A gardenia polysaccharide, a preparation method and application thereof

Abstract

This invention relates to the field of traditional Chinese medicine extracts and biomedical technology, and particularly to a gardenia polysaccharide, its preparation method, and its applications. The gardenia polysaccharide of this invention is composed of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose; the molar ratio of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose is 4.59:4.65:34.35:9.38:13.9:33.13; the total sugar content is 83.1%, the uronic acid content is 32.8%, and it contains no protein; the molecular weight is 4.3~652.9 kDa. In vitro experiments have confirmed that the gardenia polysaccharide has strong anti-complement activity and can be further used as an active ingredient in the preparation of anti-complement drugs; whole animal experiments have confirmed that the gardenia polysaccharide can effectively alleviate endotoxin-induced acute lung injury in mice and can be further used as an active ingredient in the preparation of drugs for the prevention and treatment of acute lung injury.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extracts and biomedical technology, and in particular to a gardenia polysaccharide, its preparation method and application. Background Technology

[0002] Acute lung injury (ALI) is a critical clinical condition induced by various factors such as severe infection, trauma, and shock. Its pathological feature is diffuse alveolar-capillary membrane damage, leading to pulmonary edema and increased permeability, representing an early stage of acute respiratory distress syndrome (ARDS). The core pathophysiological changes include decreased lung compliance, increased intrapulmonary shunting, and ventilation / perfusion mismatch. Clinical manifestations are primarily refractory hypoxemia and respiratory distress, with a persistently high mortality rate. Domestic and international research has confirmed that ALI is essentially an immune disease with acute immune damage and clearance as its core mechanisms, mainly manifested as infiltration of inflammatory cells such as mononuclear macrophages, overexpression of various cytokines and adhesion molecules, and excessive activation of the complement system. Previous studies have found that the allergens (such as C3a and C5a) produced after excessive complement activation can promote the accumulation of neutrophils in the lungs, damaging the alveolar capillary walls, leading to increased vascular permeability, which in turn causes pulmonary edema, decreased blood oxygen levels, and further aggravates endothelial cell damage, forming a vicious cycle of ALI. Therefore, finding highly effective and low-toxicity complement inhibitors is one of the effective ways to develop drugs for the prevention and treatment of ALI. Currently, clinical treatment for ALI primarily relies on glucocorticoids and antibiotics, but their overall efficacy is limited and has significant limitations: while glucocorticoids have some anti-inflammatory effects, they lack specificity, have significant side effects, and long-term or high-dose use can easily lead to immunosuppression and secondary infections; while complement-targeting biologics (such as sCR1 and C5 monoclonal antibodies) have achieved some clinical efficacy, their high price limits their widespread adoption. Therefore, developing ALI prevention and treatment drugs that are highly effective, low in toxicity, and economical has become an urgent clinical need.

[0003] Gardenia is a plant of the Rubiaceae family (family Rubiaceae). Gardenia jasminoidesGardenia (Ellis) is the dried, ripe fruit of the plant. It is cold in nature and bitter in taste, and enters the heart, lung, and triple burner meridians. It has the effects of purging fire and relieving irritability, clearing heat and promoting diuresis, cooling blood and detoxifying. As a traditional Chinese medicine for clearing heat and purging fire, gardenia has a long history of use in treating lung diseases within the TCM theoretical system. Clinically, it is a core ingredient in many classic formulas for treating lung diseases (such as Qingjin Huatan Decoction, Zhixing Siwu Decoction, Zhizi Chishi Decoction, and Huanglian Jiedu Decoction) and commonly used prepared Chinese medicines (such as Qingfei Yihuo Pills, Qinzhi Qingfei Granules, and Lingyang Qingfei Capsules). Modern pharmacological studies have confirmed that gardenia has various pharmacological activities, including anti-inflammatory, antibacterial, antipyretic and analgesic, antiviral, choleretic, sedative, and antihypertensive effects. Its main chemical components include iridoid glycosides (such as geniposide), volatile oils, polysaccharides, flavonoids, organic acids, and diterpenoids. Previous studies have shown that gardenia extract can effectively inhibit viral acute lung injury induced by influenza A virus in mice; geniposide can significantly inhibit lipopolysaccharide-induced acute lung injury in mice and rats, as well as viral pneumonia induced by influenza virus; and gardenia polysaccharide can delay the progression of TGF-β-induced pulmonary fibrosis. However, a review of domestic and international research has revealed no reports on gardenia's inhibition of complement activation, nor on the efficacy of gardenia polysaccharide in preventing and treating acute lung injury, or on any related drug formulations. Summary of the Invention

[0004] The purpose of this invention is to provide a gardenia polysaccharide, its preparation method, and its new uses in pharmaceuticals, specifically relating to the use of gardenia polysaccharide in the preparation of anticomplement drugs and drugs for the prevention and treatment of acute lung injury.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: One of the technical solutions of the present invention provides a gardenia polysaccharide, which is composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose; The molar ratio of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose is 4.59:4.65:34.35:9.38:13.9:33.13. The total sugar content of gardenia polysaccharide is 83.1%, the uronic acid content is 32.8%, and it does not contain protein; the molecular weight of gardenia polysaccharide is 4.3~652.9 kDa.

[0006] The second technical solution of this invention provides a method for preparing the above-mentioned gardenia polysaccharide, comprising the following steps: (1) Mix dried gardenia fruit with ethanol, extract by ultrasound, and filter to obtain the residue; (2) Soak the dregs in water, extract them, and filter to obtain the extract; (3) The extract was concentrated, and the concentrate was precipitated with ethanol. The precipitate was collected, purified and dried to obtain gardenia polysaccharide.

[0007] The third technical solution of this invention provides the application of the above-mentioned gardenia polysaccharide in the preparation of anticomplement drugs.

[0008] The fourth technical solution of the present invention provides the application of the above-mentioned gardenia polysaccharide in the preparation of drugs for the prevention and treatment of acute lung injury.

[0009] Compared with the prior art, the present invention has the following beneficial effects: This invention is derived from gardenia ( Gardenia jasminoides A total polysaccharide extract was isolated and extracted from Ellis (E. esculentus), with a yield of 6.9%, a total sugar content of 83.1%, a uronic acid content of 32.8%, and no protein. The molecular weight was 4.3~652.9 kDa. In vitro experiments confirmed that the gardenia polysaccharide has strong anti-complement activity and significantly inhibits complement activation, and can be further used as an active ingredient in the preparation of anti-complement drugs. Whole animal experiments confirmed that gardenia polysaccharide can effectively alleviate endotoxin-induced acute lung injury in mice, and can be further used as an active ingredient in the preparation of drugs for the prevention and treatment of acute lung injury. Attached Figure Description

[0010] Figure 1 This is a high-performance gel permeation chromatogram of gardenia polysaccharide of the present invention.

[0011] Figure 2 The infrared spectrum of gardenia polysaccharide of this invention.

[0012] Figure 3 This is a high-performance liquid chromatogram of the monosaccharide composition of the gardenia polysaccharide of the present invention.

[0013] Figure 4 This invention relates to the effect of gardenia polysaccharide on the lung index in mice with endotoxin (LPS)-induced acute lung injury, wherein ** p <0.01, *** p <0.001.

[0014] Figure 5 This invention relates to the effect of gardenia polysaccharide on pathological changes in lung tissue of mice with endotoxin (LPS)-induced acute lung injury.

[0015] Figure 6 This figure shows the effect of Gardenia polysaccharide of the present invention on inflammation in lung homogenate of mice with endotoxin (LPS)-induced acute lung injury, where (A) represents IL-1β (A), (B) represents IL-6 (B), and (C) represents TNF-α; Note: * p <0.05,** p <0.01, *** p <0.001. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] The room temperature described in this invention is 25±2℃.

[0022] All raw materials used in this invention can be obtained commercially or prepared using existing technologies.

[0023] This invention provides a gardenia polysaccharide composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose; The molar ratio of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose is 4.59:4.65:34.35:9.38:13.9:33.13. The total sugar content of gardenia polysaccharide is 83.1%, the uronic acid content is 32.8%, and it does not contain protein; the molecular weight of gardenia polysaccharide is 4.3~652.9 kDa.

[0024] In this invention, the gardenia refers to the Rubiaceae plant Gardenia (Gardenia jasminoides). Gardenia jasminoides The dried, ripe fruit of Ellis.

[0025] This invention also provides a method for preparing the above-mentioned gardenia polysaccharide, comprising the following steps: (1) Mix dried gardenia fruit with ethanol, extract by ultrasound, and filter to obtain the residue; (2) Soak the dregs in water, extract them, and filter to obtain the extract; (3) The extract was concentrated, and the concentrate was precipitated with ethanol. The precipitate was collected, purified and dried to obtain gardenia polysaccharide.

[0026] Step (1) of this invention involves adding dried gardenia fruit to ethanol and performing ultrasonic extraction to remove lipid components. After filtration, the residue is dried in a ventilated place at room temperature.

[0027] In this invention, the volume concentration of ethanol in step (1) is 95%; the ratio of dried gardenia fruit to ethanol is 1:15 (g:L); the ultrasonic extraction power is 80~120kW, the time is 1h, and the extraction is repeated once.

[0028] Step (2) of this invention involves soaking the dried medicinal residue in water for extraction, filtering, repeating the extraction 4 times, and combining the 4 filtrates to obtain the extract.

[0029] In this invention, the ratio of the medicinal residue to water is 1:15 (g:L); the soaking temperature is room temperature and the soaking time is 12h; the extraction temperature in step (2) is 100℃ and the extraction time is 2h, and the extraction is repeated 4 times.

[0030] Step (3) involves concentrating the extract under reduced pressure, centrifuging the concentrated solution to remove the precipitate, mixing the supernatant with ethanol, precipitating the ethanol, centrifuging after standing, collecting the precipitate, purifying the precipitate, concentrating and freeze-drying it to obtain gardenia polysaccharide.

[0031] In this invention, the concentration temperature is 50~70 ℃, the volume of the concentrate is 1 / 8 of the volume of the extract; the volume ratio of the concentrate to ethanol is 1:5; the volume concentration of ethanol in step (3) is 95%; and the alcohol precipitation time is 18~28h.

[0032] In this invention, the purification method is as follows: the precipitate is dissolved in water and then dialyzed; the molecular cutoff of the dialysis bag is 3500 Da, and the dialysis time is 2-3 days.

[0033] This invention also provides the application of the above-mentioned gardenia polysaccharide in the preparation of anticomplement drugs.

[0034] The present invention also provides the application of the above-mentioned gardenia polysaccharide in the preparation of a drug for the prevention and treatment of acute lung injury, for the treatment of acute lung injury and acute respiratory distress syndrome.

[0035] This invention discloses a total polysaccharide extract obtained from gardenia. In vitro experiments have demonstrated that the total polysaccharide extract exhibits strong anti-complement activity, and whole-animal model experiments have confirmed its strong effect in preventing and treating acute lung injury.

[0036] In this invention, given that it has similar pathological features and pathophysiological changes to the acute lung injury described above, the gardenia polysaccharide of this invention can also be used to prepare drugs for the prevention and treatment of severe atypical pneumonia, viral-bacterial co-infection pneumonia, avian influenza, H1N1 influenza, and seasonal influenza.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1 (1) Take 100 g of gardenia medicinal material, add 95% ethanol solution at a material-to-liquid ratio of 1:15 (g:L) and extract by ultrasonic extraction at 120 kW for 1 h. Repeat the extraction once to remove lipid components. After filtration, obtain the residue. (2) The dregs were dried in a ventilated place at room temperature, then water was added at a ratio of 1:15 (g:L) and soaked at room temperature for 12 hours. After that, it was extracted 4 times at 100 ℃ for 2 hours each time. The filtrates were combined and concentrated at 50~70 ℃ so that the volume of the concentrated liquid was 1 / 8 of the filtrate. The concentrated liquid was centrifuged to remove the precipitate. (3) The supernatant was precipitated with 95% ethanol by volume, with the volume ratio of extract to ethanol being 1:5 (v / v). After standing for 24 h, the extract was centrifuged and the precipitate was collected. (4) The precipitate was re-dissolved in water and dialyzed with deionized water (the molecular cutoff of the dialysis bag was 3500 Da) for 2-3 days, concentrated and freeze-dried to obtain total gardenia polysaccharide GJP (6.9 g was obtained, with a yield of 6.9%).

[0039] Test Example 1: Structural Characterization of Gardenia Polysaccharide (1) Analysis of molecular weight Sample preparation: Take 2.0 mg of gardenia polysaccharide sample into a centrifuge tube, add 0.5 mL of 0.02 mol / L ammonium acetate solution to dissolve it, filter it through a 0.22 μL filter membrane and then perform HPLC analysis.

[0040] Chromatographic conditions: Column: TSKgel G4000SWXL (300 × 7.8 mm, 8 μm); Mobile phase: 0.02 mol / L ammonium acetate; Flow rate: 0.6 mL / min; Injection volume: 20 μL; Column temperature: 40 ℃; Detector: Refractive index detector (RID).

[0041] The characteristic chromatogram of Gardenia polysaccharide GJP on high performance liquid chromatography gel permeation chromatography is as follows: Figure 1 As shown, its molecular weight ranges from 4.3 to 652.9 kDa.

[0042] (2) Determination of total sugar, uronic acid and protein content The total sugar content of Gardenia polysaccharide GJP was determined to be 83.1% using the sulfuric acid-phenol method; the uronic acid content of Gardenia polysaccharide GJP was determined to be 32.8% using the m-hydroxybiphenyl method; and the protein content of Gardenia polysaccharide GJP was determined to be 0% using the Coomassie brilliant blue method.

[0043] (3) Infrared spectral analysis 2.0 mg of dried gardenia polysaccharide was ground with KBr, compressed into tablets, and analyzed using a PerkinElmer Fourier transform infrared spectroscopy (FTIR) instrument at 4000–4000 cm⁻¹. -1 Scan within the range.

[0044] like Figure 2 As shown in the infrared spectrum, the gardenia polysaccharide of the present invention contains characteristic absorption peaks of polysaccharides.

[0045] (4) Monosaccharide composition analysis Gardenia polysaccharide was completely hydrolyzed with 2 mol / L trifluoroacetic acid (TFA) at 110 °C to obtain monosaccharide residue products. After derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and extraction with trichloroacetic acid, the product was filtered through a 0.22 μL filter membrane and then analyzed by HPLC.

[0046] like Figure 3 As shown, Gardenia polysaccharide GJP is composed of six monosaccharides with a monosaccharide molar ratio of mannose:rhamnose:galacturonic acid:glucose:galactose:arabinose = 4.59:4.65:34.35:9.38:13.90:33.13.

[0047] Test Example 2: Classical Pathway Complement Inhibition Test The complement sources for this classic pathway were: serum from 3-month-old guinea pigs, diluted 1:100 with BBS buffer (barbital buffer, pH=7.4); hemolysin: rabbit anti-sheep erythrocyte antibody diluted 1:1000 with BBS buffer; and sheep erythrocytes (SRBC) prepared as 2% SRBC.

[0048] Accurately weigh approximately 3 mg of Gardenia polysaccharide GJP, dissolve it in BBS buffer, and serially dilute it to 8 concentrations. Take 200 μL of each polysaccharide solution and 200 μL of complement diluted to 1:100, pre-incubate at 37 ℃ for 10 min, then add 100 μL of 1:1000 hemolysin and 100 μL of 2% SRBC, continue incubating at 37 ºC for 30 min, then centrifuge at 5000 rpm and 4 ºC for 10 min. Take 200 μL of the supernatant from each sample and place it in a 96-well plate, measuring the absorbance at 405 nm. The experiment also included a polysaccharide control group (200 μL of the corresponding concentration of polysaccharide solution added to 400 μL of BBS buffer), a complement control group (200 μL of BBS buffer instead of polysaccharide), and a complete hemolysis group (100 μL of 2% SRBC dissolved in 500 μL of distilled water). The hemolysis inhibition rate was calculated by subtracting the absorbance value (corresponding to the control group) from the absorbance value (for each polysaccharide concentration group). A fitted curve was obtained by plotting the logarithm of the polysaccharide concentration on the X-axis and the hemolysis inhibition rate on the Y-axis. The required concentration of the polysaccharide sample (CH) for 50% inhibition of hemolysis was then calculated. 50 (Value). Using heparin as a positive control, the results showed that Gardenia polysaccharide GJP had significant inhibitory activity against classical complement pathway activation (as shown in Table 1).

[0049] Table 1. Inhibitory effects of gardenia polysaccharides and heparin on complement activation.

[0050] CH 50 The value is expressed as: average ± SD (n=3) Test Example 3: Effects of Gardenia Polysaccharide GJP on LPS-induced Acute Lung Injury in Mice Thirty male BALB / c mice (18-22 g) were randomly assigned to five groups according to body weight: a normal group, a low-dose gardenia polysaccharide group, a high-dose gardenia polysaccharide group, and a dexamethasone positive control group. The administration of drugs to the different groups was as follows: the normal group (Normal) and the model group (LPS) received saline; the low-dose gardenia polysaccharide group (GJP-L) and the high-dose gardenia polysaccharide group (GJP-H) received 50 mg / kg and 100 mg / kg GJP, respectively; and the positive control group (DEX) received 5 mg / kg dexamethasone. Mice were administered the drugs via gavage once daily for three consecutive days. One hour after administration on day 3, except for the normal group mice which received saline via intranasal instillation, all other mice received 3 mg / kg LPS via intranasal instillation to induce acute lung injury. Twenty-four hours after LPS treatment, all mice were weighed and blood was collected by enucleation. The whole lung tissue was carefully cut out, weighed and recorded. It was placed in 10% formalin solution to prepare pathological sections for pathological evaluation. The right lung was stored at -80°C for the detection of lung-related factor indicators.

[0051] (1) Effect of Gardenia polysaccharide GJP on lung index in LPS-induced acute lung injury mice The lung index is the ratio of lung weight to body weight in mice; a higher ratio indicates a more severe degree of lung disease. Results are as follows... Figure 4 As shown, compared with the normal group, the lung index of mice in the model group was significantly increased ( p <0.001); the lung index of the low-dose (GJP-L) and high-dose (GJP-H) gardenia polysaccharide administration groups and the positive control group (DEX) was significantly lower than that of the model group ( p <0.01、 p <0.001、 p <0.001). Gardenia polysaccharide GJP significantly reduced LPS-induced pulmonary edema in mice.

[0052] (2) Effects of Gardenia polysaccharide GJP on pathological changes in the lungs of mice with LPS-induced acute lung injury like Figure 5 As shown, pathological examination results indicated that, compared with the normal group, the model group mice exhibited significant alveolar wall thickening, alveolar atrophy and deformation, pulmonary hemorrhage, and extensive inflammatory cell infiltration in the pulmonary interstitium, indicating more severe inflammation. The low-dose and high-dose Gardenia polysaccharide groups (GJP-L and GJP-H), as well as the positive control group (DEX), significantly improved pulmonary pathological tissue damage, resulting in clearer alveolar contours, intact alveolar structure, lower alveolar wall inflammatory cell content, and significantly reduced inflammatory cell infiltration.

[0053] (3) Effects of Gardenia polysaccharide GJP on lung inflammation in mice with LPS-induced acute lung injury The lung homogenate supernatant was analyzed using ELISA according to the kit instructions for IL-1β, IL-6, and TNF-α. Results are as follows: Figure 6 As shown, compared with the normal group, the levels of IL-1β, IL-6 and TNF-α in the lung homogenate of the model group mice were significantly increased ( p <0.001、 p <0.001、 p <0.001. The IL-1β levels in lung homogenates from the low-dose (GJP-L) and high-dose (GJP-H) gardenia polysaccharide treatment groups and the positive control group (DEX) were significantly lower than those in the model group. p <0.01、 p <0.001、 p <0.001); The IL-6 level in lung homogenates of the low-dose (GJP-L) and high-dose (GJP-H) gardenia polysaccharide treatment groups and the positive control group (DEX) was significantly lower than that in the model group ( p <0.05、 p <0.05、 p <0.01); Similarly, the TNF-α levels in lung homogenates from the low-dose (GJP-L) and high-dose (GJP-H) gardenia polysaccharide treatment groups and the positive control group (DEX) were significantly lower than those in the model group ( p <0.01、 p <0.001、 p <0.001). Gardenia polysaccharide GJP can significantly reduce the recruitment of inflammatory cells and alleviate the inflammatory infiltration and inflammation in the lungs of LPS-infected mice.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of gardenia polysaccharide in the preparation of an anti-complement drug, characterized in that, The gardenia polysaccharide is composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose; The molar ratio of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose is 4.59:4.65:34.35:9.38:13.9:33.

13. The total sugar content of gardenia polysaccharide is 83.1%, the uronic acid content is 32.8%, and it does not contain protein; the molecular weight of gardenia polysaccharide is 4.3~652.9 kDa.

2. The application according to claim 1, characterized in that, The preparation method of the gardenia polysaccharide includes the following steps: (1) Mix dried gardenia fruit with ethanol, extract by ultrasound, and filter to obtain the residue; (2) Soak the dregs in water, extract them, and filter to obtain the extract; (3) The extract was concentrated, and the concentrate was mixed with ethanol for alcohol precipitation. The precipitate was collected, purified and dried to obtain gardenia polysaccharide.

3. The application according to claim 2, characterized in that, In step (1), the volume concentration of ethanol is 95%; the ratio of dried gardenia fruit to ethanol is 1g:15L; the ultrasonic extraction power is 80~120kW, the time is 1h, and the extraction is repeated once.

4. The application according to claim 2, characterized in that, The ratio of the medicinal residue to water is 1g:15L; the soaking temperature is room temperature and the soaking time is 12h; the extraction temperature in step (2) is 100℃ and the extraction time is 2h, and the extraction is repeated 4 times.

5. The application according to claim 2, characterized in that, The concentration temperature is 50~70℃, the volume of the concentrate is 1 / 8 of the volume of the extract; the volume ratio of the concentrate to ethanol is 1:5; the volume concentration of ethanol in step (3) is 95%; the alcohol precipitation time is 18~28h.

6. The application according to claim 2, characterized in that, The purification method is as follows: dissolve the precipitate in water and perform dialysis; the molecular cutoff of the dialysis bag is 3500 Da, and the dialysis time is 2-3 days.

7. The application according to claim 1, characterized in that, The anticomplement drug is a drug used to prevent and treat acute lung injury.

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