective components of Citrus aurantium: The inhalation formulation is prepared according to the following proportions: 8 mg of effective components of Citrus aurantium in Example 1 is added to 2 mL of ddH2O, 0.4 mg of Tween 80 is added to aid dissolution, the pH is adjusted to 4.5 after thorough suspension, and the osmotic pressure is adjusted with sodium chloride. When used, it is diluted with 0.2‰ Tween 80.

[0087] (3) Animal experiment: Sixty mice were randomly divided into four groups: a. 100 mg / kg of the effective ingredient group of Citrus aurantium in Example 1 + LPS; b. 20 mg / kg of the effective ingredient group of Citrus aurantium in Example 1 + LPS; c. LPS (model control group); d. blank control group. Each group consisted of 15 mice. On day 1, mice in groups a, b, and c were anesthetized with 0.5% sodium pentobarbital (40 mg / kg) and then given 50 μL (20 μg) of LPS via nasal drip to induce ALI. Two hours later, groups a and b were given the effective ingredient group of Citrus aurantium in Example 1 via nebulization, and groups c and d were given 0.2‰ of Tween 80 via nebulization. The administration was repeated once on day 2.

[0088] (4) Collection and processing of bronchoalveolar lavage fluid (BALF) from ALI mice: Five mice were used in each group for BALF extraction. After euthanasia, the trachea was exposed and endotracheal intubation was performed using a disposable intravenous catheter. The intubation was secured with silk sutures. Then, 1 mL of physiological saline was used to lavage the bronchoalveolar lavage with a syringe. During lavage, 1 mL of physiological saline was used each time to flush the bronchoalveolar lavage from both sides through the intravenous catheter. This process was repeated three times before the lavage fluid was collected. The recovery rate was greater than 90% to ensure thorough lavage. After collection, the BALF was centrifuged at 1000 rpm for 5 min at 4°C in a refrigerated centrifuge. The levels of IL-6, IL-1β, TNF-α, and IFN-γ inflammatory factors in the supernatant were detected using an ELISA kit.

[0089] (5) Detection of wet / dry weight ratio (W / D) of lung tissue in ALI mice: Five mice were dissected in each group. Approximately 50g of lung tissue was taken, washed with ice-cold saline to remove residual blood, blotted dry with filter paper, and weighed to obtain the wet weight (W). The tissue was then dried in a 60℃ oven for 48 hours and weighed again to obtain the dry weight (D). The degree of edema in the lung tissue was evaluated by calculating the ratio of W to D.

[0090] (6) Pathological examination of lung tissue in ALI mice: Five mice from each group were dissected and lung tissue was fixed in 10% formaldehyde solution. After sampling, embedding and sectioning, some sections were used for HE staining for microscopic observation of inflammation damage and improvement.

[0091] The test results are as follows Figure 3-5 As shown.

[0092] Figure 2 The figure shows the effect of the effective components of Citrus aurantium in Example 1 on the inflammatory factor IL-6 in the bronchoalveolar lavage fluid of mice with acute lung injury after treatment. Figure 3 The figure shows the effect of the effective components of Citrus aurantium in Example 1 on the inflammatory factor IL-1β in the bronchoalveolar lavage fluid of mice with acute lung injury after treatment. Figure 4The figure shows the effect of the effective components of Citrus aurantium in Example 1 on the inflammatory factor TNF-α in the bronchoalveolar lavage fluid of mice with acute lung injury after treatment. Figure 5 This is a graph showing the effect of the effective components of Citrus aurantium (Quzhi Ke) on the inflammatory factor IFN-γ in the bronchoalveolar lavage fluid of mice with acute lung injury after treatment in Example 1; compared with the blank control group. Compared with the model control group, ***p<0.001, **p<0.01. Figure 3-5 It was found that after LPS-induced acute lung injury, the levels of inflammatory factors IL-6, IL-1β, TNF-α, and IFN-γ in bronchoalveolar lavage fluid were significantly increased (p<0.001 compared with the control group). After two days of inhalation treatment with the effective components of Citrus aurantium in Example 1, the levels of the above inflammatory factors were significantly reduced (p<0.01 compared with the model control group).

[0093] Figure 6 This is a graph showing the effect of the effective components of Citrus aurantium (Quzhi Ke) on the wet-to-dry weight ratio of the lungs in mice with acute lung injury after treatment in Example 1; compared with the blank control group. Compared with the model control group, *p<0.05, **p<0.01. Figure 6 It can be seen that after LPS-induced acute lung injury, the wet-to-dry weight ratio of the mouse lungs increased significantly (p<0.01 compared with the control group). In Example 1, after 2 days of inhalation treatment with the effective components of Quzhi Ke, the wet-to-dry weight ratio decreased significantly (p<0.05 compared with the model control group), and the high dose basically reached the level of the control group.

[0094] Figure 7 The figure shows the effect of the blank control group on the pathological damage of the lungs in mice with acute lung injury. Figure 8 Figure showing the effect of LPS on pathological damage to the lungs of mice with acute lung injury; Figure 9 Figure 1: Effect of LPS+100mg / kg effective component group on lung pathological damage in mice with acute lung injury. Figure 10 The effect of LPS + 20 mg / kg (Example 1) on the pathological damage of the lungs in mice with acute lung injury after treatment with the effective components of Citrus aurantium. Figure 7-10 It was found that after LPS-induced acute lung injury, the lung tissue structure of mice in the blank control group was intact, the alveoli were normal, and no large amount of inflammatory cell infiltration was observed. In the model control group, the lung tissue of mice showed a large number of alveolar ruptures, severe alveolar structural damage, severe pulmonary hemorrhage, a large amount of inflammatory cell infiltration, and thickening and widening of the pulmonary interstitium. In Example 1, after 2 days of inhalation treatment with the effective components of Citrus aurantium, the lung tissue damage was significantly improved compared with the model control group, as evidenced by reduced alveolar rupture, reduced pulmonary hemorrhage, and improved inflammatory cell infiltration, indicating that the effective components of Citrus aurantium can effectively alleviate the lung tissue damage in mice with LPS-induced acute lung injury.

[0095] Conclusion: The effective components of Citrus aurantium can be administered via inhalation to treat acute lung injury.

[0096] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A group of effective components from Citrus aurantium for treating acute lung injury, characterized in that, The effective component group of Quzhou Citrus aurantium contains 5 active ingredients, and 1g of the effective component group of Quzhou Citrus aurantium contains 0.109g of naringin, 0.402g of naringin, 0.044g of naringin 7-O-glucoside, 0.057g of hesperidin and 0.357g of neohesperidin; The preparation method of the effective component group of Citrus aurantium for treating acute lung injury specifically includes the following steps: (1) 100g of Quzhou Citrus aurantium was crushed into granules, then soaked in 60% ethanol for 12 hours, and then refluxed for 3 times, 1 hour each time. The extracts were combined and concentrated under reduced pressure to obtain the total extract of Quzhou Citrus aurantium. (2) The total extract of Quzhi Ke was suspended in 10 times the volume of water to obtain a suspension. Then the suspension was extracted three times with ethyl acetate at a volume ratio of 1:1 to obtain an extract. Finally, the extract was concentrated under reduced pressure to obtain the ethyl acetate fraction of Quzhi Ke. (3) The ethyl acetate fraction of Citrus aurantium was separated by ODS-C18 medium-pressure preparative chromatography, and eluted with different ratios of methanol-water gradient. Under HPLC-QTOF-MS tracking, the eluted fractions were combined according to the chemical composition type to obtain 7 chemical composition groups. (4) Combining chemical component groups Fr .1, 2, 3, and 4 yields the effective component group of Citrus aurantium for treating acute lung injury.

2. The application of the effective components of Citrus aurantium as described in claim 1 for treating acute lung injury in the preparation of a medicine, characterized in that, The drug is for the treatment of acute lung injury; the drug is administered orally or by inhalation.

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