CN103919757B - The medical usage of alkannin derivatives - Google Patents
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Abstract
The invention discloses the purposes of alkannin derivatives in the medicine of preparation treatment chronic obstructive pulmonary disease, the invention also discloses containing one or more the above-mentioned medical usage of Radix Arnebiae extract above-mentioned.
Description
This application is application number is " 201010132461.6 ", apply for that name is called the divisional application of " medical usage of alkannin derivatives ".
Technical field
The invention belongs to medical art, specifically refer to the purposes of alkannin derivatives in a kind of certain drug of preparation.
Background technology
Cyclic nucleotide (cAMP and cGMP) is second message,second messenger important in cell, in various cell, regulate many biological activitys, comprise cells grown, break up and divide a word with a hyphen at the end of a line, various biological respinses that gene expression, medium secretion, smooth muscle contraction, neurotransmitter cause, nerve synapse function, lipid and saccharic metabolism etc.Cyclic nucleotide phosphodiesterase (phosphodiesterases, PDE) is the only intracellular breakdown approach of cyclic nucleotide, and thus PDE inhibitor is by hindering the decomposition of cyclic nucleotide, thus regulates a series of biological function.PDE extended familys comprise l1 family (PDE1-11) altogether, there is different cAMP and/or cGMP specificity ［ FRANCISSH, TURKOIV, CORBINJD.Cyclicnucleotidephosphodiesterases:relatingstru ctureandfunction.ProgNucleicAeidResMolBio, 2001,65:1-52. ］.The distribution in vivo (expression in tissue and cell) of PDE is different.The mankind have 44 kinds of PDE at least, wherein PDE4 plays particular importance ［ CONTIM in inflammation and immunomodulating, JINSLC.Themolecularbiologyofcyclicnucleotidephosphodiest emses.ProgNucleicAcidResMolBiol, 1999,63:1-38. ］.
PDE4 inhibitor can be used as a kind of new medicine and is used for the treatment of the respiratory tract disease caused by inflammation, as asthma and chronic obstructive pulmonary disease (chronicobstructivepulmonarydisease, COPD).
Asthma is a kind of respiratory mucosa inflammation, it is characterized in that eosinophils, and TH2 increases relative to TH1 quantity, and the Amount of Mast Cells of activation increases and respiratory tract structure changes.The most effective Therapeutic Method sucks β 2-AR agonist and 17-hydroxy-11-dehydrocorticosterone at present, and this can control the disease of about 95%, but once stop treatment symptom to there will be again.There will be serious glucocorticoid dependence and opposing symptom in addition.PDE4 inhibitor can suppress recruitment and the activation of related inflammation cell, suppress Constituent cell as asm cell, the hypertrophy of epithelial cell and sensation and cholinergic nerve cell and loose ［ BarnesPJ.Newdrugsforasthma.NatRevDrugDiscov, 2004,3 (10): 831-44. ］.
COPD is the chronic inflammatory disease of a kind of bronchus and pulmonary, this inflammation is because the effect of various protease is as neutrophil elastase (neutrophilelastase, and matrix metalloproteinase (matrixmetalloproteinases NE), MMPs), bronchiolitis and emphysema are shown as.COPD is different from asthma, and its inflammation is by macrophage, neutrophilic granulocyte and CD8
+t lymphocyte causes, and therefore the treatment of COPD focuses on the antagonism of recruitment activation and their products suppressing these cells.Current medicine is very ineffective.PDE4 inhibitor can suppress inflammatory cell, stop the release of proinflammatory medium, improve the release of Anti-inflammatory mediator, reduce ASM cell mitogen, reduce bronchoconstriction, thus play the effect [BarnesPJ.COPD:istherelightattheendofthetunnel .CurrOpinPharmacol, 2004,4 (3): 263-72] for the treatment of COPD.In addition, tissue injury is relevant with the overexpression of MMPs, recent research shows that pro-MMP-1 and pro-MMP-2 that PDE4 inhibitor can suppress to be induced by TNF α effectively secretes [SotoFJ, HananiaNA.Selectivephosphodiesterase-4inhibitorsinchroni cobstructivelungdisease.CurrOpinPulmMed, 2005,11 (2): 129-34].
PDE4 inhibitor can be used as a kind of new medicine and is used for the treatment of inflammatory bowel (IBD), IBD mainly comprises ulcerative colitis (ulcerativecolitis, and clone disease (Crohn'sdisease UC), CD), the two pathogenesis is substantially identical with clinical symptoms, and medicine is also similar.
IBD is the Mucosal Immunity abnormal response caused by many factors.Current treatment is with aminosalicylate and steroid, but this treatment side effect is strong and have steroid leptin suppression.It is effective that experiment shows that PDE4 inhibitor is used for the treatment of IBD in animal model.Inflammation itself can cause tissue injury to change the contraction of blood vessel and intestinal smooth muscle, causes the blood flow of mucosa to reduce, finally causes the permeability of enteric cavity to be lost.This causes stream in intestinal contents, causes inflammatory reaction further, reduces Mucosal blood flow, and tissue injury aggravates.Much mechanism causes the blood flow flowing to intestinal to change: the mucosa angiemphraxis 1. caused by microthrombus; 2. the vasodilation ability caused by proinflammatory medium reduces; 3. the blood vessel caused by proinflammatory medium and intestinal smooth muscle constriction; 4. the vasoconstriction that the intestinal smooth muscle abnormal contraction caused by inflammatory mediator causes.PDE4 inhibitor can improve the above-mentioned situation except 1., improve blood flow, cAMP level can be increased thus the release of suppression proinflammatory medium, make intestinal and relaxing the VSM, thus suppress tissue injury ［ BannerKH, TrevethickMA.PDE4inhibition:anovelapproachforthetreatmen tofinflammatoryboweldisease.TrendsPharmacolSci, 2004,25 (8): 430-6 ］.
Except above-mentioned disease, PDE4 inhibitor also demonstrates activity in vivo on the animal model of other inflammation, as acute lung injury, septicemia, arthritis, dermatosis, multiple sclerosis, inflammatory pain, osteoporosis, kidney disease, allograft rejection reaction and systemic lupus erythematosus etc.
Radix Arnebiae (Radix Lithospermi) is conventional Chinese medicine, and be Boraginaceae herbaceos perennial, Yin Qigen is that purple is gained the name, and is recorded in Shennong's Herbal the earliest, has clearing away heat and cooling blood, invigorate blood circulation, effect of removing toxic substances rash.
After deliberation, the main component of Radix Arnebiae (Radix Lithospermi) has two large classes: a class is water soluble ingredient, and at present research is fewer, tentatively thinks the mixture of mainly polysaccharide and glycoprotein, another kind of is liposoluble constituent, comprises Gronwell naphthaquinone, phenolic acids, alkaloids, phenol, flavonoid etc.Wherein most it should be noted that alkannin derivatives.Research shows, the naphthoquinone compound in Radix Arnebiae (Radix Lithospermi) is mainly alkannin derivant, and structure is as follows:
Alkannin derivatives is the principle active component of Radix Arnebiae (Radix Lithospermi), its content accounts for 3-6.5%, there is multiple physiology and pharmacologically active, as wound healing antiinflammatory, anti-bacteria and anti-virus, antipyretic, hemostasis, antitumor, protect the liver, blood sugar lowering, the effect [VassiliosP.Papageorgiouetal such as calmness and immunity moderation, Angew.Chem.Int.Ed.1999,38,270 – 300; V.P.Papageorgiouetal, CurrentOrganicChemistry, 2006,10,2123-2142; V.P.Papageorgiou, K.C.Nicolaouetal.TheChemistryandBiologyofAlkannin, Shikonin, andRelatedNaphthazarinNaturalProducts [J] .Angew.Chem.Int.Ed.1999,38,270-300].
But alkannin derivatives is to the inhibitory action of PDE4, and the therapeutical effect to asthma and chronic obstructive pulmonary disease, to inflammatory bowel, especially ulcerative colitis, clone disease therapeutical effect have no report.
Summary of the invention
The present invention aims to provide the compound that one has a general formula (I) and is preparing the purposes in phosphodiesterase 4 inhibitors.
R represents H(AK), COCH
3(CAN1), COCHC (CH
3)
2(β, beta-dimethyl-acry-lalkannin), COCH(CH
3) CH
2cH
3(Alpha-Methyl butyryl AK), COCH
2cH (CH
3)
2(isovaleryl AK)
One or more that the present invention relates in the compound with general formula (I) are preparing the purposes in the medicine for the treatment of asthma and chronic obstructive pulmonary disease;
One or more that the present invention relates in the compound with general formula (I) treat the purposes in the medicine of inflammatory bowel in preparation treatment;
One or more that the present invention relates in the compound with general formula (I) treat the purposes in the medicine of ulcerative colitis in preparation.
One or more that the invention still further relates in the compound with general formula (I) treat the purposes in the medicine of clone disease in preparation.
Compound involved in the present invention can be separated by conventional method and obtain ［ VassiliosP.Papageorgiouetal, Angew.Chem.Int.Ed.1999,38,270 – 300 by list of references in Radix Arnebiae (Radix Lithospermi); V.P.Papageorgiouetal, CurrentOrganicChemistry, 2006,10,2123-2142; V.P.Papageorgiou, K.C.Nicolaouetal.TheChemistryandBiologyofAlkannin, Shikonin, andRelatedNaphthazarinNaturalProducts [J] .Angew.Chem.Int.Ed.1999,38,270-300 ］.
As got lithospermum euchromum Royle (Radix Arnebiae Arnebiaeuchroma (Royle) Johnst) pharmaceutical decocting piece, 15 times amount petroleum ether lixiviate 3 times under 55 DEG C of conditions, each 3 hours, extracting solution concentration and recovery solvent, obtained Radix Arnebiae extract.Radix Arnebiae extract, through silica gel column chromatography, uses petroleum ether respectively: ethyl acetate=100:1; 100:1; 100:2; 100:4 ratio eluting, again respectively with petroleum ether or petroleum ether, ethyl acetate and methanol recrystallization in certain proportion after gained eluent solvent evaporated, obtain red-brownish coloured particles shape crystallization-CAN1, cerise flake-like crystal-deoxidation AK, bronzing lamellar shape crystallization-β respectively, beta-dimethyl-acry-lalkannin.Alpha-Methyl butyryl AK cannot be separated on silica gel column chromatography with isovaleryl AK, is a speckle (colour band), and be separated by preparation liquid phase, separation condition is: mobile phase: THF:H2O=45:55; Flow velocity: 1ml/min; Determined wavelength: 515nm.The separable red thick thing Alpha-Methyl butyryl AK that obtains and red thick thing isovaleryl AK.
Accompanying drawing illustrates:
Fig. 1 rat body weight change broken line graph (* P<0.05, * * P<0.01vs.model.)
The DAI index map of Fig. 2 administration after 7 days (* P<0.05, * * P<0.01vs.model.)
The colon lengths of Fig. 3 administration after 7 days (* P<0.05, * * P<0.01vs.model.)
MPO activity (* P<0.05, * * P<0.01vs.model.) in Fig. 4 colon
TNF-alpha levels (* P<0.05, * * P<0.01vs.model.) in Fig. 5 serum
Fig. 6 rat causes scorching parapodum swelling schematic diagram
Fig. 7 rat secondary parapodum swelling schematic diagram
Fig. 8 rat body weight variation diagram
Fig. 9 arthritis index is marked
Detailed description of the invention
Embodiment 1
The mixture of the compound of tool general formula (I) also directly can be obtained by following method from Radix Arnebiae (Radix Lithospermi).
Get lithospermum euchromum Royle (Radix Arnebiae Arnebiaeuchroma (Royle) Johnst) pharmaceutical decocting piece 100Kg(purchased from medicine store, Yantai), 1500L petroleum ether lixiviate 3 times under 55 DEG C of conditions, each 3 hours, extracting solution concentration and recovery solvent, obtain about 4.2Kg extractum, with 70kg silica gel column chromatography, petroleum ether-ethyl acetate system eluting, thin layer checks to obtain Radix Arnebiae extract 2.1kg.
Analyze through HPLC, high-efficient liquid phase chromatogram condition is mobile phase: acetonitrile-water-formic acid (700:300:0.5), flow velocity: 1ml/min, determined wavelength: 518nm, column temperature: 30 DEG C.Content is calculated by areas of peak normalization method.
Content containing CAN1 in Radix Arnebiae extract is 25.1%; The content of isovaleryl AK+Alpha-Methyl butyryl AK is 30.2%; β, the content of beta-dimethyl-acry-lalkannin is 24.2%.
Embodiment 2
The preparation of Radix Arnebiae (Radix Lithospermi) naphthoquinone monomeric compound
The Radix Arnebiae extract that embodiment 1 obtains, through silica gel column chromatography, uses petroleum ether respectively: ethyl acetate=100:1; 100:1; 100:2; 100:4 ratio eluting, again respectively with petroleum ether or petroleum ether-ethyl acetate and methanol recrystallization in certain proportion after gained eluent solvent evaporated, obtain red-brownish coloured particles shape crystallization-CAN1, cerise flake-like crystal-deoxidation AK, bronzing lamellar shape crystallization-β respectively, beta-dimethyl-acry-lalkannin.Alpha-Methyl butyryl AK cannot be separated on silica gel column chromatography with isovaleryl AK, is a speckle (colour band), and be separated by preparation liquid phase, separation condition is: mobile phase: THF:H2O=45:55; Flow velocity: 1ml/min; Determined wavelength: 515nm.The separable red thick thing Alpha-Methyl butyryl AK that obtains and red thick thing isovaleryl AK.
The inhibitory action of test example 1 pair of PDE4 activity
4.1 material
Given the test agent: alkannin derivatives, prepares by embodiment 2
4.2 methods and result
The extracting method reference literature method of PDE4 enzyme obtains [Chen Wu, Jiang Daixun, willow, Deng. the extraction of pig neutrophil cell cAMP phosphodiesterase and Activity determination [J]. Beijing Agricultural College's journal, 2003,18 (4): 242-244.], namely prepare pig neutrophil cell with reference to above-mentioned literature method, granulocyte Ca/MgPBS is diluted to about 10
11individual/L, then this diluent is made homogenate with glass homogenizer in ice bath, confirm under microscope that cell is pulverized evenly, the enzyme sample of PDE4 must be rich in.
First the activity of enzyme is measured: cAMP Ca/MgPBS is made into 100 μm of ol.L
-14 sampling amounts established by enzyme sample, and be settled to 100 μ L with Ca/MgPBS, application of sample process is carried out in ice bath, concrete application of sample amount puts constant-temperature incubation 30min in 35 DEG C in pipe each after table 2. application of sample, then 2-3min cessation reaction in 100 DEG C of water-baths, each pipe, in 4 DEG C of centrifugal 30min of 15000r/min, gets supernatant 70 μ L, dilute 5 times (adding 280 μ lddH2O) with ultra-pure water, HPLC sample introduction 20 μ L, detects under 254nm wavelength, and mobile phase is methanol: phosphate buffer=10:90.Experimental result refers to Fig. 1.
Table 2: the application of sample amount of enzyme reaction
PDE activity represents with the percent hydrolysis of substrate:
Next measures Radix Arnebiae extract to the impact of PDE activity: enzyme reaction is carried out in Ca/MgPBS, and cAMP buffer is made into 100 μm of ol.L
-1, get 65 μ L during reaction, enzyme sample amount 30 μ L, medicine divides five variable concentrations, and consumption is 1 μ L, and control tube and sample cell add the PDE4 enzyme sample of extraction, and blank pipe adds the enzyme sample of deactivation, and be settled to 100 μ L with buffer, Loading sequence is in table 3.
Table 3 enzyme reaction Loading sequence and dosage
After application of sample, constant-temperature incubation 30min in 35 DEG C put by each pipe, then 2-3min cessation reaction in 100 DEG C of water-baths, and each pipe, in 4 DEG C of centrifugal 30min of 15000r/min, gets supernatant 70 μ L, dilutes 5 times (add 280 μ lddH with ultra-pure water
2o), HPLC sample introduction 20 μ L, detects under 254nm wavelength, and mobile phase is methanol: phosphate buffer=10:90.The results detailed in Table 4.
Table 4 Radix Arnebiae (Radix Lithospermi) naphthoquinone compound is on the impact of PDE4 activity
4.3 conclusion (of pressure testing)s: alkannin derivatives has inhibit activities to PDE4.
Test example 2
Radix Arnebiae (Radix Lithospermi) naphthoquinone monomeric compound and containing one or more Radix Arnebiae extract of general formula (I) to the therapeutical effect of rat inflammatory enteropathy
One, experimental apparatus and material
Animal: Wistar rat, male, SPF level, body weight 180-200g, purchased from Peking University's Experimental Animal Center, the quality certification number: SCXK(capital) 2006-2008, raises in SPF level laboratory, keep room temperature 23 scholar 2 DEG C, relative humidity 50 scholar 10%, 12 hours/day artificial lighting time, automatic wind exhaust, freely drink water and take food, adapting to use after 7 days.
Dextran sulfate (DSS): molecular weight 5000, available from Sigma;
Salazosulfamide (SASP): purchased from friendship pharmaceutical Co. Ltd of upper Hisense, purity is greater than 95%;
Radix Arnebiae extract (CAN3) is prepared by embodiment 1
CAN1 (CAN1), β, beta-dimethyl-acry-lalkannin (CAN2), prepares by embodiment 2;
Rat blood serum TNF-α measures test kit: purchased from American R & DSystem company;
Rat MPO measures test kit: build up Bioisystech Co., Ltd purchased from Nanjing
Two, experimental technique
1, the foundation of rat colitis model
DSS is dissolved in distilled water, preparation 3%(W/W) DSS aqueous solution, substitute drinking water and freely drink 7 days by Wistar rat (SPF level, male, body weight 180-200g).
2, animal grouping and administration
After the laundering period of 7 days, Wistar rat (SPF level, male) is divided into 6 groups after weighing at random, often organizes 8, is respectively normal group (normal), model group (model), positive drug group (SASP, 50mgkg
-1), CAN1 group (CAN1,10mgkg
-1), β, beta-dimethyl-acry-lalkannin group (CAN2,20mgkg
-1), Radix Arnebiae extract group (CAN3,40mgkg
-1).Normal group gives normal water, freely drinks; Model group, positive drug group, administration group give 3%DSS aqueous solution respectively, freely drink; Model group, positive drug group, administration group are by above-mentioned dosage gastric infusion simultaneously, once a day; Model group and normal group are filled with normal saline.All normally take food for each group.
3, disease (progress extent) enlivens the assessment of situation
3.1, body weight
Claim rat body weight respectively at every day before modeling and after modeling, observe each group of rat body weight change.
3.2, disease index (diseaseactivityindex, DAI)
Observe the stool of rat and situation of occulting blood, mark by following principle, draw the DAI of every rat.
Criteriaforscoringdiseaseactivityindex(DAI)
Diseaseactivityindex(DAI)=combinedscore(stoolconsistency+bleeding).
*Normal=well-formedpellets;loosestools=pastystoolthatdoesnotsticktoanus;
diarrhoea=liquidstoolsthatsticktoanus.
4, colon lengths
Administration is after 7 days, and each group rat is with 10% chloral hydrate (60mgkg
-1) intraperitoneal injection of anesthesia.After abdominal aortic blood, be separated colon, cut off enteric cavity along mesentery, with freezing normal saline cleaning twice, filter paper launches, with vernier caliper measurement colon lengths.
5, immunohistochemical staining
Get the above-mentioned colon measuring length, get the tissue near about 0.5cm ulcer, be stored in the formalin solution of 10%, dyeed by the give birth semi-annular jade pendant top hospital pathology department film-making of Affiliated Hospital of Qingdao Medical Inst and Yantai City, remaining tissue sample in ultra cold storage freezer-80 DEG C frozen.Adopt Computer digital image analysis.Tissue specimen after dyeing amplifies 40-100 doubly by microscope camera system, pickup image.
6, the mensuration that colon's myeloperoxidase (MPO) (MPO) is active
Get the colon of above-mentioned sampling, weigh, add by 1: 19 the tissue homogenate that homogenate medium makes 5%, detect by test kit description and calculate MPO vigor.The ability of degraded per minute 1 μm of ol hydrogen peroxide when MPO activity is defined as 37 DEG C, unit is: Ug
-1.
Suppression ratio computing formula is as follows:
7, the mensuration of TNF-α content
In rat blood serum, the mensuration of TNF-α measures according to the description of R & D company commercial ELISA kits, and unit is ngL
-1.
8, date processing
Data average ± standard deviation (MEAN ± SEM) represents, adopt SPSS16.0 software to carry out one factor analysis of variance (ANOVA), the comparison in difference Tukey ' st thereupon between each group checks, and P<0.05 is that difference has significance.
Three, experimental result
1, body weight
As shown in Figure 1, after DSS induced synthesis ulcerative colitis, rat body weight increasess slowly, and along with the prolongation of the course of disease, body weight declines gradually.Compare with model group, CAN3 group (40mgkg
-1) decline (P<0.05) of rat body weight significantly can be suppressed from the 3rd day, CAN1, CAN2 group significantly can suppress the decline (P<0.01) of rat body weight from the 5th day.
2, DAI index
As shown in Figure 2, administration is after 7 days, and positive drug group and CAN3, CAN2 group compare with model group, and DAI score obviously reduces,
3, colon lengths
Administration is after 7 days, and model group colon lengths compared with normal group obviously shortens.Positive drug group and administration group compare with model group, and colon lengths has significant difference.
4, colon's myeloperoxidase (MPO) (MPO) is active
Compared with normal group, the MPO activity in model group colon obviously increases, but administration group CAN3, CAN2 group and positive drug group all can significantly reduce MPO content in colon.
5, TNF-alpha levels in rat blood serum
Compare with normal group, the TNF-alpha levels given in each group of rat blood serum of 3%DSS aqueous solution obviously raises, but compare with model group, positive drug group and administration group TNF-alpha levels significance reduce, the rising of the content of TNF-α in the rat blood serum that DSS can be suppressed to induce.
Four, conclusion
Radix Arnebiae (Radix Lithospermi) naphthoquinone compound and the rat inflammatory enteropathy tool significant protective effect of dextran sulfate being induced containing one or more Radix Arnebiae extract of general formula (I).Result shows, oral said medicine significantly can reduce the order of severity of ulcer, alleviates the atrophy degree of colon, and its effect is suitable with positive drug salazosulfamide.
Test example 3. Radix Arnebiae (Radix Lithospermi) naphthoquinone compound and Radix Arnebiae extract are on the impact of collagen-induced rat arthritis
3.1 material
Given the test agent: Radix Arnebiae extract, prepares by embodiment 1.Alpha-Methyl butyryl AK+isovaleryl AK β, beta-dimethyl-acry-lalkannin, prepares by embodiment 2.
3.2 methods and result
70 male Wistar rats are divided into 7 groups at random by body weight, be respectively: normal group, model group, benefit match general group of (0.8mg/kg), Tripterygium glycosides group (30mg/kg), Alpha-Methyl butyryl AK+isovaleryl AK group (CAN1,10mg/kg), β, beta-dimethyl-acry-lalkannin group (CAN2,10mg/kg), Radix Arnebiae extract group (CAN3,20mg/kg), often 10 are organized.In left whole pad intradermal injection cattle II Collagen Type VI (CIA) the Emulsion 0.1ml of every rat during modeling; Every rat is apart from the subcutaneous Isodose booster immunization in 2-3cm place of root of the tail portion once afterwards for initial immunity 10 days; After 14d, modeling successfully starts administration, successive administration 28d; Survey the toes volume of foot about all rats before modeling with toes capacity measurer, after modeling, every day measures the left sufficient volume of CIA rat (former swell swollen), and while after modeling 10d start to survey the whole toe volume of offside every other day; After modeling, 12d starts arthritis index scoring, marks 1 time every 2d, continuous 32d; 1h after last administration, takes the weight of rat, abdominal aortic blood, measures TNF-ɑ content.
Experimental result shows, Radix Arnebiae extract can suppress the sufficient pawl swelling (Fig. 6,7) of CIA rat, improves rat body weight and reduces (Fig. 8), reduce polyarthritis index score (Fig. 9), alleviate the inflammation performance of CIA rat, alleviate TNF-ɑ content in rat blood serum.
Radix Arnebiae extract is on the impact of TNF-ɑ content in CIA rat blood serum
n=10)
Δ P<0.01 is compared with normal group; * P<0.05 is compared, * * P<0.01. with model group
Conclusion: Radix Arnebiae extract has obvious therapeutical effect to collagen-induced rat arthritis.
Test example 4:
Radix Arnebiae extract and alkannin derivatives are to the effect of rat copd
1 materials and methods
1.1 material
Healthy SD rat 40, Mus 4 ~ 8W in age, body weight (210 ± 20) g, male and female are not limit, and are provided by Shandong Traditional Chinese Medicine University's Experimental Animal Center.Medicated cigarette is that fire-cured tobacco type eight likes board (Qingzhou Cigarette Factory produces, tar content 15mg, nicotine content in smoke 1.1mg); LPS (Sigma Co., USA); Tumor necrosis factor TNF-alpha test kit (Tianjin Jiuding Medical Biological Engineering Co., Ltd); Radix Arnebiae extract (preparing by embodiment 1), CAN1, β, β '-dimethyl allene acyl AK (preparing by embodiment 2).
1.2 animal groupings and model are set up
Healthy SD rat 40, male and female are not limit, be divided into 5 groups at random, often organize 8, Normal group (matched group): raise in normal circumstances, by rat in the 1st, 14 day tracheal strips saline injection 0.2ml, within the 8th day, play intraperitoneal injection of saline 0.05ml/kg every day, put to death to 28 days; Chronic obstructive pulmonary disease (COPD) model group (model group): rat is injected lipopolysaccharide (LPS) each 200 μ g/200 μ l in the 1st, 14 day tracheal strips, 2nd ~ 13 days, 5 ~ 28 days at self-control smoking toxicant exposure box (70cm × 50cm × 50cm, upper right side is with 1.5cm × 1.5cm passage) interior incense cigarette 2 times/d, smoking capacity is 14/time, wherein each smoking period 1h, twice smoking is spaced apart 1h.Within 8th day, play intraperitoneal injection of saline 0.05ml/kg every day, put to death to the 28th day; Treatment group 1 (Radix Arnebiae extract group): tracheal strips injects LPS and the same model group of incense cigarette process, plays oral administration gavage administration every day 160mg/kg on the 8th day, put to death to the 28th day.Treatment group 2 (CAN1 group), treatment group 3 (β, β '-dimethyl allene acyl AK group): tracheal strips injects LPS and the same model group of incense cigarette process, within the 8th day, plays oral administration gavage administration every day 80mg/kg, put to death to the 28th day.
The collection of 1.3 bronchoalveolar lavage fluid (BALF)
Rat is fixed on operating board with lying on the back after pentobarbital (25mg/kg) anesthesia of 2%, cut chest, expose trachea and two lung, ligation right principal bronchus, with trocar puncturing extremely left lung in knuckle, slow injection physiological saline solution 3ml, after each injection, resorption obtains BALF immediately, repeats 3 times.Irrigating solution filtered through gauze, the response rate is 60% ~ 70%.Draw this BALF of 1ml on hematimeter, counted for total White and M7 (PMN) number.The remaining BALF4 DEG C of centrifugal 10min of 1500r/min, gets its supernatant and inserts EP pipe and be stored in TNF-α concentration to be checked in-20 DEG C of refrigerators.
1.4TNF-α Concentration Testing
Application TNF-α detection kit, by specification enzyme-linked immunosorbent assay (ELISA) measures the concentration of TNF-α.
1.5 Pathologic specimen preparation and Morphological Quantitative Analysis
The trachea at 2 ~ 3mm place and middle period lung tissue 5mm in the knuckle of getting every rat
3size, 10% formalin is fixed, ethanol serial dehydration, waxdip, embedding, section, and HE dyes, om observation.By the 1mm fixed through 4% glutaraldehyde
3right lung organizes rinsing, with fixing after 2% Osmic acid., acetone serial dehydration, embedding, polymerization, section, acetic acid uranium-lead citrate electron staining, transmission electron microscope observing, and apply micro--microcomputer image processing system, measure following index: the 1. average liner interval (MLI) of lung, its numerical value reflection alveolar average diameter.
2. average alveolar number (MAN), its numerical value reflection alveolar density.
1.6 statistical procedures
With SPSS12.0 statistical software, measurement data with
represent, compare between two groups with q inspection, compare between three groups and use one factor analysis of variance.
2 results
2.1 lung morphology quantitative analyses
Compare with matched group, model group MLI raises, and MAN reduces, difference highly significant (equal P<0.01); Compare with model group, treatment group MLI reduces, and MAN raises, and difference highly significant (equal P<0.01), in table 5.
2.2BALF cell counting and PMN number
Compare with matched group, in model group BALF, total white blood cells and PMN number increase, difference highly significant (equal P<0.01); Compare with model group, in treatment group BALF, total white blood cells and PMN number obviously decline, and difference highly significant (equal P<0.01), in table 5.
The change of TNF-alpha levels in 2.3BALF
Compare with matched group, in model group BALF, TNF-α concentration raises, difference highly significant (P<0.01);
Compare with model group, treatment group TNF-α concentration reduces, and difference highly significant (P<0.01), in table 5.
Level and lung tissue MLI, the MAN of table 5 each group BALF of Rats total cellular score, PMN number, TNF-α compare
n=8)
Compare with matched group: * P<0.01,
#p<0.01
3 conclusions
This experiment sets up COPD rat model by injecting LPS method in smoked cigarette gas-adding pipe of enfleuraging, and can reflect the pathology forming process of human diseases similarly.Meanwhile, analyze display, the MLI of model group reflection alveolar average diameter comparatively matched group increases, and the MAN reflecting alveolar density comparatively matched group significantly reduce, the significant difference of two indices, illustrates that animal model is successful.
This result shows, treatment group rat pathological examination prompting lung injury comparatively model group rats alleviates, and MLI comparatively model group reduces, and MAN comparatively model group raises, illustrate that treatment group can alleviate COPD injury of lung to a certain extent, certain control, mitigation are served to the reconstruction of emphysema structure.Simultaneously in treatment group BALF of Rats TNF-α concentration, total white blood cells, PMN number comparatively model group obviously decline; prompting treatment group can reduce the infiltration of PMN in lung; the Tissue-cell culture (ICAM-1) caused by TNF-α and LPS is suppressed to be expressed; TNF-alpha levels is declined; alleviate pulmonary edema; protection PMEC (Pulmonary Microvascular Endothelial Cells), improves microcirculation.Experiment proves, Radix Arnebiae extract and alkannin derivatives have certain curative effect for treatment chronic obstructive pulmonary disease.
Test example 5:
Radix Arnebiae extract and alkannin derivatives are to the therapeutical effect of experimental rat model of asthma
1 materials and methods
1.1 material
Wistar rat is provided by Shanghai Slac Experimental Animal Co., Ltd., body weight 180 ~ 200g, ovalbumin (OVA) is produced by Sigma Co., USA, IgE, IL-4, IL-5, IL-13 and IFN-γ detection kit is purchased from Jing Mei biological engineering company limited, Radix Arnebiae extract is prepared by embodiment 1, CAN1, β, β '-dimethyl allene acyl AK are prepared by embodiment 2.
1.2 animal groupings and model preparation
60 healthy male rats of SPF level adopt random digits table to be divided into normal healthy controls group (I group), asthmatic model group (II group), Radix Arnebiae extract treatment group (III group), CAN1 treatment group (IV group), β, β '-dimethyl allene acyl AK (V group), treating asthma negative control group (VI group), 10/group.II-VI group rat is with OVA aluminium hydroxide mixed liquor 1ml (OVA100mg+ aluminium hydroxide 100mg+ normal saline lm1).1st day lumbar injection sensitization, within 7th day, repeat to strengthen sensitization 1 time, within 14th day, insert colourless, transparent, in closed glass case, excite (mist particle diameter 2.5-5 μm) with 1%OVA (OVA100mg+ normal saline 10m1) aerosol inhalation, 1 time/d, 30min/ time, continuous 2 weeks, I group rats by intraperitoneal injection sensitization equal-volume normal saline replaces OVA aluminium hydroxide mixed liquor, Ultrasonic atomising taring excites and replaces OVA with equal-volume normal saline, III group rat excites front 1h gastric infusion 120mg/kg at every turn, IV, V group rat excites front 1h gastric infusion 60mg/kg at every turn.Treatment group gastric infusion is replaced with equal-volume normal saline before VI group rat excites at every turn.All rats are 6h 3% pentobarbital sodium (1ml/kg) intraperitoneal injection of anesthesia after last excites, breast is opened after dorsal position is fixing, get central vein blood 2ml and measure IgE content, an osculum is cut in left lung main bronchus, fixing after inserting diameter 3mm plastic flexible pipe, with 4 DEG C of capable bronchoalveolar lavage of normal saline, each consumption 2ml, continuous lavation 3 times, response rate >70%, by centrifugal for bronchoalveolar lavage fluid (BALF) 4 DEG C, 1200r/min, 10min, get cell sediment for cell counting and classification, supernatant-20 DEG C preserves the detection being used for cytokine.
1.3BALF cell counting and classification
BALF is centrifugal, and rear cell sediment 2mlHanks dilutes, and gets lm1 and measures total white blood cells in hematimeter, get 0.2ml and make cell smear, Wright's staining, and 400 power microscopes are down to minority 200 cells, and row classified counting of leucocyte, calculates various types of cells content.
1.4 IL-4, IL-5, IL-13, IFN-γ assay in venous blood IgE and BALF
All adopt Double antibody sandwich-ELISA (SandwichELISA) to measure, operate by reagent description.
1.5 statistical procedures
Adopt SPSS13.0 statistical software to carry out statistical procedures to data, data are used
represent, employing independent samples t test compares between organizing respectively, and P<0.05 is that difference has statistical significance.
2 results
2.1 rats excite rear reaction
II, VI group rat excites rear about 5min to start dysphoria, grabs nose, and exaggerated respiration is subsequently accelerated, breathing of nodding, mouth and nose cyanosis, and activity obviously reduces, and I group rat is without above-mentioned reaction, and the reaction of III, IV, V group rat is lighter.
2.2BALF cell counting and classification
In II group BALF, neutrophilic granulocyte, lymphocyte, eosinophil count are significantly higher than I group (P<0.01), compare with II group, III, IV, V group significantly can reduce neutrophilic granulocyte in BALF, lymphocyte, eosinophilic granulocyte (P<0.01), II group and VI group neutrophilic granulocyte, lymphocyte, eosinophilic granulocyte and mononuclear phagocyte count the equal not statistically significant of difference (P>0.05), in table 6.
Table 6 is group BALF of Rats cell divide and counting (× 10 respectively
5individual/L,
Compare with I group,
*p<0.01; Compare with II group,
aMP.AMp.Ampp<0.01
2.3 IL-4, IL-5, IL-13, IFN-γ content in venous blood IgE and BALF
In II group venous blood IgE and BALF, IL-4, IL-5, IL-13 content is significantly higher than I group (P<0.01), compare with II group, III-V group significantly can to reduce in venous blood IL-4, IL-5, IL-13 in IgE and BALF, significantly raises the IFN-γ (P<0.01) in BALF; II group and VI group IgE, IL-4, IL-5, IL-13, IFN-γ content difference not statistically significant (P>0.05), in table 7.
Table 7 respectively organizes IL-4, IL-5, IL-13, IFN-γ comparision contents in rat vein blood IgE and bronchoalveolar lavage fluid
Compare with I group,
*p<0.01; Compare with II group,
aMP.AMp.Ampp<0.01
3 discuss
Asthmatic rats model has successfully been prepared in this research, research finds, in experimental rat model of asthma acute stage lung tissue, IL-4, IL-5, IL-13 produce and increase, and IFN-γ produces minimizing, with neutrophilic granulocyte, lymphocyte, eosinophils, IgE synthesis increases; And gastric infusion Radix Arnebiae (Radix Lithospermi) treatment group (III, IV, V) can reduce the generation of IL-4, IL-5, IL-13 in Asthmatic Rat Lung, promote the generation of IFN-γ, alleviate neutrophilic granulocyte, lymphocyte, eosinophils, IgE is synthesized reduce, obviously alleviate rat asthma attack degree.After gastric infusion Radix Arnebiae (Radix Lithospermi), Th cell regulating factor IL-4 declines, and IFN-γ rises, and prompting Radix Arnebiae (Radix Lithospermi) may stop Th0 cell differentiation to be Th2 cell, promotes that Th0 cell differentiation is Thl cell; The generation of IL-5, IL-13 and IgE reduces prompting Radix Arnebiae (Radix Lithospermi) and can also regulate inflammatory reaction, alleviates the generation of I type allergy.
In sum, Radix Arnebiae extract and alkannin derivatives can alleviate airway inflammation, reduce airway hyperreactivity, thus reach the object of control asthma attack.
Claims (2)
1. containing one or more the purposes of Radix Arnebiae extract in preparation treatment chronic obstructive pulmonary disease medicine of general formula (I),
R represents COCH
3, COCHC (CH
3)
2, COCH (CH
3) CH
2cH
3, COCH
2cH (CH
3)
2.
2. one kind containing, for example one or more the purposes of Radix Arnebiae extract in preparation treatment chronic obstructive pulmonary disease medicine of general formula according to claim 1 (I), it is characterized in that described Radix Arnebiae extract is prepared by following methods: get lithospermum euchromum Royle pharmaceutical decocting piece 100Kg, 1500L petroleum ether lixiviate 3 times under 55 DEG C of conditions, each 3 hours, extracting solution concentration and recovery solvent, obtain about 4.2Kg extractum, with 70kg silica gel column chromatography, petroleum ether-ethyl acetate system eluting and get final product.
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