CN103690518A - Medical application of lithospermum naphthoquinone compounds - Google Patents

Abstract

The invention discloses application of lithospermum naphthoquinone compounds in preparing a drug for treating a chronic obstructive pulmonary disease, and further discloses medical application of one or more lithospermum extracts.

Description

The medical usage of alkannin derivatives

This application is that application number is that " 201010132461.6 ", application name are called dividing an application of " medical usage of alkannin derivatives ".

Technical field

The invention belongs to medical technical field, specifically refer to the purposes of alkannin derivatives in a kind of certain drug of preparation.

Background technology

Cyclic nucleotide (cAMP and cGMP) is important second message,second messenger in cell, in various cells, regulate many biological activitys, various biological respinses, nerve synapse function, lipid and the saccharic metabolism etc. that comprise that cell is grown up, broken up and divides a word with a hyphen at the end of a line, gene expression, medium secretion, smooth muscle contraction, neurotransmitter cause.Cyclic nucleotide phosphodiesterase (phosphodiesterases, PDE) is decomposition approach in the only cell of cyclic nucleotide, thereby PDE inhibitor is by hindering the decomposition of cyclic nucleotide, thereby regulates a series of biological function.PDE extended familys comprise 11Ge family (PDE1-11) altogether, there is different cAMP and/or cGMP specificity ［ FRANCIS SH, TURKO IV, CORBIN JD.Cyclic nucleotide phosphodiesterases:relating structure and function.Prog Nucleic Aeid Res Mol Bio, 2001,65:1-52. ］.Distribution in the body of PDE (expression in tissue and cell) is different.The mankind have 44 kinds of PDE at least, wherein PDE4 plays particular importance ［ CONTI M in inflammation and immunomodulating, JIN SLC.The molecular biology of cyclic nucleotide phosphodiestemses.Prog Nucleic Acid Res Mol Biol, 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 being caused by inflammation, as asthma and chronic obstructive pulmonary disease (chronic obstructive pulmonary disease, COPD).

Asthma is a kind of respiratory mucosa inflammation, it is characterized in that eosinophils, and TH2 increases with respect to TH1 quantity, in the Amount of Mast Cells increase of activation and respiratory tract structure, changes.The most effective Therapeutic Method is to suck β at present ₂-AR agonist and 17-hydroxy-11-dehydrocorticosterone, this can control approximately 95% disease, once but stopping treating symptom there will be again.There will be in addition serious glucocorticoid dependence and opposing symptom.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 hypertrophy ［ Barnes P J.New drugs for asthma.Nat Rev Drug Discov, 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 (neutrophil elastase, NE) and matrix metalloproteinase (matrix metallop roteinases, MMPs), show as obstructive bronchiolitis and emphysema.COPD is different from asthma, and its inflammation is by macrophage, neutrophilic granulocyte and CD8 ⁺t lymphocyte causes, so the treatment of COPD focuses on the recruitment activation of these cells of inhibition and the antagonism of their products.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, thereby play the effect [Barnes P J.COPD:is there light at the end of the tunnel .Curr Opin Pharmacol, 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 PDE4 inhibitor can suppress pro-MMP-1 and pro-MMP-2 secretion [the Soto F J being induced by TNF α effectively, Hanania N A.Selective phosphodiesterase-4inhibitors in chronic obstructive lung disease.CurrOpin Pulm Med, 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 (ulcerative colitis, UC) and clone disease (Crohn's disease, CD), the two pathogenesis and clinical symptoms are basic identical, and medicine is also similar.

IBD is the Mucosal Immunity abnormal response being caused by many factors.Current treatment is with aminosalicylate and steroid, but this treatment side effect is strong and have steroid to resist phenomenon.Experiment shows that it is effective that PDE4 inhibitor is used for the treatment of IBD in animal model.Inflammation itself can cause that tissue injury changes the contraction of blood vessel and intestinal smooth muscle, causes the blood flow of mucosa to reduce, and finally causes the permeability of enteric cavity to be lost.This causes stream in intestinal contents, further causes inflammatory reaction, reduces mucosa blood flow, tissue injury's aggravation.Much mechanism causes the blood flow that flows to intestinal to change: the mucosa angiemphraxis 1. being caused by microthrombus; 2. the vasodilation ability being caused by proinflammatory medium reduces; 3. the blood vessel being caused by proinflammatory medium and intestinal smooth muscle constriction; 4. the vasoconstriction that the intestinal smooth muscle abnormal contraction being caused by inflammatory mediator causes.PDE4 inhibitor can improve the above-mentioned situation except 1., improve blood flow, thereby can increase the release that cAMP level suppresses proinflammatory medium, make intestinal and to relax the VSM, thereby suppress ［ the Banner K H of tissue injury, Trevethick M A.PDE4inhibition:a novel approach for the treatment of inflammatory bowel disease.Trends Pharmacol Sci, 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, is Boraginaceae herbaceos perennial, and Yin Qigen is purple and gains the name, be recorded in the earliest < < Sheng Nong's herbal classic > >, there is clearing away heat and cooling blood, invigorate blood circulation, the 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, studies at present fewerly, tentatively thinks it is mainly the mixture of polysaccharide and glycoprotein, another kind of is liposoluble constituent, comprises Gronwell naphthaquinone, phenolic acids, alkaloids, phenol, flavonoid etc.Wherein it should be noted that most 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 main effective ingredient 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, calmness and regulate effect [the Vassilios P.Papageorgiou et al such as immunity, Angew.Chem.Int.Ed.1999,38,270 – 300; V.P.Papageorgiou et al, Current Organic Chemistry, 2006,10,2123-2142; V.P.Papageorgiou, K.C.Nicolaou et al.The Chemistry and Biology of Alkannin, Shikonin, and Related Naphthazarin Natural Products[J] .Angew.Chem.Int.Ed.1999,38,270-300].

But the inhibitory action of alkannin derivatives to 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 purposes of a kind of compound with general formula (I) in preparing 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)

The purposes of one or more in the present invention relates to the to there is general formula compound of (I) in the medicine of preparation treatment asthma and chronic obstructive pulmonary disease;

The purposes of one or more in the present invention relates to the to there is general formula compound of (I) in the medicine of preparation treatment treatment inflammatory bowel;

The purposes of one or more in the present invention relates to the to there is general formula compound of (I) in the medicine of preparation treatment ulcerative colitis.

The invention still further relates to one or more in the there is general formula compound of (I) purposes in the medicine of preparation treatment clone disease.

Compound involved in the present invention can obtain ［ Vassilios P.Papageorgiou et al, Angew.Chem.Int.Ed.1999,38,270 – 300 by conventional method separation in Radix Arnebiae (Radix Lithospermi) by list of references; V.P.Papageorgiou et al, Current Organic Chemistry, 2006,10,2123-2142; V.P.Papageorgiou, K.C.Nicolaou et al.The Chemistry and Biology of Alkannin, Shikonin, and Related Naphthazarin Natural Products[J] .Angew.Chem.Int.Ed.1999,38,270-300 ］.

As get lithospermum euchromum Royle (Radix Arnebiae Arnebia euchroma (Royle) Johnst) pharmaceutical decocting piece, and 55 ℃ of lower 15 times of amount petroleum ether of condition lixiviate 3 times, each 3 hours, extracting solution concentration and recovery solvent, obtained Radix Arnebiae extract.Radix Arnebiae extract, through silica gel column chromatography, is used respectively petroleum ether: ethyl acetate=100:1; 100:1; 100:2; 100:4 ratio eluting, after gained eluent solvent evaporated more respectively with petroleum ether or petroleum ether, ethyl acetate and methanol recrystallization in certain proportion, obtain respectively bronzing crystalline particulate-CAN1, cerise flake-like crystal-deoxidation AK, bronzing lamellar shape crystallization-β, beta-dimethyl-acry-lalkannin.Alpha-Methyl butyryl AK cannot be separated on silica gel column chromatography with isovaleryl AK, is a speckle (colour band), can carry out separation by preparation liquid phase, and separation condition is: mobile phase: THF:H2O=45:55; Flow velocity: 1ml/min; Detect wavelength: 515nm.The separable red dope Alpha-Methyl butyryl AK obtaining and red dope isovaleryl AK.

Accompanying drawing explanation:

Fig. 1 rat body weight changes broken line graph (* P<0.05, * * P<0.01vs.model.)

The DAI index map (* P<0.05, * * P<0.01vs.model.) of Fig. 2 administration after 7 days

The colon length (* P<0.05, * * P<0.01vs.model.) of Fig. 3 administration after 7 days

MPO active (* P<0.05, * * P<0.01vs.model.) in Fig. 4 colon

TNF-alpha levels in Fig. 5 serum (* P<0.05, * * P<0.01vs.model.)

Fig. 6 rat causes scorching parapodum swelling schematic diagram

Fig. 7 rat secondary parapodum swelling schematic diagram

Fig. 8 rat body weight variation diagram

The scoring of Fig. 9 arthritis index

The specific embodiment

Embodiment 1

The mixture of the compound of tool general formula (I) also can directly be obtained by following method from Radix Arnebiae (Radix Lithospermi)

Get lithospermum euchromum Royle (Radix Arnebiae Arnebia euchroma (Royle) Johnst) pharmaceutical decocting piece 100Kg(purchased from medicine store, Yantai), under 55 ℃ of conditions, the lixiviate of 1500L petroleum ether is 3 times, 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.

Through HPLC, analyze, high-efficient liquid phase chromatogram condition is mobile phase: acetonitrile-water-formic acid (700:300:0.5), flow velocity: 1ml/min, detects wavelength: 518nm, column temperature: 30 ℃.Press areas of peak normalization method and calculate content

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, is used respectively petroleum ether: ethyl acetate=100:1; 100:1; 100:2; 100:4 ratio eluting, after gained eluent solvent evaporated more respectively with petroleum ether or petroleum ether-ethyl acetate and methanol recrystallization in certain proportion, obtain respectively bronzing crystalline particulate-CAN1, cerise flake-like crystal-deoxidation AK, bronzing lamellar shape crystallization-β, beta-dimethyl-acry-lalkannin.Alpha-Methyl butyryl AK cannot be separated on silica gel column chromatography with isovaleryl AK, is a speckle (colour band), can carry out separation by preparation liquid phase, and separation condition is: mobile phase: THF:H2O=45:55; Flow velocity: 1ml/min; Detect wavelength: 515nm.The separable red dope Alpha-Methyl butyryl AK obtaining and red dope isovaleryl AK.

The inhibitory action of 1 pair of PDE4 activity of test example

4.1 material

Given the test agent: alkannin derivatives, by embodiment 2 preparations

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 active detect [J]. Beijing Agricultural College's journal, 2003,18 (4): 242-244.], with reference to above-mentioned literature method, prepare pig neutrophil cell, granulocyte is diluted to approximately 10 with Ca/Mg PBS ¹¹individual/L, then this diluent is made to homogenate with glass homogenizer in ice bath, under microscope, confirm that cell is pulverized evenly, must be rich in the enzyme sample of PDE4.

First measure the activity of enzyme: cAMP and be made into 100 μ mol.L with Ca/Mg PBS ^-1enzyme sample is established 4 sampling amounts, with Ca/Mg PBS, is settled to 100 μ L, and application of sample process is carried out in ice bath, concrete application of sample amount in Table 2. application of samples after each pipe put constant-temperature incubation 30min in 35 ℃, then 2-3min cessation reaction in 100 ℃ of water-baths, each pipe, in 4 ℃ of centrifugal 30min of 15000r/min, is got supernatant 70 μ L, with ultra-pure water, dilute 5 times (adding 280 μ lddH2O), HPLC sample introduction 20 μ L, detect 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 hydrolysis percentage rate of substrate:

Next is to measure the impact of Radix Arnebiae extract on PDE activity: enzyme reaction is carried out in Ca/Mg PBS, and cAMP is made into 100 μ mol.L with buffer ^-1, during reaction, get 65 μ L, 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, with buffer, is settled to 100 μ L, and application of sample order is in Table 3.

Table 3 enzyme reaction application of sample order and dosage

After application of sample, each pipe is put constant-temperature incubation 30min in 35 ℃, 2-3min cessation reaction in 100 ℃ of water-baths then, and each pipe, in 4 ℃ of centrifugal 30min of 15000r/min, is got supernatant 70 μ L, with 5 times of ultra-pure water dilutions, (adds 280 μ lddH ₂o), HPLC sample introduction 20 μ L, detect under 254nm wavelength, and mobile phase is methanol: phosphate buffer=10:90.The results detailed in Table 4.

The impact of table 4 Radix Arnebiae (Radix Lithospermi) naphthoquinone compound on PDE4 activity

4.3 conclusion (of pressure testing)s: alkannin derivatives has and suppresses active PDE4.

Test example 2

Radix Arnebiae (Radix Lithospermi) naphthoquinone monomeric compound and contain the therapeutical effect of one or more Radix Arnebiae extract of general formula (I) to rat inflammatory bowel

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 23 ± 2 ℃ of room temperatures, relative humidity 50 ± 10%, 12 hours/day artificial lighting time, automatic wind exhaust, freely drink water and take food, adapting to 7 days and use afterwards.

Dextran sulfate (DSS): molecular weight 5000, purchased from Sigma company;

Salazosulfamide (SASP): purchased from friendship pharmaceutical Co. Ltd of upper Hisense, purity is greater than 95%;

Radix Arnebiae extract (CAN3) is pressed embodiment 1 preparation

CAN1 (CAN1), β, beta-dimethyl-acry-lalkannin (CAN2), by embodiment 2 preparations;

Rat blood serum TNF-α measures test kit: purchased from U.S. R & D System company;

Rat MPO measures test kit: purchased from Nanjing, build up Bioisystech Co., Ltd

Two, experimental technique

1, the foundation of rat enteritis model

DSS is dissolved in distilled water to preparation 3%(W/W) DSS aqueous solution, substitute drinking water and freely drunk 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, 8 every group, 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 is given normal drinking-water, freely drinks; Model group, positive drug group, administration group are given respectively 3%DSS aqueous solution, freely drink; Model group, positive drug group, administration group are by above-mentioned dosage gastric infusion, once a day simultaneously; Model group and normal group are filled with normal saline.Each group is normally feed all.

3, disease (progress degree) is enlivened the assessment of situation

3.1, body weight

Respectively at claiming rat body weight every day before modeling and after modeling, observe that respectively organizing rat body weight changes.

3.2, disease index (disease activity index, DAI)

Observe the stool character of rat and the situation of occulting blood, by following principle, mark, draw the DAI of every rat.

Criteria?for?scoring?disease?activity?index(DAI)

Disease?activity?index(DAI)=combined?score(stool?consistency+bleeding).

*Normal=well-formed?pellets;loose?stools=pasty?stool?that?does?not?stick?to?anus;diarrhoea=liquid?stools?that?stick?to?anus.

4, colon length

After administration 7 days, each organizes rat with 10% chloral hydrate (60mgkg ^-1) intraperitoneal injection of anesthesia.After abdominal aortic blood, separated colon, cuts off enteric cavity along mesentery, with freezing normal saline, cleans twice, launches, with vernier caliper measurement colon length on filter paper.

5, immunohistochemical staining

Get the above-mentioned colon that measures length, get near the tissue of about 0.5cm ulcer, be stored in 10% formalin solution, by the semi-annular jade pendant top hospital pathology department film-making of giving birth of Affiliated Hospital of Qingdao Medical Inst and Yantai City, dyeed, remaining tissue sample in ultra cold storage freezer-80 ℃ frozen.Adopt Computer digital image analysis.Tissue specimen after dyeing amplifies 40-100 doubly by microscope camera system, pickup image.

6, the active mensuration of colon's myeloperoxidase (MPO) (MPO)

Get the colon of above-mentioned sampling, weigh, by 1: 19, add homogenate medium and make 5% tissue homogenate, by test kit description, detect and calculate MPO vigor.The ability of degraded 1 μ mol hydrogen peroxide per minute when MPO activity is defined as 37 ℃, 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 is measured according to the description of the commercial ELISA test kit of R & D company, and unit is ngL ^-1.

8, date processing

Average ± standard deviation for data (MEAN ± SEM) represents, adopts SPSS16.0 software to carry out one factor analysis of variance (ANOVA), and the diversity ratio between each group is with Tukey ' s t check thereupon, and P<0.05 is that difference has significance.

Three, experimental result

1, body weight

As shown in Figure 1, after DSS induction forms ulcerative colitis, rat body weight increasess slowly, and along with the prolongation of the course of disease, body weight declines gradually.With model group comparison, CAN3 group (40mgkg ^-1) from the 3rd day, can significantly suppress the decline (P<0.05) of rat body weight, CAN1, CAN2 group can significantly 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 organize and model group comparison, and DAI score obviously reduces,

3, colon length

After administration 7 days, model group colon length compared with normal group obviously shortens.Positive drug group and administration group and model group comparison, colon length has significant difference.

4, colon's myeloperoxidase (MPO) (MPO) activity

Compare 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

With normal group comparison, the TNF-alpha levels of respectively organizing in rat blood serum of giving 3%DSS aqueous solution obviously raises, but with model group comparison, positive drug group and administration group TNF-alpha levels significance reduce, can suppress the rising of the content of TNF-α in the rat blood serum of DSS induction.

Four, conclusion

Radix Arnebiae (Radix Lithospermi) naphthoquinone compound and contain the rat inflammatory bowel tool significant protective effect of one or more Radix Arnebiae extract of general formula (I) to dextran sulfate induction.Result shows, oral said medicine can significantly 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 compounds and the impact of Radix Arnebiae extract on collagen-induced rat arthritis

3.1 material

Given the test agent: Radix Arnebiae extract, by embodiment 1 preparation.Alpha-Methyl butyryl AK+isovaleryl AK β, beta-dimethyl-acry-lalkannin, by embodiment 2 preparations.

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), 10 every group.During modeling, in every rat left side, pad fully intradermal injection cattle II Collagen Type VI (CIA) Emulsion 0.1ml; After 10 days every rat is apart from the subcutaneous Isodose booster immunization in the 2-3cm of root of the tail portion place once for initial immunity; After 14d, modeling successfully starts administration, successive administration 28d; With toes capacity measurer, surveys the toes volume of all rats left and right foot before modeling, after modeling, measure the left sufficient volume of CIA rat (former swell swollen) every day, and while 10d after modeling starts to survey every other day the whole toe volume of offside; After modeling, 12d starts arthritis index scoring, every 2d scoring 1 time, continuously 32d; 1h after last administration, takes the weight of rat, and abdominal aortic blood is measured 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), reduces polyarthritis index scoring (Fig. 9), the inflammation performance that alleviates CIA rat, alleviates TNF-ɑ content in rat blood serum.

The impact of Radix Arnebiae extract on TNF-ɑ content in CIA rat blood serum

Compare Δ P<0.01 with normal group; Compare * P<0.05, * * P<0.01. with model group

Conclusion: Radix Arnebiae extract has obvious therapeutical effect to collagen-induced rat arthritis.

Test example 4:

The effect to rat copd of Radix Arnebiae extract and alkannin derivatives

1 materials and methods

1.1 material

40 of healthy SD rats, Mus 4～8W in age, body weight (210 ± 20) g, male and female are not limit, and by Shandong Traditional Chinese Medicine University's Experimental Animal Center, are provided.Medicated cigarette is fire-cured tobacco type eight happiness boards (Qingzhou Cigarette Factory produces, tar content 15mg, nicotine content in smoke 1.1mg); LPS (U.S. Sigma company); Tumor necrosis factor TNF-alpha test kit (Tianjin Jiuding Medical Biological Engineering Co., Ltd); Radix Arnebiae extract (pressing embodiment 1 preparation), CAN1, β, β '-dimethyl allene acyl AK (pressing embodiment 2 preparations).1.2 animal groupings and model are set up

40 of healthy SD rats, male and female are not limit, and are divided at random 5 groups, every group 8, Normal group (matched group): raise under home, by rat saline injection 0.2ml in the 1st, 14 days tracheas, within the 8th day, play intraperitoneal injection of saline 0.05ml/kg every day, to 28 days, put to death; Chronic obstructive pulmonary disease (COPD) model group (model group): rat was injected to each 200 μ g/200 μ l of lipopolysaccharide (LPS) in the 1st, 14 days in trachea, 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) 2 times/d of interior incense cigarette, smoking capacity is 14/time, each smoking time 1h wherein, twice smoking is spaced apart 1h.Within the 8th day, play intraperitoneal injection of saline 0.05ml/kg every day, to the 28th day, put to death; Treatment group 1 (Radix Arnebiae extract group): inject LPS and the same model group of incense cigarette process in trachea, play 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): in trachea, inject LPS and the same model group of incense cigarette process, within the 8th day, play oral administration gavage administration every day 80mg/kg, to the 28th day, put to death.

The collection of 1.3 bronchoalveolar lavage fluid (BALF)

After rat is anaesthetized with 2% pentobarbital (25mg/kg), lie on the back and be fixed on operating board, cut chest, expose trachea and two lung, ligation right principal bronchus, in knuckle with trocar puncturing to left lung, slowly inject 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 to hematimeter, counting total white blood cells and M7 (PMN) number.The remaining BALF4 ℃ 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 ℃ of refrigerators.

1.4TNF-α concentration detects

Application TNF-α detection kit, enzyme-linked immunosorbent assay for by specification (ELISA) is measured the concentration of TNF-α.

1.5 Pathologic specimen preparation and Morphological Quantitative Analysis

Get trachea and the middle period lung tissue 5mm at 2～3mm place in the knuckle of every rat ³size, 10% formalin is fixed, the dehydration of ethanol gradient, waxdip, embedding, section, HE dyeing, om observation.By through the fixing 1mm of 4% glutaraldehyde ³right lung is organized rinsing, with fixing after 2% Osmic acid., the dehydration of acetone gradient, 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 of lung interval (MLI), 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, between two groups, relatively with q check, between three groups, relatively use one factor analysis of variance.

2 results

2.1 lung morphology quantitative analyses

With matched group comparison, model group MLI raises, and MAN reduces, difference highly significant (all P<0.01); With model group comparison, treatment group MLI reduces, and MAN raises, and difference highly significant (all P<0.01), in Table 5.

2.2BALF cell counting and PMN number

With matched group comparison, in model group BALF, total white blood cells and PMN number increase, difference highly significant (all P<0.01); With model group comparison, in treatment group BALF, total white blood cells and PMN number obviously decline, and difference highly significant (all P<0.01), in Table 5.

The variation of TNF-alpha levels in 2.3BALF

With matched group comparison, in model group BALF, TNF-α concentration raises, difference highly significant (P<0.01); With model group comparison, treatment group TNF-α concentration reduces, and difference highly significant (P<0.01), in Table 5.

Table 5 is respectively organized the level of BALF of Rats total cellular score, PMN number, TNF-α and lung tissue MLI, MAN comparison (± s, n=8)

With matched group comparison: * P<0.01, ^#p<0.01

3 conclusions

This experiment is set up COPD rat model by injecting LPS method in the smoked cigarette gas-adding pipe of enfleuraging, and can reflect similarly the pathology forming process of human diseases.Meanwhile, analyze and show, the MLI of model group reflection alveolar average diameter increases compared with matched group, and the MAN of reflection alveolar density significantly reduces compared with matched group, and the significant difference of two indexs, illustrates that animal model is successful.

This result shows, treatment group rat pathological examination prompting lung injury alleviates compared with model group rat, and MLI reduces compared with model group, and MAN raises compared with model group, illustrate that treatment group can alleviate COPD injury of lung to a certain extent, has played certain control, mitigation to the reconstruction of emphysema structure.In treatment group BALF of Rats, TNF-α concentration, total white blood cells, PMN number obviously decline compared with model group simultaneously; prompting treatment group can reduce the infiltration of PMN in lung; express cell adhesion molecule-1 (ICAM-1) that inhibition is caused by TNF-α and LPS; TNF-alpha levels is declined; alleviate pulmonary edema; protection PMEC (Pulmonary Microvascular Endothelial Cells), improves microcirculation.Experiment showed, that Radix Arnebiae extract and alkannin derivatives have certain curative effect for treatment chronic obstructive pulmonary disease.

Test example 5:

Radix Arnebiae extract and the alkannin derivatives therapeutical effect to 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 U.S. Sigma company, IgE, IL-4, IL-5, IL-13 and IFN-γ detection kit are purchased from Jing Mei biological engineering company limited, Radix Arnebiae extract is pressed embodiment 1 preparation, CAN1, β, β '-dimethyl allene acyl AK is pressed embodiment 2 preparations.

1.2 animal groupings and model preparation

60 healthy male rats of SPF level adopt table of random number method 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 OVA aluminium hydroxide mixed liquor 1ml (OVA100mg+ aluminium hydroxide 100mg+ normal saline 1ml) for rat.The 1st day lumbar injection sensitization, within the 7th day, repeat to strengthen sensitization 1 time, within the 14th day, insert in colourless, transparent, airtight glass box, with 1%OVA (OVA100mg+ normal saline 10ml) aerosol inhalation, excite (mist particle diameter 2.5-5 μ m), 1 time/d, 30min/ time, continuous 2 weeks, equal-volume physiologic saline for substitute OVA aluminium hydroxide mixed liquor for I group rats by intraperitoneal injection sensitization, ultrasonic atomizatio sucks and excites with equal-volume physiologic saline for substitute OVA, III group rat excites front 1h gastric infusion 120mg/kg at every turn, and IV, V group rat excites front 1h gastric infusion 60mg/kg at every turn.VI group rat excites front with equal-volume physiologic saline for substitute treatment group gastric infusion at every turn.All rats 3% pentobarbital sodium (1ml/kg) intraperitoneal injection of anesthesia for 6h after last excites, after dorsal position is fixing, open breast, get central vein blood 2ml and measure IgE content, in left lung main bronchus, cut an osculum, after inserting diameter 3mm plastic flexible pipe, fix, with 4 ℃ of capable bronchoalveolar lavage of normal saline, each consumption 2ml, lavation is 3 times continuously, response rate >70%, 4 ℃ of bronchoalveolar lavage fluid (BALF) is centrifugal, 1200r/min, 10min, get cell sediment for cell counting and classification, supernatant-20 ℃ preservation is for the detection of cytokine.1.3BALF cell counting and classification

BALF 2ml Hanks dilution for centrifugal rear cell sediment, gets 1ml and measures total white blood cells in hematimeter, and get 0.2ml and make cell smear, Wright's staining, 400 power microscopes are down to 200 cells of minority, 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 DASELISA immunoabsorption (Sandwich ELISA) to measure, by reagent description, operate.

1.5 statistical procedures

Adopt SPSS13.0 statistical software to carry out statistical procedures to data, data are used

represent, between organizing respectively relatively, P<0.05 is that difference has statistical significance to the check of employing independent sample t.

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, the breathing of nodding, and mouth and nose cyanosis, activity obviously reduces, and I group rat is without above-mentioned reaction, and III, IV, V group rat react 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 can significantly reduce neutrophilic granulocyte in BALF, lymphocyte, eosinophilic granulocyte (P<0.01), II group and VI group neutrophilic granulocyte, lymphocyte, eosinophilic granulocyte and the mononuclear phagocyte counting equal not statistically significant of difference (P>0.05), in Table 6.

Table 6 is respectively organized BALF of Rats cell divide and counting

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 are significantly higher than I group (P<0.01), compare with II group, III-V group can significantly reduce in venous blood IL-4, IL-5, IL-13 in IgE and BALF, the IFN-γ (P<0.01) significantly raising 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 is respectively organized IL-4, IL-5 in rat vein blood IgE and bronchoalveolar lavage fluid, IL-13, the comparison of IFN-γ content

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 discovery, 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, Ig E is synthetic to be increased; And gastric infusion Radix Arnebiae (Radix Lithospermi) treatment group (III, IV, V) can reduce the generation of IL-4 in Asthmatic Rat Lung, IL-5, IL-13, promote the generation of IFN-γ, alleviate neutrophilic granulocyte, lymphocyte, eosinophils, make the synthetic minimizing of Ig E, 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 it is Th2 cell that prompting Radix Arnebiae (Radix Lithospermi) may stop Th0 cell differentiation, promotes that Th0 cell differentiation is Th1 cell; The generation of IL-5, IL-13 and Ig E reduces prompting Radix Arnebiae (Radix Lithospermi) 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, thereby reach the object of control asthma attack.

Claims (2)

1. the compound with following general formula (I) is treated the purposes in chronic obstructive pulmonary disease medicine in preparation,

R represents H, COCH ₃, COCHC (CH ₃) ₂, COCH (CH ₃) CH ₂cH ₃, COCH ₂cH (CH ₃) ₂.

2. one kind contains the purposes of one or more Radix Arnebiae extract of general formula (I) in preparation treatment chronic obstructive pulmonary disease medicine.

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