CN104042658A - Application of Eupatorium lindleyanum flavone part to preparation of medicament for resisting acute lung injury - Google Patents

Abstract

The invention discloses application of Eupatorium lindleyanum flavone part to preparation of a medicament for resisting acute lung injury. The Eupatorium lindleyanum flavonoid in the invention contains jaceosidin and eupafolin. The eupatorium lindleyanum flavonoid part is prepared into an animal medicament, and administered to mice in vivo; and the wet / dry weight ratio of the lung of mice with induced acute lung injury and the contents of nitric oxide (NO) and protein in the bronchoalveolar lavage fluid (BALF) are measured; myeloperoxidase (MPO) and superoxide dismutase (SOD) activity in lung tissue homogenate are detected; contents of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 beta (IL-1beta) in bronchoalveolar lavage fluid are measured; contents of C3 and C3c complements in serum are measured; and mouse lung tissue pathological change is observed. The results show that the Eupatorium lindleyanum flavone part has the effect of resisting acute lung injury.

Description

The application of Herba Eupatorii Lindleyani flavone position in preparing resisting acute lung injury medicine

Technical field

The present invention relates to the application of a kind of Herba Eupatorii Lindleyani flavone position in preparing resisting acute lung injury medicine.

Background technology

Acute lung injury (acute lung injury, ALI) be to take the damage of diffusivity pneumonocyte as basis, pulmonary edema due to injury of pulmonary vessels and lung tissue inflammatory cell infiltration are its pathological characters, and clinical main manifestations is that serious hypoxemia, diffusivity lung infiltrates and pulmonary edema; Some patients were finally will form adult respiratory distress syndrome (acute respiratory distress syndrome, ARDS), causes the irreversible acute respiratory failure of body and multiple organ dysfunction, and case fatality rate is up to 30%-40%; ALI pathogenesis is intricate, and ALI risk factors can be the coup injury from lung, can be also the indirect injury that lung other factor produces lung by systemic inflammatory response.(referring to: Baffert F, Le T, Thurston G, et al.Angiopoietin-1decreases plasma leakage by reducing number and size of endothelial gaps in venules.Am J Physiol Heart Circ Physiol, 2006,290 (1): H107-H118; Ware L B, Matthay M A.Medical progress-The acute respiratory distress syndrome.N Engl J Med, 2000,342 (18): 1334-1349; Matthay M A, Zimmerman G A, Esmon C, et al.Future research directions in acute lung injury:summary of a National Heart, Lung, and Blood Institute working group.Am J Respir Crit Care Med, 2003,167 (7): 1027-1035.)

At present, the scheme of domestic and international accepted treatment ALI: (1) removes the cause of disease; (2), on the basis of Cure the Primary Disease, mechanical ventilation (Mechanical ventilation, MV) is corrected anoxia and oxygen supply is organized in improvement as early as possible; MV is one of ALI classic treatment mode, can correct intractable anoxia, and control alveolar withers, and antagonism pulmonary edema, improves gas exchange, reduces autonomous respiration work done, prevents respiratory muscle fatigue; But only can maintain organism physiology, breathe, still can not solve ALI whole issue completely; (3) Drug therapy; Be widely used clinically the treatment acute lung injurys such as Adrenal Glucocorticoid, glucocorticoid, cyclophosphamide, MTX, can reduce the formation that blood capillary oozes out and suppress pulmonary fibrosis, pulmonary function is improved fast; But the immunosuppressant such as glucocorticoid are to acute lung injury poor selectivity, prolonged application can produce multiple complications and side effect (referring to Yang Yongtao, Wang Zhengqing. complement inhibitor progress. Chinese Journal of New Drugs, 2008,17 (24): 2093; Xu Han, Zhang Yun is firm, Zhang Jianwen, etc. the anticomplementary activity composition in natural product. Chinese natural drug, 2007,5 (5): 322.).Therefore, find selectivity treatment acute lung injury medicine high, that toxic and side effects is little and there is important clinical meaning.

Herba Eupatorii Lindleyani is the herb of feverfew point Herba Eupatorii Eupatorium lindleyanum DC., Jiangsu genuine medicinal materials.Bitter in the mouth, property are put down, return liver, spleen channel, the function with preventing phlegm from forming and stopping coughing, heat-clearing and toxic substances removing, inducing diuresis to remove edema, blood pressure lowering, for flu, cough ant phlegm, headache, tonsillitis, bacillary dysentery and hypertension (referring to State Administration of Traditional Chinese Medicine, China's book on Chinese herbal medicine, Shanghai: Science and Technology of Shanghai publishing house, 1999:7,6876).

The research such as Jiang Zhou, Yang Hui shows that Herba Eupatorii Lindleyani extracting solution has protective effect to acute lung injury induced by oleic acid rat, can obviously reduce emigrated cell number, protein content, lung coefficient and the moisture content of lung of acute lung injury induced by oleic acid rat and pulmonary vascular permeability (referring to Jiang Zhou, Yang Hui, He Haixia, Deng. Eupatorium Lindleyanum on Acute Lung Injury in Rats protective effect research, China Dispensary, 2007,18 (27): 2094; Yang Hui, Zhou Yuan great, He Haixia. the impact of Herba Eupatorii Lindleyani on acute acidity of oil induced lung injury in rats pulmonary vascular permeability. Chinese Pharmaceutical, 2010,19 (9): 5.).But above-mentioned research is all the Herba Eupatorii Lindleyani extracting solution that adopt drugmaker directly to provide, and Herba Eupatorii Lindleyani is indefinite to the active component of acute lung injury protective effect, and mechanism of action is unclear.Jaceosidin and eupafolin are flavone compound, from Herba Eupatorii Lindleyani separation, obtain (referring to Wu Shuanqing, Sun Qun, Chu Chunjuan, Zhang Jian, Herba Eupatorii Lindleyani chemical composition, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2012,37 (7): 937), Jaceosidin and eupafolin are inhibited (referring to Huang Mengchu, Liao Zhixin, Chen Daofeng to human colon carcinoma HCT8 cell and people's lung cancer A549 cell, the chemical composition of Saussurea arenaria Maxim. and cytotoxic activity thereof, Chinese herbal medicine, 2007,38 (10): 1463).But whether Jaceosidin, eupafolin have resisting acute lung injury effect, have no report, the present invention finds that the flavone position (composition of Jaceosidin and eupafolin) of preparing from Herba Eupatorii Lindleyani has the activity of resisting acute lung injury.

Summary of the invention

The object of the invention is to provide the application of a kind of Herba Eupatorii Lindleyani flavone position (EUP-FLA) in preparing resisting acute lung injury medicine.

Technical scheme of the present invention is: the present invention is prepared into animal-use drug by Herba Eupatorii Lindleyani flavone position, then to mice vivo medicine-feeding, measure the wet/dry weight ratio of lung of mice with acute lung injury, nitric oxide (NO) and protein content in bronchoalveolar lavage fluid (BALF); Detect myeloperoxidase (MPer) (MPO) and superoxide dismutase (SOD) activity in lung tissue homogenate; Tumor necrosis factor-alpha in bronchoalveolar lavage fluid (TNF-α), interleukin-6 (IL-6) and il-1 β (IL-1 β) content; Complement C3 and C3c content in serum; Observation mouse lung histo pathological change.Chmice acute injury of lung experimental result shows, Herba Eupatorii Lindleyani flavone position can alleviate mouse lung edema symptom, the content of nitric oxide (NO) and protein in reduction bronchoalveolar lavage fluid, strengthened significantly the activity of superoxide dismutase (SOD) in mouse lung tissue, the activity that suppresses myeloperoxidase (MPer) (MPO), reduced tumor necrosis factor-alpha (TNF-α), the content of interleukin-6 (IL-6) and il-1 β (IL-1 β), reduce the content of complement C3 and complement fragment C3c in serum and improve complement fragment C3c in the deposition of lung tissue, lung tissue to mice, pneumonocyte, blood capillary all has protective effect.Experimental result is definite, and in Herba Eupatorii Lindleyani, flavone position has resisting acute lung injury effect.

In the present invention, Herba Eupatorii Lindleyani flavone position preparation method is: get the dry herb of Herba Eupatorii Lindleyani, and cutting 2～3cm, the volume fraction that adds 8～15 times of volumes is 60%～95% ethanol water, reflux, extract, 1～3 hour, repeat 1～3 time, 40 ℃～60 ℃ decompression and solvent recoveries, obtain Herba Eupatorii Lindleyani ethanol extraction; This extract is soluble in water, be prepared into the solution that concentration is 0.5～1g/ml, through low pole absorption with macroporous adsorbent resin, first with water elution, remove macromolecular compound, then by volume fraction, be 90%～95% ethanol water eluting, 40 ℃～60 ℃ decompression and solvent recoveries, obtain macroporous resin position; Adsorb through ODS reverse phase silica gel chromatographic column at gained macroporous resin position, first take volume fraction as 50% methanol aqueous solution eluting, then use 70%～75% methanol aqueous solution eluting, after 40 ℃～60 ℃ decompression and solvent recoveries, through 30～60 order polyamide chromatographic column absorption, first take volume fraction as 70%～75% ethanol water eluting, rear use 95%～100% ethanol elution again, 40 ℃～60 ℃ decompression and solvent recoveries, obtain Herba Eupatorii Lindleyani flavone position.

Further, preferred, described low pole macroporous adsorbent resin is a kind of in AB-8, D101, HPD-100, HPD-200A, HPD-200B, HPD-300, HPD-700, HPD-722 macroporous adsorbent resin.

Further, preferred, described ODS reverse phase silica gel chromatographic column is a kind of in DAISOGEL ODS reverse phase silica gel packing material size 50～70um, Cosmsil ODS reverse phase silica gel packing material size 50～70um, YMCODS-A reverse phase silica gel packing material size 50～70um.

Further, preferred, described water and macroporous adsorbent resin volume ratio are 8～15:1; Described 90%～95% ethanol water and macroporous adsorbent resin volume ratio are 8～15:1; Described reverse phase silica gel chromatographic column column volume and gained macroporous resin position mass ratio are 20～30:1; The volume ratio of described 50% methanol aqueous solution and 70%～75% methanol aqueous solution and reverse phase silica gel chromatographic column is 5～8:1; Mass ratio 20～the 30:1 of described polyamide chromatographic column column volume and adsorption sample; The volume ratio of described 70%～75% ethanol water and 95%～100% ethanol and polyamide chromatographic column is 4～10:1.

Through efficient liquid phase chromatographic analysis, draw, Jaceosidin and eupafolin composition are contained in Herba Eupatorii Lindleyani flavone of the present invention position.

Advantage of the present invention is to provide a kind of new medicament sources for resisting acute lung injury.

Accompanying drawing explanation

Below in conjunction with drawings and Examples, the invention will be further described:

Fig. 1 is the comparison diagram of sample 1 and 2 Herba Eupatorii Lindleyani flavone position high-efficient liquid phase chromatograms in embodiment 1

1 (a) wherein, 1 (b) corresponds to respectively the chromatogram of sample 1, sample 2;

Fig. 2 is the high-efficient liquid phase chromatogram of embodiment 1 reference substance eupafolin;

Fig. 3 is the high-efficient liquid phase chromatogram of embodiment 1 reference substance Jaceosidin;

Fig. 4 is that the wet dry weight of embodiment 2 each experimental group lungs is than the contrast of W/D ratio;

Fig. 5 is nitric oxide (NO) content balance in embodiment 2 each experimental group bronchoalveolar lavage fluids;

Fig. 6 is the contrast of total protein content in embodiment 2 each experimental group bronchoalveolar lavage fluids;

Fig. 7 is the contrast of inflammatory factor TNF-alpha levels in embodiment 2 each experimental group bronchoalveolar lavage fluids;

Fig. 8 is the contrast of inflammatory factor IL-6 level in embodiment 2 each experimental group bronchoalveolar lavage fluids;

Fig. 9 is the contrast of inflammatory factor IL-1 β level in embodiment 2 each experimental group bronchoalveolar lavage fluids;

Figure 10 is the activity contrast of MPO in embodiment 2 each experimental group lung tissues;

Figure 11 is the activity contrast of SOD in embodiment 2 each experimental group lung tissues;

Figure 12 is C3 content balance in embodiment 2 each experimental group serum;

Figure 13 is C3c content balance in embodiment 2 each experimental group serum;

Wherein, in Fig. 4-13, corresponding Control is blank group, and EUP-FLA is matched group, and LPS is model group, and LPS+EUP-FLA group is the medicine group of various dose, positive group of LPS+DXM;

Figure 14 is the comparison diagram of the sectional drawing (X400) after embodiment 2 each experimental group lung tissue H & E dyeing;

Figure 15 is the comparison diagram of the sectional drawing (X400) of embodiment 2 each experimental group lung tissue C3c deposition SABC;

Figure 16 is that the wet dry weight of embodiment 3 each experimental group lungs is than the contrast of W/D ratio;

Figure 17 is the contrast of total protein content in embodiment 3 each experimental group bronchoalveolar lavage fluids;

Figure 18 is the contrast of inflammatory factor TNF-alpha levels in embodiment 3 each experimental group bronchoalveolar lavage fluids;

Figure 19 is the contrast of content of complement C 3 in embodiment 3 each experimental group serum;

Figure 20 is the contrast of complement fragment C3c content in embodiment 3 each experimental group serum;

Wherein, in Figure 16-20, corresponding Control is blank group, and LPS is model group, and EtOH is Herba Eupatorii Lindleyani ethanol extraction group, and FLA is Herba Eupatorii Lindleyani flavone position group, positive group of LPS+DXM;

The specific embodiment

Embodiment 1:

The preparation at Herba Eupatorii Lindleyani flavone position (EUP-FLA) and the detection of active component

(1) preparation of Herba Eupatorii Lindleyani flavone position (EUP-FLA)

Sample 1: take the dry herb of 300g Herba Eupatorii Lindleyani, cutting 2～3cm, the volume fraction that adds 10 times of volumes is 80% ethanol water, reflux, extract, 2 hours, repeat 2 times, 40 ℃ of decompression and solvent recoveries, obtain Herba Eupatorii Lindleyani ethanol extraction 45g, this ethanol extraction is dissolved in about 90ml water, through D101 macroporous adsorbent resin 500ml absorption, first with 5000ml water elution, remove macromolecular compound, rear is 95% ethanol water eluting by 5000ml volume fraction, decompression and solvent recovery, obtains 17g macroporous resin position; Adsorb through 350mlYMCODS-A reverse phase silica gel packing material size 60um reverse phase silica gel chromatographic column at gained macroporous resin position, first take 1750ml volume fraction as 50% methanol aqueous solution eluting, then 1750ml70% methanol aqueous solution eluting, 40 ℃ of decompression and solvent recoveries, obtain 6g extract, this extract adsorbs through 150ml30～60 order polyamide chromatographic column again, first take 600ml volume fraction as 70% ethanol water eluting, after use 600ml95% ethanol elution, 40 ℃ of decompression and solvent recoveries, obtain Herba Eupatorii Lindleyani flavone position (EUP-FLA) 0.8g.

Sample 2: take the dry herb of 1000g Herba Eupatorii Lindleyani, cutting 2～3cm, the volume fraction that adds 15 times of volumes is 80% ethanol water, reflux, extract, 2 hours, repeat 2 times, 60 ℃ of decompression and solvent recoveries, obtain Herba Eupatorii Lindleyani ethanol extraction 158g, be dissolved in about 150ml water, through 1800ml AB-8 absorption with macroporous adsorbent resin, first with 20000ml water elution, remove macromolecular compound, rear with 20000ml volume fraction 95% ethanol water eluting, 60 ℃ of decompression and solvent recoveries, obtain 61g macroporous resin position; Adsorb through 1500ml Cosmsil ODS reverse phase silica gel packing material size 70um reverse phase silica gel chromatographic column at gained macroporous resin position, take 10000ml volume fraction as 50% methanol aqueous solution eluting, then 10000ml70% methanol aqueous solution eluting, 60 ℃ of decompression and solvent recoveries, obtain 20g extract, this extract adsorbs through 600ml30～60 order polyamide chromatographic column again, first with 4000ml70% ethanol water eluting, after use 4000ml100% ethanol elution, 60 ℃ of decompression and solvent recoveries, obtain Herba Eupatorii Lindleyani flavone position (EUP-FLA) 2.8g.

(2) Herba Eupatorii Lindleyani flavone position (EUP-FLA) active component detects

The present embodiment adopts high efficiency liquid phase chromatographic analysis method to measure its composition.High performance liquid chromatograph (Agilent1260Infinity, U.S. Agilent company), its high-efficient liquid phase chromatogram condition is: mobile phase A is acetonitrile (containing 0.1% formic acid); Mobile phase B is water (containing 0.1% formic acid); Mobile phase condition: in Table 1; Chromatographic column: YMC-PACK ODS-A (Ф 4.6mm * 250mm, 5 μ m); Column temperature: 28 ℃; Detect wavelength: 365nm; Sample size: 20 μ l; Flow velocity: 1mlmin ^-1.

Table 1HPLC chromatographiccondition

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	36	64
15	36	64
			30	90	10

Precision takes reference substance eupafolin 4.67mg and Jaceosidin 8.17mg respectively; And be settled to 10ml by mobile phase A, as storing solution, add mobile phase A two-fold dilution and be made into 1:2,1:4,1:8,1:16 and 1:32, by 0.22 μ m membrane filtration, filtrate for later use.The reference substance of getting 5 concentration carries out the mensuration of chromatograph, and drawing standard curve, each concentration point parallel analysis 3 times.Record each chromatograph peak-to-peak area, with reference substance peak area (y), its concentration (x) is carried out to linear regression, obtain the standard curve of reference substance, eupafolin y=52964x+19.30, Jaceosidin y=55161x+210.79.

Precision takes Herba Eupatorii Lindleyani flavone position EUP-FLA sample 1, sample 2 each 6mg of preparation in embodiment 1, and is settled to 10ml volumetric flask by mobile phase A, 0.22 μ m membrane filtration, and filtrate for later use, gets respectively filtrate and carries out chromatographic determination.

As sample 1 and the 2 Herba Eupatorii Lindleyani flavone positions of Fig. 1-3 for obtaining; The high-efficient liquid phase chromatogram of reference substance eupafolin, reference substance Jaceosidin.

Through HPLC (high performance liquid chromatography), analysis draws in two groups of Herba Eupatorii Lindleyani flavone position samples and all contains eupafolin and Jaceosidin, by the chromatographic signal value substitution standard curve of sample 1 and 2, further draws the concrete content of each component, as shown in table 2.

The content of 2 kinds of flavone in table 2 Herba Eupatorii Lindleyani flavone position

Sample	Eupafolin (%)	Jaceosidin (%)	Toatl proportion (%)
				1	25.2	65.7	90.9
2	26.0	66.1	92.1

Embodiment 2:

The anti-chmice acute injury of lung experiment at Herba Eupatorii Lindleyani flavone position (EUP-FLA)

1, experimental drug and reagent: Herba Eupatorii Lindleyani flavone position (EUP-FLA); 0.5% sodium carboxymethyl cellulose; Lipopolysaccharide (LPS), U.S. Sigma-Aldrich company; Contrast medicine, dexamethasone acetate tablets, Zhejiang Province XianJu Pharmacy stock Co., Ltd; The anti-human C3c complement of multi-resistance rabbit, Shanghai Rui Qi Bioisystech Co., Ltd; Distilled water; Normal saline; 20% urethane-normal saline; 4% formalin, H-E dyeing liquor etc.

2, experiment mice: Balb/c mice, male, 20～24 grams of body weight, are provided by University Of Suzhou's Experimental Animal Center, laboratory animal production licence: XCYK (Soviet Union) 2002-0008.Experimental animal feeding is 24 ± 1 ℃ of temperature, under the environment of relative humidity 40%～80%, and free choice feeding and drinking-water.Before experiment, adaptability is raised 7 days.

3, other materials: mouse tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and il-1 β (IL-1 β) test kit, Shanghai boatman trade Co., Ltd; Nitric oxide (NO), BCA, superoxide dismutase (SOD) and myeloperoxidase (MPer) (MPO) test kit, Bioengineering Research Institute is built up in Nanjing; Mice Complement3 (C3) ELISA test kit, Suzhou Saden Bioisystech Co., Ltd, mice complement fragment C3c ELISA test kit, Shanghai Yuan Ye Bioisystech Co., Ltd; Knife blade, syringe, filter paper, refiner etc.

4, experimental technique:

Balb/c mice random packet is dosage group in the heavy dose of group of blank group, matched group, model group, sample, sample, sample small dose group and positive group, and 20 every group, in the heavy dose of group of sample, sample, dosage group, sample small dose group are referred to as medicine group.Blank group and model group give distilled water 7 days continuously, and dosage is 0.5ml/25g; Positive group gives distilled water 6 days continuously, and dosage is 0.5ml/25g; Matched group and medicine group give the medicine of Herba Eupatorii Lindleyani flavone position preparation according to dosage, gastric infusion is 7 days continuously, wherein, Herba Eupatorii Lindleyani flavone position (EUP-FLA) concrete dosage: matched group 40mg/kg, Herba Eupatorii Lindleyani flavone position (EUP-FLA) be little, in, heavy dose of group be respectively 10mg/kg, 20mg/kg, 40mg/kg.Positive group gave dexamethasone 5mg/kg at the 7th day; The 7th day medicine group and positive group of 2h after administration, model group gives 2h after distilled water, medicine group, positive group and model group, with the slight anesthetized mice of 20% urethane-normal saline (4ml/kg) lumbar injection, open cervical region, in trachea, inject LPS (2ml/kg, 1mg/ml LPS-normal saline), blank group and matched group are with method trachea saline injection (2ml/kg).After 6h, excessive anesthetized mice, collect the bronchoalveolar lavage fluid (BALF) of right lung for measuring nitric oxide (NO) and protein content, collect left lung for measuring wet/dry weight of lung than W/D ratio, collect mouse blood sample and be used for measuring serum C3 and C3c content.After 24h, excessive anesthetized mice, collects inferior lobe of right lung fixing in 4% formalin, and pathology to be done and immunohistochemical observation, collect superior lobe of right lung BALF, for measuring Inflammatory Factors Contents, collects left lung and makes and do tissue homogenate, active for measuring MPO and SOD.

The preparation method of above-mentioned bronchoalveolar lavage fluid (BALF) is: separated trachea, from cricoid cartilage below, with knife blade, make slant cutting edge, homemade tack pin is inserted to trachea, with stitching thread, fix separated cardiopulmonary, ligation left side bronchus, separated right lung, with syringe absorption 0.8ml normal saline, slowly inject right lung and slowly extract lavation back and forth out again, repeat 3 times, obtaining total amount is the bronchoalveolar lavage fluid BALF of 1.5ml.

The wet dry weight of lung than the assay method of W/D ratio is: 6h after modeling, and mice is put to death in excessive anesthesia, collects left lung, with filter paper, blots its surperficial tissue fluid and blood, weigh, weight is " weight in wet base ", is then placed in 80 ℃ of drying baker, after 48h, take out, weigh, weight is " dry weight ".With the ratio of lung " weight in wet base " and " dry weight ", being the wet dry weight of lung is used for the situation of assess pulmonary edema than W/D size.

The assay method of nitric oxide in bronchoalveolar lavage fluid (NO) and total protein content is: 6h after modeling, mice is put to death in excessive anesthesia, collect BALF, centrifugal (4 ℃, 1400 * g, 10min), draw supernatant, according to nitric oxide NO and total protein content BCA test kit explanation requirement, measure nitric oxide (NO) and total protein content.

In bronchoalveolar lavage fluid, the assay method of Inflammatory Factors Contents level is: 24h after modeling, mice is put to death in excessive anesthesia, collect BALF, centrifugal (4 ℃, 1400 * g, 10min), draw supernatant, according to ELISA test kit explanation requirement, measure inflammatory factor TNF-α, IL-6 and IL-1 β contents level.

Being determined as of MPO and SOD activity in lung tissue: 24h after modeling, mice is put to death in excessive anesthesia, collects left lung, by homogenizer, makes 10% tissue homogenate, according to MPO test kit explanation requirement, measures MPO active; By 10% tissue homogenate centrifugal (4 ℃, 1400 * g, 20min), draw supernatant, according to SOD test kit explanation requirement, measure SOD active.

Being determined as of complement C3 and complement fragment C3c content in serum: 6h after modeling, mice is put to death in excessive anesthesia, the mouse blood sample of collecting, the lower standing 10min of room temperature (25 ℃), centrifugal (4 ℃, 1400 * g, 20min), draw serum, according to ELISA test kit explanation requirement, measure C3 and C3c content.

Lung tissue's section pathological observation: 24h after modeling, mice is put to death in excessive anesthesia, collects inferior lobe of right lung, is placed in 4% formalin and fixes 3 days, and conventional method paraffin is fixed, section, H-E dyeing; Adopt horseradish peroxidase (horseradishperoxidase, HRP) system to detect structural complement C3c deposition.5 μ m pulmonary paraffin sections dewax in dimethylbenzene, ethanol gradient aquation, with the anti-human complement C3c of rabbit night incubation at 4 ℃, and then 3,3'-diaminobenzidine (DAB) colour developing, microscope amplifies 400 times of observations and takes pictures.

5, experimental result

All average ± standard deviation (mean ± S.D.) expressions for data, application SPSS19 software carries out one factor analysis of variance to data, relatively adopts Fisher ' s PLSD method between group, when P<0.05 thinks, has significant difference.

After experimental data adopts said method to process, between each experimental group, the index of evaluation acute lung injury contrasts, as shown in Fig. 4-15.

From Fig. 4-6, can find out, with respect to blank group, in the W/D anharmonic ratio of model group, bronchoalveolar lavage fluid (BALF), the content of nitric oxide (NO) and protein all has remarkable rising, and its difference has statistical significance (P<0.01).Comparative control group (40mg/kg) with blank group, find that Herba Eupatorii Lindleyani flavone position (EUP-FLA) does not make significant difference at this three aspects: to normal mouse.EUP-FLA under 20mg/kg and 40mg/kg dosage to pulmonary edema, BALF in the content of NO and protein have the effect (P<0.01) of significant inhibition and minimizing.Wherein ## represents compared to blank group, P<0.01; * represents compared to model group, P<0.01; * represent compared to model group P<0.05

From Fig. 7-9, can find out, with respect to blank group, in model group bronchoalveolar lavage fluid (BALF), BTNF-α, IL-6 and IL-1 β level all have remarkable rising, and its difference has statistical significance (P<0.01).Comparative control group (40mgkg ^-1) with blank group, find that EUP-FLA does not make significant difference aspect two at this to normal mouse.EUP-FLA is at 40mgkg ^-1under dosage, inflammatory factor TNF-α, IL-6 in BALF and IL-1 β level are had to significant inhibition (P<0.01).Wherein ## represents compared to blank group, P<0.01; * represents compared to model group, P<0.01; * represent compared to model group P<0.05

From Figure 10-11, can find out, with respect to blank group, the MPO in model group lung tissue has remarkable rising, and SOD has significant reduction, and its difference has statistical significance (P<0.01).Comparative control group (40mgkg ^-1) and blank group, find that EUP-FLA does not make significant difference in these areas to normal mouse.EUP-FLA is at 40mgkg ^-1under dosage, MPO in lung tissue is had to significant inhibition (P<0.01), EUP-FLA under 20mgkg-1 dosage to lung tissue in the SOD effect (P<0.01) that increases significantly.Wherein ## represents compared to blank group, P<0.01; * represents compared to model group, P<0.01; * represent compared to model group P<0.05.

From Figure 12-13, can find out, with respect to blank group, in model group serum, C3 and C3c level all have remarkable rising, and its difference has statistical significance (P<0.01).Comparative control group (40mgkg-1) with blank group, find that EUP-FLA does not make significant difference in these areas to normal mouse.EUP-FLA is at 20mgkg ^-1and 40mgkg ^-1under dosage, complement C3 in serum and complement fragment C3c level are all had to significant inhibition (P<0.01).Wherein ## represents compared to blank group, P<0.01; * represents compared to model group, P<0.01; * represent compared to model group P<0.05.

From Figure 14-15, can find out, with respect to blank group, the lung tissue of model group is imperfect, cell becomes greatly, breaks, and capillary tube inner wall thickens, lung cilium hollow deformation, cell interior is flooded with medium, with brown complement precipitation speckle (seeing brown speckle in Figure 15).Yet the situation of medicine group makes moderate progress, and EUP-FLA is at 20mgkg ^-1and 40mgkg ^-1pulmonary condition and matched group (40mgkg under dosage ^-1) and blank group difference very little.Wherein ## represents compared to blank group, P<0.01; * represents compared to model group, P<0.01; * represent compared to model group P<0.05.

Above-mentioned experimental result shows, in the acute lung injury experimental model of lipopolysaccharide LPS induction, Herba Eupatorii Lindleyani flavone position (EUP-FLA) has and has significantly alleviated mouse lung edema symptom, reduce the content of the middle nitric oxide (NO) of mice bronchoalveolar lavage fluid (BALF) and protein, strengthened the activity of superoxide dismutase (SOD) in mouse lung tissue, the activity that suppresses myeloperoxidase (MPer) (MPO), reduced tumor necrosis factor-alpha (TNF-α), the content of interleukin-6 (IL-6) and il-1 β (IL-1 β), reduce the content of complement C3 and complement fragment C3c in serum and improve complement fragment C3c in the deposition of lung tissue.

Embodiment 3:

Herba Eupatorii Lindleyani flavone position and the experiment of Herba Eupatorii Lindleyani ethanol extract resisting acute lung injury Contrast on effect

The preparation of Herba Eupatorii Lindleyani ethanol extraction: take the dry herb of Herba Eupatorii Lindleyani, cutting 2～3cm, adds 80% ethanol water of 10 times of volumes, and reflux, extract, 2 hours, repeats 2 times, and decompression and solvent recovery, obtains Herba Eupatorii Lindleyani ethanol extraction (EtOH).

This contrast experiment, experiment reagent, experiment mice and experiment material are with embodiment 2.

Experiment mice is divided into totally 5 groups of blank group, model group, Herba Eupatorii Lindleyani ethanol extraction EtOH group, Herba Eupatorii Lindleyani flavone position EUP-FLA group and positive groups, 10 every group at random.Herba Eupatorii Lindleyani ethanol extraction EtOH group administration EtOH, dosage is 30mgkg ^-1; Herba Eupatorii Lindleyani flavone position EUP-FLA group gives EUP-FLA, and dosage is 30mgkg ^-1; Positive group gives dexamethasone, dosage 2mgkg ^-1,

All experiment mices are first used 20% urethane-normal saline (4mlkg ^-1) the slight anesthetized mice of lumbar injection, open cervical region, except blank group, all the other 5 groups are injected LPS (2mlkg in mice trachea ^-1, 1mgml ^-1lPS-normal saline); And blank group with method trachea saline injection 2mlkg ^-1.After modeling 0.5h, carry out administration for the first time respectively by above-mentioned dosage, blank group gives equal-volume distilled water; After modeling 1h, EtOH group and EUP-FLA group are carried out administration for the second time by above-mentioned dosage, and positive group, blank group and model group give equal-volume distilled water.After modeling 6h, excessive anesthetized mice, eyeball is got blood, and every about 1ml blood sample of mice, with the bronchoalveolar lavage fluid (BALF) of embodiment 2 methods collection right lungs, is collected left lung.

Data Processing in Experiment is with embodiment 2, and its result is as shown in Figure 16-20.

As can be seen from Figure 16, with respect to blank group, the W/D anharmonic ratio of model group has remarkable rising, and its difference has statistical significance (P<0.01); Compare the pulmonary edema situation of Herba Eupatorii Lindleyani EtOH, EUP-FLA group and positive group be all significantly improved (P<0.05) with model group; Compared to Herba Eupatorii Lindleyani EtOH group, the pulmonary edema of EUP-FLA group mice is significantly suppressed (P<0.01).Herba Eupatorii Lindleyani flavone position (EUP-FLA) has the effect of better inhibition mouse lung edema than Herba Eupatorii Lindleyani alcohol extract (EtOH).Wherein ## represents compared to blank group, P<0.01; * represents the group compared to EtOH, P<0.01; Not and model group comparison, P value is not all less than 0.05 and has significant difference, in figure, does not indicate for EtOH, FLA and positive component.

From Figure 17-18, can find out, as shown in Figure 2, with respect to blank group, in model group mice BALF, total protein and TNF-alpha content all have remarkable rising, and its difference has statistical significance (P<0.01); Compare the total protein of Herba Eupatorii Lindleyani EtOH, FLA group and positive group and TNF-alpha content be all significantly improved (P<0.05) with model group; Compared to Herba Eupatorii Lindleyani EtOH group, FLA group significantly suppresses total protein content (P<0.01) in BALF; Compared to Herba Eupatorii Lindleyani EtOH group, FLA group significantly suppresses TNF-alpha content (P<0.01) in BALF.Herba Eupatorii Lindleyani flavone position (FLA) can better suppress total protein and TNF-alpha content in mice BALF than Herba Eupatorii Lindleyani alcohol extract (EtOH).Wherein ## represents compared to blank group, P<0.01; * represents the group compared to EtOH, P<0.01; Not and model group comparison, P value is not all less than 0.05 and has significant difference, in figure, does not indicate for EtOH, FLA and positive component.

From Figure 19-20, can find out, with respect to blank group, in model group mice serum, C3 and C3c content all have remarkable rising, and its difference has statistical significance (P<0.01); Compare the C3 of Herba Eupatorii Lindleyani EtOH, FLA group and positive group and C3c content be all significantly improved (P<0.05) with model group; Compared to Herba Eupatorii Lindleyani EtOH group, C3 and the C3c content of FLA group are significantly suppressed (P<0.05).Herba Eupatorii Lindleyani flavone position (FLA) can better reduce C3 and C3c content in mice serum than Herba Eupatorii Lindleyani alcohol extract (EtOH).Wherein ## represents compared to blank group, P<0.01; * represents the group compared to EtOH, P<0.01; Not and model group comparison, P value is not all less than 0.05 and has significant difference, in figure, does not indicate for EtOH, FLA and positive component.

From the present embodiment result, can find out, Herba Eupatorii Lindleyani flavone position EUP-FLA has better effect in resisting acute lung injury than Herba Eupatorii Lindleyani ethanol extraction.

By above-described embodiment, obtain, Herba Eupatorii Lindleyani flavone position all has protective effect to the lung tissue of mice, pneumonocyte, blood capillary.Herba Eupatorii Lindleyani flavone position (EUP-FLA) is by improving body radical scavenging activity, the release of inflammation-inhibiting medium and the factor, reduce level of complement, improve chmice acute injury of lung, and Herba Eupatorii Lindleyani flavone position EUP-FLA has better drug effect than Herba Eupatorii Lindleyani ethanol extraction.Herba Eupatorii Lindleyani flavone position can be applied and be prepared resisting acute lung injury medicine.

Claims (6)

1. the Herba Eupatorii Lindleyani flavone position application in preparing resisting acute lung injury medicine.

2. application according to claim 1, is characterized in that, described Herba Eupatorii Lindleyani flavone position refers to the Herba Eupatorii Lindleyani flavone position containing Jaceosidin and eupafolin.

3. application according to claim 1, it is characterized in that, the preparation method at described Herba Eupatorii Lindleyani flavone position is: get the dry herb of Herba Eupatorii Lindleyani, cutting 2～3cm, the volume fraction that adds 8～15 times of volumes is 60%～95% ethanol water, and reflux, extract, 1～3 hour repeats 1～3 time, 40 ℃～60 ℃ decompression and solvent recoveries, obtain Herba Eupatorii Lindleyani ethanol extraction; This extract is soluble in water, be prepared into the solution that concentration is 0.5～1g/ml, through low pole absorption with macroporous adsorbent resin, first with water elution, remove macromolecular compound, then by volume fraction, be 90%～95% ethanol water eluting, 40 ℃～60 ℃ decompression and solvent recoveries obtain macroporous resin position; Adsorb through ODS reverse phase silica gel chromatographic column at gained macroporous resin position, first take volume fraction as 50% methanol aqueous solution eluting, then use 70%～75% methanol aqueous solution eluting, after 40 ℃～60 ℃ decompression and solvent recoveries, through 30～60 order polyamide chromatographic column absorption, first take volume fraction as 70%～75% ethanol water eluting, rear use 95%～100% ethanol elution again, 40 ℃～60 ℃ decompression and solvent recoveries, obtain Herba Eupatorii Lindleyani flavone position.

4. application according to claim 3, is characterized in that, described low pole macroporous adsorbent resin is a kind of in AB-8, D101, HPD-100, HPD-200A, HPD-200B, HPD-300, HPD-700, HPD-722 macroporous adsorbent resin.

5. application according to claim 3, it is characterized in that, described ODS reverse phase silica gel chromatographic column is a kind of in DAISOGEL ODS reverse phase silica gel packing material size 50～70um, Cosmsil ODS reverse phase silica gel packing material size 50～70um, YMCODS-A reverse phase silica gel packing material size 50～70um.

6. application according to claim 3, is characterized in that, described water and low pole macroporous adsorbent resin volume ratio are 8～15:1; Described 90%～95% ethanol water and macroporous adsorbent resin volume ratio are 8～15:1; Described ODS reverse phase silica gel chromatographic column column volume and gained macroporous resin position mass ratio are 20～30:1; The volume ratio of described 50% methanol aqueous solution and 70%～75% methanol aqueous solution and ODS reverse phase silica gel chromatographic column is 5～8:1; The mass ratio of described polyamide chromatographic column column volume and adsorption sample is 20～30:1; Described 70%～75% ethanol water and 95%～100% ethanol and polyamide chromatographic column volume ratio are 4～10:1.