CN104161783B - Application of the eupatorium lindleynun var. trifoliolatum sequiterpene position in resisting acute lung injury medicine is prepared - Google Patents

Application of the eupatorium lindleynun var. trifoliolatum sequiterpene position in resisting acute lung injury medicine is prepared Download PDF

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CN104161783B
CN104161783B CN201410239824.4A CN201410239824A CN104161783B CN 104161783 B CN104161783 B CN 104161783B CN 201410239824 A CN201410239824 A CN 201410239824A CN 104161783 B CN104161783 B CN 104161783B
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trifoliolatum
eupalinolide
sequiterpene
lindleynun var
eupatorium lindleynun
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CN104161783A (en
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张健
褚纯隽
任慧玲
陈道峰
严彪
李显伦
吴天威
徐乃玉
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Suzhou University
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Abstract

The invention discloses a kind of application of eupatorium lindleynun var. trifoliolatum sequiterpene position in resisting acute lung injury medicine is prepared.Eupatorium lindleynun var. trifoliolatum sequiterpene position is prepared into animal-use drug by the present invention, take the mode to mouse vivo medicine-feeding, using lipopolysaccharides LPS inducing mouse ALIs, afterwards determine mice with acute lung injury lung it is wet/dry weight ratio, nitric oxide (NO) and protein content in bronchoalveolar lavage fluid (BALF);MPer (MPO) and superoxide dismutase (SOD) activity in detection lung homogenate;Tumor necrosis factor α (TN α), interleukin 6 (IL 6) and interleukin-11 β (β of IL 1) content in bronchoalveolar lavage fluid;Complement C_3 and C3c contents in serum;Mouse lung tissue pathological change after observation H&E dyeing.Result shows that the eupatorium lindleynun var. trifoliolatum sequiterpene position containing Eupalinolide F, Eupalinolide G, Eupalinolide H, Eupalinolide I and Eupalinolide K has the activity of resisting acute lung injury.

Description

Application of the eupatorium lindleynun var. trifoliolatum sequiterpene position in resisting acute lung injury medicine is prepared
Technical field
The present invention relates to a kind of application of eupatorium lindleynun var. trifoliolatum sequiterpene position in resisting acute lung injury medicine is prepared.
Background technology
ALI (acute lung injury, ALI) is that based on diffusivity lung cellular damage, Pulmonary Vascular is damaged Wound caused by pulmonary edema and lung tissue inflammatory cell infiltration be its pathological characters, clinic be mainly shown as serious hypoxemia, Diffusivity lung infiltrates and pulmonary edema;Some patientss will eventually form ARDS (acute respiratory Distress syndrome, ARDS), the irreversible acute respiratory failure of body and multiple organ dysfunction are caused, die of illness Rate is up to 30-40%;ALI pathogenesis is intricate, and ALI risk factors can be the coup injury from lung, also may be used With the indirect injury that to be lung other factor produced by systemic inflammatory response to lung.(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) cause of disease is removed;(2) on the basis of Cure the Primary Disease, Mechanical ventilation (Mechanical ventilation, MV) organizes oxygen supply to correct anoxic and improve as early as possible;MV is that ALI classics are controlled One for the treatment of mode, can correct intractable anoxic, prevent and treat alveolar collapse, resist pulmonary edema, improve gas exchanges, and reduction is independently exhaled Work done is inhaled, respiratory muscle fatigue is prevented;But it is only capable of maintaining organism physiology breathing, can not be still fully solved ALI whole issues;(3) medicine Thing is treated;Clinically widely use the treatment such as Adrenal Glucocorticoid, glucocorticoid, endoxan, MTX acute Injury of lungs, can reduce the formation that capillary oozes out and suppress pulmonary fibrosis, PFT is quickly improved;But sugared cortex The immunodepressant such as hormone to ALI poor selectivity, prolonged application can produce multiple complications and side effect (referring to:Poplar Great waves, Wang Zhengqing forever.Complement inhibitor progress Chinese Journal of New Drugs, 2008,17 (24):2093;Xu Han, Zhang Yunyi, Zhang Jian Text, waits the ACA composition China natural drug in natural products, 2007,5 (5):322.).Therefore, selectivity is found The small treatment ALI medicine of high, toxic and side effect has important clinical meaning.
Eupatorium lindleynun var. trifoliolatum is the herb of feverfew point eupatorium Eupatorium lindleyanum DC., Jiangsu genunie medicinal materials. Bitter, mild-natured, Return liver, the spleen channel, with preventing phlegm from forming and stopping coughing, clearing heat and detoxicating, inducing diuresis to remove edema, the function of step-down, for catching a cold, coughing many 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 goes out Version society, 1999:7,6876).
The research such as Jiang Zhou, Yang Hui shows that eupatorium lindleynun var. trifoliolatum extract solution has protective effect, energy to acute lung injury induced by oleic acid rat Emigrated cell number, protein content, paragonimus cyst and the moisture content of lung and Pulmonary Vascular for substantially reducing acute lung injury induced by oleic acid rat are logical Permeability (referring to:Jiang Zhou, Yang Hui, He Haixia, wait the protective effect of Eupatorium Lindleyanum on Acute Lung Injury in Rats to study China Dispensaries, 2007,18(27):2094;Yang Hui, Zhou Yuan great, what extra large rosy clouds eupatorium lindleynun var. trifoliolatum is to acute acidity of oil induced lung injury in rats pulmonary vascular permeability Influence China medicine company, 2010,19 (9):5.).But the studies above is that the eupatorium lindleynun var. trifoliolatum directly provided using drugmaker is extracted Liquid, eupatorium lindleynun var. trifoliolatum is indefinite to the active component of ALI protective effect, and mechanism of action liquid is unclear.eupalinolide F (Eupalinolide F), eupalinolide G (Eupalinolide G), eupalinolide H (Eupalinolide H), Eupalinolide I (Eupalinolide I), eupalinolide K (Eupalinolide K) are sesquiterpenoids, It is strong wait it is isolated from eupatorium lindleynun var. trifoliolatum (referring to:Shuang-Qing Wu,Nai-Yu Xu,Qun Sun,Hai-Yan Han,and Jian Zhang*.Six New Sesquiterpenes from Eupatorium lindleyanum.Helvetica Chimica Acta,2012,95:1637).Whether make with resisting acute lung injury on this five sesquiterpenoids at present With having no report.Present invention discover that the sequiterpene position of its composition has the activity of resisting acute lung injury.
The content of the invention
The present invention seeks to:A kind of eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) is provided and is preparing resisting acute lung injury medicine In application.
The technical scheme is that:Eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) is configured to animal-use drug, by small The mode of mouse vivo medicine-feeding, determine inducing acute injury of lungs mouse lung it is wet/dry weight ratio, in bronchoalveolar lavage fluid (BALF) one oxidation Nitrogen (NO) and protein content;MPer (MPO) and superoxide dismutase (SOD) activity in detection lung homogenate; Tumor necrosis factor-alpha (TN- α), interleukin-6 (IL-6) and Interleukin -1β (IL-1 β) content in bronchoalveolar lavage fluid;In serum Complement C_3 and C3c contents;Mouse lung tissue pathological change after observation H&E dyeing and DAB develop the color respectively.Test result indicate that, it is wild Horse chases after sequiterpene position can mitigate mouse lung edematous condition, reduce nitric oxide (NO) and protein in bronchoalveolar lavage fluid Content, significantly enhances the activity of superoxide dismutase in mouse lung tissue (SOD), suppresses MPer (MPO) Activity, reduces the content of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and Interleukin -1β (IL-1 β), reduces The content of Complement C_3 and complement fragment C3c in serum, and improve depositions of the complement fragment C3c in lung tissue, to the lung group of mouse Knit, pneumonocyte, capillary are respectively provided with protective effect.Experimental result determines that there is resisting acute lung to damage at eupatorium lindleynun var. trifoliolatum sequiterpene position Wound is acted on.
The preparation method at eupatorium lindleynun var. trifoliolatum sequiterpene position is in the present invention:Take eupatorium lindleynun var. trifoliolatum and dry herb, 2~3cm of segment plus 8 ~15 times of volume fractions of volume are 60%~95% ethanol water, refluxing extraction 1~3 hour, repeat 1~3 time, 40 DEG C~ 60 DEG C are recovered under reduced pressure solvent, obtain eupatorium lindleynun var. trifoliolatum ethanol extract;The extract is soluble in water, concentration is prepared into for 0.5~1g/ml Solution, through low pole macroporous absorbent resin adsorb, first with water elution remove macromolecular compound, be then with volume fraction 90%~95% ethanol water is eluted, and 40 DEG C~60 DEG C are recovered under reduced pressure solvent, obtain macroreticular resin position;Gained macroreticular resin portion Position is adsorbed through ODS reverse phase silica gels chromatographic column, is eluted by 50% methanol aqueous solution of volume fraction, obtains eupatorium lindleynun var. trifoliolatum sequiterpene position.
Further, the low pole macroporous absorbent resin be AB-8, D101, HPD-100, HPD-200A, HPD-200B, One kind in HPD-300, HPD-700, HPD-722 macroporous absorbent resin.
Further, the ODS reverse phase silica gels chromatographic column be 50~70um of DAISOGEL ODS reverse phase silica gel packing material sizes, One kind in 50~70um of Cosmsil ODS reverse phase silica gel packing material sizes, 50~70um of YMCODS-A reverse phase silica gel packing material sizes.
Further, the water and low pole macroporous absorbent resin volume ratio are 8~15:1;90%~95% ethanol The aqueous solution is 8~15 with macroporous absorbent resin volume ratio:1;The ODS reverse phase silica gels chromatographic column column volume and gained macroreticular resin Position mass ratio is 20~30:1;50% methanol aqueous solution is 5~8 with the volume ratio of ODS reverse phase silica gel chromatographic columns:1.
Obtained using high efficiency liquid phase chromatographic analysis method, following composition is contained at eupatorium lindleynun var. trifoliolatum sequiterpene position in the present invention: Eupalinolide F (Eupalinolide F), eupalinolide G (Eupalinolide G), eupalinolide H (eupatorium lindleynun var. trifoliolatums Lactone H), eupalinolide I (Eupalinolide I) and eupalinolide K (Eupalinolide K), its chemical structural formula For:
It is an advantage of the invention that for resisting acute lung injury provides a kind of new medicament sources.
Brief description of the drawings
Below in conjunction with the accompanying drawings and embodiment the invention will be further described:
Fig. 1 is the high-efficient liquid phase chromatogram at the eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) of sample 1 and 2 in embodiment 1
Wherein 1 (a), 1 (b) difference counter sample 1 and sample 2;
Fig. 2 is the high-efficient liquid phase chromatogram of reference substance eupalinolide F (Eupalinolide F) in embodiment 1;
Fig. 3 is the high-efficient liquid phase chromatogram of reference substance eupalinolide G (Eupalinolide G) in embodiment 1;
Fig. 4 is the high-efficient liquid phase chromatogram of reference substance eupalinolide H (Eupalinolide H) in embodiment 1;
Fig. 5 is the high-efficient liquid phase chromatogram of reference substance eupalinolide I (Eupalinolide I) in embodiment 1;
Fig. 6 is the high-efficient liquid phase chromatogram of reference substance eupalinolide K (Eupalinolide K) in embodiment 1;
Fig. 7 is each contrast of the wet dry weight of experimental group lung than W/D ratio of embodiment 2;
Fig. 8 is nitric oxide (NO) content balance in each experimental group bronchoalveolar lavage fluid of embodiment 2;
Fig. 9 is the contrast of total protein content in each experimental group bronchoalveolar lavage fluid of embodiment 2;
Figure 10 is the contrast of inflammatory factor TNF-α level in each experimental group bronchoalveolar lavage fluid of embodiment 2;
Figure 11 is the contrast of inflammatory factor IL-6 levels in each experimental group bronchoalveolar lavage fluid of embodiment 2;
Figure 12 is the contrast of inflammatory factor IL-1 β levels in each experimental group bronchoalveolar lavage fluid of embodiment 2;
Figure 13 is the activity contrast of MPO in each experimental group lung tissue of embodiment 2;
Figure 14 is the activity contrast of SOD in each experimental group lung tissue of embodiment 2;
Figure 15 is the contrast of content of complement C 3 in each experimental group serum of embodiment 2;
Figure 16 is the contrast of complement fragment C3c contents in each experimental group serum of embodiment 2;
In wherein Fig. 7-16, corresponding Control is blank group, and EUP-SQT is control group, and LPS is model group, LPS+ EUP-SQT groups are the medicine group of various dose, and LPS+DXM is positive group;
Figure 17 is the comparison diagram of the sectional drawing (X400) after each experimental group lung tissue H&E dyeing of embodiment 2;
Figure 18 is the comparison diagram of the sectional drawing (X400) of each experimental group lung tissue C3c depositions SABC of embodiment 2;
Figure 19 is each contrast of the wet dry weight of experimental group lung than W/D ratio of embodiment 3;
Figure 20 is the contrast of total protein content in each experimental group bronchoalveolar lavage fluid of embodiment 3;
Figure 21 is the contrast of inflammatory factor TNF-α level in each experimental group bronchoalveolar lavage fluid of embodiment 3;
Figure 22 is the contrast of content of complement C 3 in each experimental group serum of embodiment 3;
Figure 23 is the contrast of complement fragment C3c contents in each experimental group serum of embodiment 3;
In wherein Figure 19-23, corresponding Control is blank group, and LPS is model group, and EtOH is extracted for eupatorium lindleynun var. trifoliolatum ethanol Thing group, SQT is eupatorium lindleynun var. trifoliolatum sequiterpene position group, and LPS+DXM is positive group;
Specific embodiment
Embodiment 1:
The preparation at eupatorium lindleynun var. trifoliolatum sequiterpene position and the detection of active component
(1) preparation at eupatorium lindleynun var. trifoliolatum sequiterpene position:
Sample 1:Weigh 300g eupatorium lindleynun var. trifoliolatums and dry herb, 2~3cm of segment, plus 10 times of volume fractions of volume are 80% second Alcohol solution, refluxing extraction 2 hours, is repeated 2 times, and 40 DEG C are recovered under reduced pressure solvent, obtains eupatorium lindleynun var. trifoliolatum ethanol extract 45g;By the second Alcohol extracting thing is dissolved in about 90ml water, is adsorbed through D101 macroporous absorbent resins 500ml, first removes macromolecular with 5000ml water elutions Compound, is recovered under reduced pressure solvent with 5000ml volume fractions for 95% ethanol water is eluted afterwards, obtains 17g macroreticular resins position; Gained macroreticular resin position is adsorbed through 350mlYMCODS-A reverse phase silica gel packing material size 60um reverse phase silica gels chromatographic column, with 1750ml volume fractions are eluted for 50% methanol aqueous solution, and 40 DEG C are recovered under reduced pressure, and obtain eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) 5.6g。
Sample 2:Weigh 1000g eupatorium lindleynun var. trifoliolatums and dry herb, 2~3cm of segment, plus 15 times of volume fractions of volume are 80% second Alcohol solution, refluxing extraction 2 hours, is repeated 2 times, and 60 DEG C are recovered under reduced pressure solvent, obtains eupatorium lindleynun var. trifoliolatum ethanol extract 158g, is dissolved in about In 150ml water, adsorbed through 1800ml AB-8 macroporous absorbent resins, macromolecular compound is first removed with 20000ml water elutions, after Eluted with the ethanol water of 20000ml volume fractions 95%, 60 DEG C are recovered under reduced pressure solvent, obtain 61g macroreticular resins position;Gained is big Hole resin position is adsorbed through 1500ml Cosmsil ODS reverse phase silica gel packing material size 70um reverse phase silica gels chromatographic column, with 10000ml volume fractions are eluted for 50% methanol aqueous solution, and 60 DEG C are recovered under reduced pressure to obtain eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) 21g。
(2) eupatorium lindleynun var. trifoliolatum sequiterpene position active component detection
Its composition is determined using high efficiency liquid phase chromatographic analysis method in the present embodiment.High performance liquid chromatograph (Agilent1260Infinity, Agilent company of the U.S.), wherein mobile phase A are acetonitrile;Mobile phase B is water;Mobile phase condition It is shown in Table 1.Chromatographic column:YMC-PACK ODS-A(Ф4.6mm×250mm,5μm);Column temperature:28℃;Detection wavelength:210nm;Sample introduction Amount:20μl;Flow velocity:1ml·min-1
Table 1HPLC chromatographicconditions
Time (min) Mobile phase A (%) Mobile phase B (%)
0 30 70
5 30 70
15 32 68
25 36 64
40 36 64
Precision weighs reference substance eupalinolide F (Eupalinolide F) 6.02mg, eupalinolide G (open countries respectively Horse chases after lactone G) 14.94mg, eupalinolide H (Eupalinolide H) 5.08mg, eupalinolide I (Eupalinolides I) 10.04mg, eupalinolide K (Eupalinolide K) 13.98mg, 10ml is settled to mobile phase A, used as storing solution. Every group of reference substance adds mobile phase A two-fold dilution to be made into 1:2、1:4、1:8、1:16 and 1:32,0.22 μm of membrane filtrations, filtrate is standby With.Taking 5 solution of concentration of each group reference substance respectively carries out the measure of chromatogram, and draws standard curve, and each concentration point is parallel Analysis 3 times.Each chromatographic peak peak area is recorded, linear regression is carried out to its concentration (x) with reference substance peak area (y), compareed The standard curve of product, eupalinolide F, y=63403x-15.408;Eupalinolide G, y=24720x-20.065; Eupalinolide H, y=79063x+6.7811;Eupalinolide I, y=239018x+53.303;eupalinolide K, y=7913.5x+62.857.
Precision weighs the eupatorium lindleynun var. trifoliolatum sequiterpene position EUP-SQT sample 1 and each 6mg of sample 2 prepared in embodiment 1, is used in combination Mobile phase A is settled to 10ml volumetric flasks, and 0.22 μm of membrane filtration, filtrate is standby, and filtrate is taken respectively carries out chromatographic determination.
Eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT), the reference substance eupalinolide of sample 1 and 2 are respectively such as Fig. 1-6 F, eupalinolide G, eupalinolide H, eupalinolide I and eupalinolide K high performance liquid chromatography Figure.
By the analysis to high performance liquid chromatography result, it can be deduced that three groups of eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) samples In product containing eupalinolide F (Eupalinolide F), eupalinolide G (Eupalinolide G), Eupalinolide H (Eupalinolide H), eupalinolide I (Eupalinolide I) and eupalinolide K (wild horses Chase after lactone K) composition, the chromatographic signal value of sample 1 and 2 is substituted into standard curve, the concrete content of each component is from which further followed that, such as Shown in table 2.
5 kinds of Eupalinolide contents in 2 two groups of eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) samples of table
Sample eupalinolide F eupalinolide G eupalinolide H eupalinolide I eupalinolide K Ratio
1 3.8% 31.4% 6.6% 3.3% 46.3% 91.4%
2 4.1% 31.8% 6.8% 3.4% 47.7% 93.8%
Embodiment 2:
(EUP-SQT) the anti-mouse ALI experiment of eupatorium lindleynun var. trifoliolatum sequiterpene position
1st, experimental drug and reagent:0.5% sodium carboxymethylcellulose;Lipopolysaccharides (LPS), U.S. Sigma-Aldrich is public Department;Dexamethasone acetate tablets, Zhejiang Province XianJu Pharmacy stock Co., Ltd;Many anti-human C3c complements of anti-rabbit, the auspicious neat biotechnology in Shanghai Co., Ltd;Distilled water;Physiological saline;20% urethane-physiological saline;4% formalin, H-E dyeing liquors, 3,3'- diaminos Base benzidine (DAB) nitrite ion etc..
2nd, experiment mice:Balb/c mouse, male, 24~28 grams of body weight is provided by University Of Suzhou's Experimental Animal Center, real Test animal productiong licensing:XCYK (Soviet Union) 2002-0008.Experimental animal feeding in 24 ± 1 DEG C of temperature, relative humidity 40%~ In the environment of 80%, free choice feeding and drinking-water.Before experiment, adaptability is raised 7 days.
3rd, other materials:Murine tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), Interleukin -1β (IL-1 β), Shanghai boatman trade Co., Ltd;Mouse Complement3 (C3) ELISA kit, Suzhou Saden Bioisystech Co., Ltd, Mouse complement fragment C3c ELISA kits, Shanghai Yuan Ye Bioisystech Co., Ltd;Nitric oxide (NO), BCA, super oxygen Thing mutase (SOD) and MPer (MPO) kit, Bioengineering Research Institute is built up in Nanjing;Knife blade, syringe, Filter paper, refiner etc..
4th, experimental technique
Balb/c mouse are grouped into blank group, control group, model group, sample heavy dose group, sample middle dose group, sample at random Product small dose group and the positive organize totally 7 experimental groups, every group 20.Sample heavy dose group, sample middle dose group, sample small dose group It is referred to as medicine group.Blank group, model group continuously give distilled water 7 days, and dosage is 0.5ml/25g;Continuous the giving of positive group is steamed Distilled water 6 days, dosage is 0.5ml/25g;Control group and medicine group give eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) and match somebody with somebody according to dosage The medicine of system, continuous gavage is administered 7 days.Wherein control group, eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) it is small, in, the agent of heavy dose of group Amount is respectively 60mg/kg, 15mg/kg, 30mg/kg and 60mg/kg.Positive group gave dexamethasone 5mg/kg at the 7th day;7th Its medicine group and the positive organize 2h upon administration, and model group gives 2h after distilled water, medicine group, positive group and model group, with 20% Urethane-physiological saline (4ml/kg) intraperitoneal injection light anaesthesia mouse, opens neck, toward tracheal strips injection lipopolysaccharides LPS (2ml/kg, 1mg/ml LPS- physiological saline), blank group and control group are with method tracheae saline injection (2ml/kg).After 6h, Excessive anesthetized mice, collecting the bronchoalveolar lavage fluid (BALF) of right lung is used to determine NO and protein content, and collecting left lung is used to survey The wet dry weight of lung is determined than W/D ratio.After 24h, excessive anesthetized mice is collected inferior lobe of right lung and is fixed in 4% formalin, waits to do Pathology and immunohistochemical observation, collect superior lobe of right lung BALF, for determining Inflammatory Factors Contents, collect left lung and are made that to do tissue even Slurry, for determining MPO and SOD activity;The mouse blood sample of collection, determines C3 and C3c contents.
The preparation method of above-mentioned bronchoalveolar lavage fluid (BALF) is:Tracheae is separated, is made with knife blade from annular cartilage lower section Slant cutting edge, tracheae is inserted by homemade tack pin, is fixed with suture, separates cardiopulmonary, and ligation left side bronchus is separated right Lung, is slowly injected into right lung and slowly extracts lavation back and forth out again with syringe absorption 0.8ml physiological saline, is repeated 3 times, and obtains total amount It is the BALF of 1.5ml.
Lung assay method of the wet dry weight than W/D ratio be:Mouse is put to death in 6h after modeling, excessive anesthesia, collects left lung, is used Filter paper blots the tissue fluid and blood on its surface, weighs, and weight is " weight in wet base ", is then placed in 80 DEG C of drying boxes, after 48h Take out, weigh, weight is " dry weight ".It is used for assessing the situation of pulmonary edema with the ratio size of lung " weight in wet base " and " dry weight ".
The assay method of nitric oxide (NO) and total protein content is in bronchoalveolar lavage fluid:6h after modeling, at excessive anesthesia Dead mouse, collects BALF, is centrifuged (4 DEG C, 1400 × g, 10min), Aspirate supernatant, illustrates to require according to NO and BCA kits, Determine NO and total protein content.
The assay method of middle Inflammatory Factors Contents level is in bronchoalveolar lavage fluid:24h after modeling, excessive anesthesia puts to death small Mouse, collects BALF, is centrifuged (4 DEG C, 1400 × g, 10min), Aspirate supernatant, illustrates to require according to ELISA kit, determines scorching Inflammation factor TNF-α, IL-6 and IL-1 β contents levels.
The assay method of MPO and SOD activity is in lung tissue:Mouse is put to death in 24h after modeling, excessive anesthesia, collects left lung, The homogenate of 10% tissue is made of homogenizer, illustrates to require according to MPO kits, determine MPO activity;By 10% tissue homogenate centrifugation (4 DEG C, 1400 × g, 20min), Aspirate supernatant illustrates to require that measure SOD is active according to SOD kits.
The assay method of Complement C_3 and complement fragment C3c contents is in serum:After modeling after 24h, the mouse blood of collection Sample, stands 10min under room temperature (25 DEG C), be centrifuged (4 DEG C, 1400 × g, 20min), draws serum, is said according to ELISA kit Bright requirement, determines C3 and C3c contents.
Lung tissue section pathological observation method be:Mouse is put to death in 24h after modeling, excessive anesthesia, collects inferior lobe of right lung, is put 3 days are fixed in 4% formalin, conventional method paraffin is fixed, cut into slices, H-E dyeing, 400X basis of microscopic observation is taken pictures.Adopt Structural Complement C_3 c depositions are detected with horseradish peroxidase (horseradishperoxidase, HRP) system.5μm Lung's paraffin section through dewaxing, alcohol gradient aquation in dimethylbenzene, with rabbit-anti human complement c 3 c at 4 DEG C night incubation, then 3, 3'- diaminobenzidines (DAB) are developed the color, and X400 basis of microscopic observation is taken pictures.
5th, experimental result
The above-mentioned all data of experimental result are represented with average ± standard deviation (mean ± S.D.), using SPSS19 softwares pair Data compare between one-way analysis of variance, group and use Fisher ' s PLSD methods, work as P<0.05 thinks there is significant difference.
After experimental data is processed using the above method, between each experimental group, the index for evaluating ALI is entered Contrast is gone.
As can be seen that relative to blank group, the content of the W/D anharmonic ratioes, NO and protein of model group has aobvious from Fig. 7-9 Write and raise, its difference has statistical significance (P<0.01).Compare control group and blank group, find eupatorium lindleynun var. trifoliolatum sequiterpene position EUP-SQT does not make significant difference in these areas to normal mouse.EUP-SQT is under 30mg/kg and 60mg/kg dosage to lung's water The content of NO and protein has the significant effect (P for suppressing and reducing in swollen, BALF<0.01).In Fig. 7-9 ## represent compared to Blank group, P<0.01;* is represented compared to model group, P<0.01;* represent compared to model group, P<0.05.
As can be seen that relative to blank group, the TNF-α of model group, IL-6 and IL-1 β levels have aobvious from Figure 10-12 Write and raise, its difference has statistical significance (P<0.01).Compare control group (60mg/kg) and blank group, find EUP-SQT pairs Normal mouse does not make significant difference in these areas.Eupatorium lindleynun var. trifoliolatum sequiterpene portion EUP-SQT under 60mg/kg dosage to BALF in inflammation Factor TNF-α, IL-6 and IL-1 β levels have significant inhibition (P<0.01).## is represented compared to blank in Figure 10-12 Group, P<0.01;* is represented compared to model group, P<0.01;* represent compared to model group, P<0.05.
As can be seen that relative to blank group, the MPO in model group lung tissue has significantly rising from Figure 13-14, SOD has Significant to reduce, its difference has statistical significance (P<0.01).Compare control group (60mg/kg) and blank group, find EUP- SQT does not make significant difference in these areas to normal mouse.EUP-SQT under 60mg/kg dosage to lung tissue in MPO have significantly Inhibition (P<0.01) SOD increases significantly effect (P during, EUP-SQT is under 30mg/kg dosage to lung tissue<0.01). ## is represented compared to blank group, P in Figure 13-14<0.01;* is represented compared to model group, P<0.01;* represent compared to model Group, P<0.05.
As can be seen that relative to blank group, C3 the and C3c levels of model group have significantly rising, and it is poor from Figure 15-16 It is different with statistical significance (P<0.01).Compare control group (60mg/kg) and blank group, it is found that EUP-SQT exists to normal mouse These aspects do not make significant difference.EUP-SQT under 60mg/kg dosage to serum in Complement C_3 and complement fragment C3c levels have Significant inhibition (P<0.05).## is represented compared to blank group, P in Figure 15-16<0.01;* represented compared to model group, P<0.01;* represent compared to model group, P<0.05.
As can be seen that relative to blank group, the lung tissue of model group is imperfect from Figure 17-18, cell becomes big, rupture, Capillary tube inner wall is thickened, and lung cilium hollow deformation, cell interior is flooded with medium, (brown in figure with brown complement precipitation spot Color spot point).But the situation of medicine group makes moderate progress, EUP-SQT under 30mg/kg, 60mg/kg dosage pulmonary condition with compare Group (60mg/kg) and blank group difference are very small.
Drawn by above-described embodiment, Eupalinolide composition is contained in eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) Eupalinolide F (Eupalinolide F), eupalinolide G (Eupalinolide G), eupalinolide H (eupatorium lindleynun var. trifoliolatums Lactone H), eupalinolide I (Eupalinolide I) and eupalinolide K (Eupalinolide K), in mouse lipopolysaccharide In the acute lung injury model of induction, eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) alleviates mouse lung edematous condition, reduces small The content of nitric oxide (NO) and protein, enhances superoxide dismutase in mouse lung tissue in mouse bronchoalveolar lavage fluid (BALF) The activity of enzyme (SOD), suppresses the activity of MPer (MPO), reduces tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and Interleukin -1β (IL-1 β) content, lung tissue, pneumonocyte to mouse, capillary be respectively provided with protective effect, Reduce the content of Complement C_3 and complement fragment C3c in serum and improve depositions of the complement fragment C3c in lung tissue.Eupatorium lindleynun var. trifoliolatum sesquialter Terpene position (EUP-SQT) suppresses the release of inflammatory mediator and the factor by improving body radical scavenging activity, reduces complement water It is flat, improve chmice acute injury of lungs.Therefore eupatorium lindleynun var. trifoliolatum sequiterpene position can be used to prepare the medicine of resisting acute lung injury.
Embodiment 3:
Tested with eupatorium lindleynun var. trifoliolatum ethanol extract resisting acute lung injury Contrast on effect at eupatorium lindleynun var. trifoliolatum sequiterpene position
The preparation of eupatorium lindleynun var. trifoliolatum ethanol extract:Weigh eupatorium lindleynun var. trifoliolatum and dry herb, 2~3cm of segment, plus 10 times the 80% of volume Ethanol water, refluxing extraction 2 hours, is repeated 2 times, and solvent is recovered under reduced pressure, and obtains eupatorium lindleynun var. trifoliolatum ethanol extract (EtOH).
This contrast experiment, experiment reagent, experiment mice and experiment material are with embodiment 2.
Experiment mice is randomly divided into blank group, model group, eupatorium lindleynun var. trifoliolatum ethanol extract EtOH groups, eupatorium lindleynun var. trifoliolatum sequiterpene position Totally 5 groups, every group 10 of EUP-SQT groups and positive group.Eupatorium lindleynun var. trifoliolatum ethanol extract EtOH groups are administered EtOH, and dosage is 30mg kg-1;Eupatorium lindleynun var. trifoliolatum sequiterpene position SQT groups give EUP-SQT, and dosage is 30mgkg-1;Positive group gives dexamethasone, dosage 2mg·kg-1,
All experiment mices are first with 20% urethane-physiological saline (4mlkg-1) intraperitoneal injection light anaesthesia mouse, beat Neck is opened, remaining 5 groups are injected LPS (2mlkg toward mouse tracheal strips in addition to blank group-1, 1mgml-1LPS- physiological saline); And blank group is with method tracheae saline injection 2mlkg-1.After modeling 0.5h, first time administration is carried out by above-mentioned dosage respectively, Blank group gives isometric distilled water;EtOH groups and EUP-SQT groups carry out second administration by above-mentioned dosage after modeling 1h, positive Group, blank group and model group give isometric distilled water.After modeling 6h, excessive anesthetized mice, eyeball takes blood, and every mouse is about 1ml blood samples, the bronchoalveolar lavage fluid (BALF) of right lung is collected with the method for embodiment 2, collects left lung.
, with embodiment 2, its result is as shown in Figure 19-23 for Data Processing in Experiment.
As can be seen from Figure 19, relative to blank group, the W/D anharmonic ratioes of model group have significantly rising, and its difference has system Meter learns meaning (P<0.01);Compared with model group, the pulmonary edema situation of eupatorium lindleynun var. trifoliolatum EtOH groups, EUP-SQT groups and positive group has Significantly improve (P<0.05);Compared to eupatorium lindleynun var. trifoliolatum EtOH groups, the pulmonary edema of EUP-SQT group mouse is significantly inhibited (P< 0.05).Eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) has preferably suppression mouse pulmonary edema than eupatorium lindleynun var. trifoliolatum alcohol extracting thing (EtOH) Effect.Wherein ## is represented compared to blank group, P<0.01;* represent compared to EtOH groups, P<0.05;EtOH groups, EUP-SQT groups Do not compare with model group with positive component, P values are respectively less than 0.05 has significant difference, is indicated in figure.
As can be seen that relative to blank group, total protein and TNF-α content are equal in model group mouse BALF from Figure 20-21 There is significantly rising, its difference has statistical significance (P<0.01);Compared with model group, eupatorium lindleynun var. trifoliolatum EtOH groups, EUP-SQT groups and The total protein and TNF-α content of positive group are significantly improved (P<0.05);Compared to eupatorium lindleynun var. trifoliolatum EtOH groups, EUP-SQT groups Significantly inhibit total protein content (P in BALF<0.05);Compared to eupatorium lindleynun var. trifoliolatum EtOH groups, EUP-SQT groups are significantly inhibited in BALF TNF-α content (P<0.01).Eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) can preferably suppress than eupatorium lindleynun var. trifoliolatum alcohol extracting thing (EtOH) Total protein and TNF-α content in mouse BALF.Wherein ## is represented compared to blank group, P<0.01;* represented compared to EtOH groups, P<0.01;* represent compared to EtOH groups, P<0.05;EtOH groups, EUP-SQT groups and positive component do not compare with model group, P values Respectively less than 0.05 has significant difference, is indicated in figure.
As can be seen that relative to blank group, there be significantly C3 and C3c contents in model group mice serum from Figure 22-23 Raise, its difference has statistical significance (P<0.01);Compared with model group, eupatorium lindleynun var. trifoliolatum EtOH, EUP-SQT group and positive group C3 and C3c contents are significantly improved (P<0.05);Compared to eupatorium lindleynun var. trifoliolatum EtOH groups, C3 the and C3c contents of EUP-SQT groups Significantly inhibited (P<0.05).Eupatorium lindleynun var. trifoliolatum sequiterpene position (EUP-SQT) can be preferably than eupatorium lindleynun var. trifoliolatum alcohol extracting thing (EtOH) Reduce C3 and C3c contents in mice serum.Wherein ## is represented compared to blank group, P<0.01;* represent compared to EtOH groups, P< 0.05;EtOH groups, EUP-SQT groups and positive component do not compare with model group, and P values are respectively less than 0.05 has significant difference, figure In indicate.
Eupatorium lindleynun var. trifoliolatum sequiterpene position is can be seen that from the present embodiment result to be damaged in resisting acute lung than eupatorium lindleynun var. trifoliolatum ethanol extract There is more preferable effect in wound.
Obtained by above-described embodiment, eupatorium lindleynun var. trifoliolatum sequiterpene position has to the lung tissue of mouse, pneumonocyte, capillary There is protective effect.Eupatorium lindleynun var. trifoliolatum sequiterpene position suppresses releasing for inflammatory mediator and the factor by improving body radical scavenging activity Put, reduce level of complement, improve chmice acute injury of lungs, and eupatorium lindleynun var. trifoliolatum sequiterpene position has more preferably than eupatorium lindleynun var. trifoliolatum ethanol extract Drug effect.Eupatorium lindleynun var. trifoliolatum sequiterpene position can apply and prepare resisting acute lung injury medicine.

Claims (5)

1. application of a kind of eupatorium lindleynun var. trifoliolatum sequiterpene position in resisting acute lung injury medicine is prepared, the eupatorium lindleynun var. trifoliolatum sequiterpene position Prepared using following preparation methods:Take eupatorium lindleynun var. trifoliolatum and dry herb, 2~3cm of segment plus 8~volume fraction of 15 times of volumes It is 60%~95% ethanol water, refluxing extraction 1~3 hour is repeated 1~3 time, and 40 DEG C~60 DEG C are recovered under reduced pressure solvent, obtain Eupatorium lindleynun var. trifoliolatum ethanol extract;The extract is soluble in water, the solution that concentration is 0.5~1g/ml is prepared into, through low pole macropore Polymeric adsorbent is adsorbed, and first removes macromolecular compound with water elution, is then 90%~95% ethanol water with volume fraction Wash-out, 40 DEG C~60 DEG C are recovered under reduced pressure solvent, obtain macroreticular resin position;Gained macroreticular resin position is through ODS reverse phase silica gel chromatograms Post is adsorbed, and is eluted by 50% methanol aqueous solution of volume fraction, obtains eupatorium lindleynun var. trifoliolatum sequiterpene position.
2. application according to claim 1, it is characterised in that the eupatorium lindleynun var. trifoliolatum sequiterpene position refers to containing in eupatorium lindleynun var. trifoliolatum The eupatorium lindleynun var. trifoliolatum sequiterpene position of ester F, Eupalinolide G, Eupalinolide H, Eupalinolide I and Eupalinolide K, its chemistry Structural formula is:
3. application according to claim 1, it is characterised in that the low pole macroporous absorbent resin be AB-8, D101, One kind in HPD-100, HPD-200A, HPD-200B, HPD-300, HPD-700, HPD-722 macroporous absorbent resin.
4. application according to claim 1, it is characterised in that the ODS reverse phase silica gels chromatographic column is DAISOGEL ODS 50~70um of reverse phase silica gel packing material size, 50~70um of Cosmsil ODS reverse phase silica gel packing material sizes, YMCODS-A reverse phase silica gels One kind in 50~70um of packing material size.
5. application according to claim 1, it is characterised in that the water is 8 with low pole macroporous absorbent resin volume ratio ~15:1;90%~95% ethanol water is 8~15 with macroporous absorbent resin volume ratio:1;The ODS reverse phase silica gels Chromatographic column column volume is 20~30 with gained macroreticular resin position mass ratio:1;50% methanol aqueous solution and the anti-phase silicon of ODS The volume ratio of glue chromatographic column is 5~8:1.
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