CN103830662B

CN103830662B - A kind of application of Chinese medicine composition in treatment medicine for treating rheumatoid arthritis is prepared

Info

Publication number: CN103830662B
Application number: CN201410014362.6A
Authority: CN
Inventors: 魏聪; 王宏涛; 张会欣; 常丽萍; 肖维刚; 安军永
Original assignee: Shijiazhuang Yiling Pharmaceutical Co Ltd
Current assignee: Shijiazhuang Yiling Pharmaceutical Co Ltd
Priority date: 2012-05-26
Filing date: 2012-05-26
Publication date: 2017-09-26
Anticipated expiration: 2032-05-26
Also published as: CN103830662A

Abstract

The invention discloses a kind of application of Chinese medicine composition in treatment medicine for treating rheumatoid arthritis is prepared.It is formed by Chinese traditional medicine compositions such as the Radix Astragali, the fruit of glossy privets.The Chinese medicine composition has effects that supplementing qi and nourishing yin, strengthening spleen, tonifying kidney, dissipating bind dredging collateral, the tangent interpretation of the cause, onset and process of an illness, and experiment confirms that Chinese medicine composition of the present invention can effectively suppress angiogenesis, can be clinically used for rheumatoid arthritis.Rationally, adverse drug reaction is small, is available for patient to be used for a long time for compatibility of the present invention.

Description

A kind of application of Chinese medicine composition in treatment medicine for treating rheumatoid arthritis is prepared

This present patent application is divisional application, and original bill information is as follows：

Application number：201210166200.5

The applying date：On May 26th, 2012

Denomination of invention：A kind of application of Chinese medicine composition in the medicine for suppressing angiogenesis is prepared.

Technical field

The present invention relates to a kind of new application of Chinese medicine composition, in particular it relates to which a kind of Chinese medicine composition is preparing suppression Application in the medicine of angiogenesis, category application in TCM field.

Background technology

Angiogenesis (i.e. new vessels is formed) includes angiogenesis and angiogenesis；Angiogenesis refers to by existing blood vessel New blood vessel is formed by way of budding, angiogenesis, which refers to raise by angioblast/endothelial progenitor cells, arrives new green blood Pipe formation site is simultaneously divided into endothelial cell.Angiogenic process is promoting angiogenesis and is suppressing angiogenesis under normal circumstances The fine adjustments of the various factors be issued to poised state.Angiogenesis is in human development, tissue repair, menstrual cycle of female etc. Played a significant role in normal physiological processes.Meanwhile, a variety of advancings of disease are also closely related with blood vessel angiogenesis.

Compared with normal blood vessels, tumor vascular structure, which lacks between integrality, endothelial cell, to be present compared with big gap, penetrating Property it is strong, plexus structure disturbance has substantial amounts of blood vessel cecum and arteriovenous shunt, and the exception of this structure causes to ooze out increase And high pressure between tissue, shifted while being also easy to cancer cell.The growth of tumour has two different stages, i.e., from avascular Slow growth phase is changed into the fast breeding stage for having blood vessel.If without angiogenesis, the growth of primary tumo(u)r will not surpass Cross 1 ~ 2 mm³.Suppress the new strategy that angiogenesis is antineoplaston.Generally except in menstrual period, inflammation or wound When, the vascular endothelial cell of adult is in inactive state.VEGF (vascular endothelial Growth factor, VEGF), basic fibroblast growth factor (bFGF) be important angiogenesis factor, it is thin to endothelium Born of the same parents have high degree of specificity, and the medicine using them as target is proved to few side effect.Cell surface receptor and The process that extracellular factors can be generated with modulating vascular, this prompting medicine can not be absorbed by cell and play interference angiogenesis Effect.

Except excessively unconfined growth, the feature of tumour cell includes adhesion, migration, invasion and attack, and this is also that tumour cell leaves Primary tumor, by blood fortune and body cavity to body part or remote organ send out the reason for.Distal end of the angiogenesis to tumour cell Send out also critically important.The function of tumour cell and the transfer ability of tumour cell are closely related.Focal adhesion kinase (FAK) is thin Born of the same parents' sticks and vital effect is played in migrating, and focal adhesion kinase (FAK) inhibitor can effectively prevent tumour cell Transfer.

Phosphatidyl-inositol 3-kinase (PI3K) signal participates in the various kinds of cell work(such as propagation, differentiation, apoptosis and glucose transport The regulation of energy.Discovered in recent years, IA types PI3K and molecule protein kinase b (Akt) is constituted downstream signal path and the mankind The generation development of tumour is closely related.The propagation of path regulation tumour cell and survival, its activity is abnormal can not only to be caused carefully Born of the same parents' vicious transformation, and migration to tumour cell, stick, the degraded of Tumor Angiongesis and extracellular matrix etc. it is related, The ideas of cancer therapy using PI3K-Akt signal paths key molecule as target spot is developing at present.Wortmannin (Wortmannin) it is exactly wherein most typical representative, clinical investigation phase is come at present.Wortmannin structural formula is such as Under：

CAS accession number：19545-26-7.

The migration of tumour cell and being attached in tumor invasion and metabasis plays an important role.PI3K can transmit integrin institute The invasion and attack signal of mediation, especially to integrin alpha₂β₁、α₆β₄And α_Vβ₃Invasion and attack behavior be it is necessary, such as PI3K mediate prostate cancer Middle integrin alpha_Vβ₃The invasion and attack characteristic of driving, in breast cancer and oophoroma, Akt2 overexpression can be by IV collagen types Adjust integrin beta₁So as to increase the invasion and attack and transfer of cell.

The etiology unknown of current rheumatoid arthritis, rheumatoid arthritis is mainly shown as the destruction of joint of progressive, most After cause to be mainly shown as that synovial membrane breeds that to form synovial tissue's villiform into articular cavity raised on dysfunction, morphology, increase Raw synovial tissue produces a variety of factors, and substantial amounts of vascular components are contained in the effect with destruction cartilage, the synovial tissue of hyperplasia. Data shows that angiogenesis plays an important role in the sick progress.For patient with rheumatoid arthritis, angiogenesis It is a complex process by Angiogensis and suppression angiogenesis agent modulates.

Diabetic retinopathy (diabetic retinopathy, DR) is a kind of diabetes chronic blood for jeopardizing eyesight Pipe complication, whether insulin-dependent or non-insulin-depending type patient, DR finally influences nearly all patient of diabetes Person.The characteristics of DR, shows as the retinal microvascular disease progressively aggravated, and specific pathological manifestations have microcirculation disorder, retina micro- Blood vessel neoplasia, blood-retina barrier destruction, capillary occlusion, retinal nonperfusion area and new vessels formation etc., also Vitreous hemorrhage and detachment of retina may be triggered, the infringement of visual function irreversibility is ultimately resulted in.Eyeground regard pars papillaris or other There is new vessels in position, more with connective tissue proliferation, visual function is seriously damaged and causes that blind person is quite a few to see, therefore Suppress new vessels and occur being the important step for treating BDR (DR).

The unstable of atherosclerosis (AS) patch causes plaque rupture, and then occurs acute coronary artery syndrome (ACS).Recent study finds that angiogenesis can promote the development of atherosclerotic lesion in patch, increases the unstable of patch Property, therefore angiogenesis may play key effect to stablizing vulnerable plaque in suppression patch.Therapeutic Angiogenesis can be with Promote local vascular newborn, improve myocardium compensatory capacity, be finally reached the purpose for the treatment of ischemic heart disease.

The content of the invention

The present invention relates to a kind of new application of Chinese medicine composition, in particular it relates to which a kind of Chinese medicine composition is preparing suppression Application in angiogenesis drug.

Chinese medicine of the present invention can be according to《National Chinese medicine preparation specification》Or《Dictionary of medicinal plant》Processed.The present invention Medicine is made up of the bulk drug of following weight ratio:

Radix Astragali 120-360, fruit of glossy privet 100-300, ginseng 30-95, ganoderma lucidum 30-95, curcuma zedoary 65-195, bighead atractylodes rhizome 30-90, Sculellaria barbata 65-195, gynostemma pentaphylla 120-360, Poria cocos 30-95, the membrane of a chicken's gizzard 15-45, mock-strawberry 65-195, bittersweet 65-195, oriental wormwood 65-195, paniculate swallowwort 65-195, ground bettle 10-30, oldenlandia diffusa 65-195.

It is preferred that, the Chinese medicine composition is made up of the bulk drug of following parts by weight:

The Radix Astragali 120, the fruit of glossy privet 300, ginseng 30, ganoderma lucidum 95, curcuma zedoary 65, the bighead atractylodes rhizome 90, Sculellaria barbata 65, gynostemma pentaphylla 360, Poria cocos 30th, the membrane of a chicken's gizzard 45, mock-strawberry 65, bittersweet 195, oriental wormwood 65, paniculate swallowwort 195, ground bettle 10, oldenlandia diffusa 195.

Or

The Radix Astragali 360, the fruit of glossy privet 100, ginseng 95, ganoderma lucidum 30, curcuma zedoary 195, the bighead atractylodes rhizome 30, Sculellaria barbata 195, gynostemma pentaphylla 120, Poria cocos 95, the membrane of a chicken's gizzard 15, mock-strawberry 195, bittersweet 65, oriental wormwood 195, paniculate swallowwort 65, ground bettle 30, oldenlandia diffusa 65.

Or

The Radix Astragali 250, the fruit of glossy privet 200, ginseng 65, ganoderma lucidum 65, curcuma zedoary 132, the bighead atractylodes rhizome 64, Sculellaria barbata 128, gynostemma pentaphylla 256, Poria cocos 65, the membrane of a chicken's gizzard 30, mock-strawberry 128, bittersweet 128, oriental wormwood 128, paniculate swallowwort 128, ground bettle 20, oldenlandia diffusa 128.

It is preferred that, in the raw materials used medicine of Chinese medicine composition, the bighead atractylodes rhizome is rhizoma atractylodis macrocephalae.

It is made up present invention also offers the active component of the Chinese medicine composition of following steps:

(1) Chinese medicine, is weighed according to bulk drug part by weight, is cleaned；

(2), the fruit of glossy privet, people participate in 6-10 times and measure 50-90% ethanol extraction 1-3 times, each 1-4 hours, merge extract solution, Filtering, filtrate recycling ethanol is to without alcohol taste, and filtrate and the dregs of a decoction are standby；

(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort add 4-8 times to measure water and extract volatile oil, collect volatile oil, another device is collected, residue and The aqueous solution is standby;

(4), ground bettle, the membrane of a chicken's gizzard, Poria cocos are ground into fine powder；

(5), the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with gained in step (2) Gained curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort carry residue after oil and merged in residue after ginseng, the alcohol extracting of the fruit of glossy privet, and step (3), plus 7-10 Amount water, heats and decocts 1-3 times again, each 1-3 hours, merges ginseng, fruit of glossy privet filtrate obtained by decoction liquor, addition step (2) And step（3）In the aqueous solution, be concentrated into decoction 65 DEG C survey when relative density be 1.15-1.30, dry, pulverize, it is standby;

Step（4）Gained comminuted powder, step（3）Gained volatile oil and step（5）The dried cream powder of gained is collectively formed in this The active component of drug composition.

The formulation of medicine of the present invention is capsule, tablet, powder, oral liquid, soft capsule, pill, tincture, syrup, bolt Agent, gel, spray or injection.

Wherein the preparation method of capsule, is made up of following steps：

(2), ginseng, the fruit of glossy privet, plus 8 times of 70% alcohol refluxs of amount are extracted 2 times, 3 hours for the first time, second 2 hours, are closed And extract solution, ethanol is reclaimed to without alcohol taste, and filtrate and ginseng, fruit of glossy privet residue are standby;

(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort, merge and extract volatile oil, carry the oily time no less than 8 hours, and the another device of volatile oil is received Collection, residue and the aqueous solution are standby;

(4), ground bettle, the taste animal drugs of the membrane of a chicken's gizzard two, washing, 60 DEG C of drying merge with Poria cocos, are ground into 100 mesh powder, It is standby after sterilizing;

(5) institute in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2) Obtain gained curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort in residue after ginseng, the alcohol extracting of the fruit of glossy privet, and step (3) and carry residue merging after oil, plus 9 times Water is measured, heating is decocted 2 times, 2 hours every time, merges decoction liquor, adds gained ginseng, fruit of glossy privet alcohol extract and step in step (2) Suddenly（3）In the aqueous solution, be condensed into relative density 1.20-1.25 clear cream, dry, pulverize, get dry extract powder；By gained dried cream powder Add appropriate pharmaceutically acceptable auxiliary material granulation；

(6) gained volatile oil in step (3), is sprayed into the fine powder of gained Poria cocos, ground bettle, the membrane of a chicken's gizzard in step (4) In, mix, with step（5）The particle of middle gained is mixed, closed half an hour, encapsulated to produce.

Other formulations of medicine of the present invention are weighed after bulk drug in proportion, are prepared using conventional preparation method, for example, model Bi Ting《Pharmacy of Chinese materia medica》（Shanghai Science Press December the 1st edition in 1997）The preparation technology of record, pharmacy, which is made, to be connect The regular dosage form received.

To enable above-mentioned formulation to realize, pharmaceutically acceptable auxiliary material need to be added when preparing these formulations, for example：Filling Agent, disintegrant, lubricant, suspending agent, adhesive, sweetener, flavouring, preservative, matrix etc..Filler includes：It is starch, pre- Gelling starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose etc.；Disintegrant includes：Starch, pregelatinized starch, crystallite Cellulose, sodium carboxymethyl starch, PVPP, low-substituted hydroxypropyl cellulose, Ac-Di-Sol etc.； Lubricant includes：Magnesium stearate, lauryl sodium sulfate, talcum powder, silica etc.；Suspending agent includes：Polyvinylpyrrolidine Ketone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methyl cellulose etc.；Adhesive includes, starch slurry, polyvinylpyrrolidone, Hydroxypropyl methyl cellulose etc.；Sweetener includes：Saccharin sodium, aspartame, sucrose, honey element, enoxolone etc.；Flavouring bag Include：Sweetener and various essence；Preservative includes：Parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzene prick bromine Ammonium, acetic acid chloroethene are fixed, eucalyptus oil etc.；Matrix includes：PEG6000, PEG4000, insect wax etc..In above-mentioned formulation is realized Medicine pharmacy, need to add pharmaceutically acceptable other auxiliary materials when preparing these formulations（Fan Biting《Pharmacy of Chinese materia medica》, Shanghai section Learn the auxiliary material that each formulation is recorded in publishing house December the 1st edition in 1997）.

It is experimentally confirmed that pharmaceutical composition of the present invention has directly to angiogenesis and vascular endothelial cell with migration in vitro Inhibitory action, has direct repression, and have aobvious to capilary, Cell-matrix adhesion and cell migration for angiogenesis The inhibitory action of work.Meanwhile, it is capable to significantly suppress FAK phosphorylations, collaboration Fak inhibitor, which has, suppresses angiogenesis function.

It is experimentally confirmed that pharmaceutical composition of the present invention can cooperate with Fak inhibitor to prevent metastases, PI3K can also be cooperateed with to press down Preparation and AKT (serine/threonine kinase) inhibitor prevent metastases.

Experiment also demonstrates that the Chinese medicine composition can directly suppress tumour cell, and can prevent cancer metastasis, special It is not to play the role of preferably to prevent transfer to osteosarcoma, lung cancer, human breast carcinoma, prostate cancer, colon cancer or stomach cancer.

The Chinese medicine composition can effectively suppress the blood vessel of rheumatoid arthritis, diabetic retinopathy and artery plaque Generation, and effectively treatment rheumatoid arthritis, diabetic retinopathy and prevention of arterial patch increase and come off.

Compatibility of the present invention is rationally, simple and easy to apply, is pure Chinese medicinal preparation, adverse reaction is small, is available for patient to be used for a long time.

Brief description of the drawings

The effect that Fig. 1 DME25 are generated to external HECV cells capilary tubule.HECV cells be located at basilar memebrane interlayer it Between, with HECV as a control group（A）, Fak inhibitor intervention group（25nM）（B）, DME25 intervention groups（1:2000）（C）, FAK suppressions Preparation and DME25 Combination intervention groups（D）.Put micro- sem observation tubule image（A-D）, length is quantified with Digital image analysis technique （E）.DME25 can significantly inhibit tubule formation and there is synergy between Fak inhibitor.

Fig. 2 DME25 are not good enough to the action effect of vitro endothelial cell growth.It is thin with the DME25 processing of different diluted concentrations Born of the same parents 72h.Cell count is carried out with crystal violet.There is no obvious effect to HECV cell growths.

Fig. 3 A：DME25 dose-dependent inhibition HECV Cell-matrix adhesions, DME25 is diluted from 1:40 to 1:125, 000 (A).Dilution is less than 1:25,000 display inhibitory action.B：The three-dimensional imaging of adhesion.Left side：Blank control.Right side： 1: The cell of the DME25 processing of 1,000 dilutions.X-axis：Frequency.Y-axis：Resistance.Z axis：Time.C and D：Conventional method research is thin Cytoplasmic matrix is adhered to.C：Gentian violet dyeing adherent cell imaging.D：Adherent cell number under per high power field.With blank control group ratio Compared with * p<0.01；Compared with independent DME and independent Fak inhibitor,^#p<0.01。

Concentration-dependent relation between Fig. 4 DME25 and cell adhesion（A）With the dependence of FAK approach（B）.Shown data are come Come from Rb models.

The effect of Fig. 5 .DME25 inhibitory action and FAK.DME25, which can be substantially reduced, to be sticked, and it is acted on suppresses with FAK It is enhanced when agent is combined, migration path shown in figure（A）And threedimensional model（B）.i：Control group；ii:Fak inhibitor intervenes cell （25nM）;iii：DME25 intervenes cell（1:1000）；ⅳ：Ii and iii Combination interventions.X-axis：Frequency；Y-axis：Resistance；Z axis： Time.

The inhibitory action of Fig. 6 DME25 cell migrations and FAK effect.DME25 can substantially reduce cell migration, and it is made It is enhanced used in when being combined with Fak inhibitor, migration path shown in figure（A）And threedimensional model（B）.i：Control group；ii:FAK Inhibitor intervenes cell（25nM）;iii：DME25 intervenes cell（1:1000）；ⅳ：Ii and iii Combination interventions.X-axis：Frequency；Y Axle：Resistance；Z axis：Time.The time point of arrow prompting electric injury induction in A figures.

Fig. 7 DME25 suppress HECV endothelial cells FAK and Paxillin phosphorylation.After serum starvation 2h, DME25（2 kinds Various concentrations, 1:1000 and 1:5000）, Fak inhibitor（25nM）Or the Combination intervention HECV cells 60min of the two. Separated on SDS- polyacrylamide gel electrophoresises after equal protein, with FAK and paxillin phospho-specific antibody to phosphorylation FAK and Paxillin carries out probe in detecting.

Focal adhesion kinase in Fig. 8 HECV cells（FAK）With pFAK immunofluorescence dyeings.20,000 HECV cells add respectively To blank cultures, DME25 (1:1,000), Fak inhibitor and the two combination in.Cell is rinsed and consolidated after 2 hours It is fixed, after being handled respectively with anti-FAK and anti-pFAK antibody, total FAK（Left side）With FAK phosphorylations（Right side）Contaminated deeply.Pass through 100ms FAK imagings are obtained after irradiation, and pFAK imagings are obtained after 400ms irradiations.

Fig. 9 DME25 to MCF-7 (on), MG-63 (in) and A549 cells (under) matrix attachment must influence.It is dense Degree is higher than 1:125,000 have inhibitory action.

Figure 10 widebands detection DME25 suppresses cell adherence.3D ideographs confirm effect of the extract to cell adherence, X-axis For frequency；Y-axis is resistance；Z axis is the time.

Figure 11 A549 cell Rb mode datas.

Figure 12 DME25 suppress A549 cell migrations.

Effect of Figure 13 PI3K/AKT paths in DME25 induces DU-145 cell adherences.A:Add Wortmannin Influence to cell adherence.B：AKT suppresses and effects of the DME25 to cell adherence.

Influences of Figure 14 DME25 to A549 cells AKT (Ser 473) phosphorylation.A:West-blot detects AKT (Ser 473) phosphorylation；B:ImageJ. Bar graphical analyses pAKT/GAPDH band ratios.

Embodiment

Embodiment 1：

Raw material medicine composition is：

Radix Astragali 250g, fruit of glossy privet 200g, ginseng 65g, ganoderma lucidum 65g, curcuma zedoary 132g, rhizoma atractylodis macrocephalae 64g, Sculellaria barbata 128g, strand Stock indigo plant 256g, Poria cocos 65g, the membrane of a chicken's gizzard 30g, mock-strawberry 128g, bittersweet 128g, oriental wormwood 128g, paniculate swallowwort 128g, ground bettle 20g, Oldenlandia diffusa 128g.

Preparation method is：

(3), curcuma zedoary, rhizoma atractylodis macrocephalae, paniculate swallowwort, merge and extract volatile oil, carry the oily 8 hours time, the another device of volatile oil is collected, residual Slag and the aqueous solution are standby;

(4), ground bettle, the membrane of a chicken's gizzard, washing, 60 DEG C of drying merge with Poria cocos, 100 mesh powder are ground into, in 3KGY⁶⁰CO-r It is standby after radiation sterilization;

(5), the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with gained in step (2) Gained curcuma zedoary, rhizoma atractylodis macrocephalae, paniculate swallowwort propose the merging of the residue after oil in residue after ginseng, the alcohol extracting of the fruit of glossy privet, and step (3), plus 9 Times amount water, heating is decocted 2 times, 2 hours every time, merges decoction liquor, add gained ginseng in step (2), fruit of glossy privet alcohol extract and Step（3）In the aqueous solution, be condensed into the clear cream of relative density 1.25, dry, pulverize, it is standby;

(6) dried cream powder obtained by step (5), is added into 134 grams of starch, 85% alcohol granulation is used；

(7), gained volatile oil in step (3) dissolve with 85% ethanol, spray into step (4) it is middle obtained by Poria cocos, ground bettle, In the fine powder of the membrane of a chicken's gizzard, mix, with step（6）The particle of middle gained is mixed, closed half an hour, is loaded 1000 capsules and is produced.

Embodiment 2：

Raw material medicine composition is：

Radix Astragali 360g, fruit of glossy privet 100g, ginseng 95g, ganoderma lucidum 30g, curcuma zedoary 195g, rhizoma atractylodis macrocephalae 30g, Sculellaria barbata 195g, strand Stock indigo plant 120g, Poria cocos 95g, the membrane of a chicken's gizzard 15g, mock-strawberry 195g, bittersweet 65g, oriental wormwood 195g, paniculate swallowwort 65g, ground bettle 30g, spend in vain HERBA HEDYOTIS DIFFUSAE 65g.

Preparation method is：

(2), ginseng, the fruit of glossy privet, plus 6 times of 90% alcohol refluxs of amount are extracted 4 hours, and extract solution reclaims ethanol to without alcohol taste, filter Liquid and ginseng, fruit of glossy privet residue are standby；

(3), curcuma zedoary, rhizoma atractylodis macrocephalae, paniculate swallowwort merge, plus 4 times of amount water extract volatile oil, carry oily 10 hours time, volatile oil Another device is collected, and residue and the aqueous solution are standby;

(4), ground bettle, the membrane of a chicken's gizzard, washing, 60 DEG C of drying merge with Poria cocos, 100 mesh powder are ground into, in 3KGY⁶⁰CO-r It is standby after radiation sterilization；

(5), the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with gained in step (2) Gained curcuma zedoary, rhizoma atractylodis macrocephalae, paniculate swallowwort propose the merging of the residue after oil in residue after ginseng, the alcohol extracting of the fruit of glossy privet, and step (3), plus 7 Amount water, heating decoction 3 times again, 1 hour for the first time, 2 hours for the second time, third time 3 hours merged decoction liquor, addition step (2) Middle gained ginseng, fruit of glossy privet alcohol extract and step（3）In the aqueous solution, be condensed into the clear cream of relative density 1.15, dry, powder It is broken, it is standby；

(6) dried cream powder obtained by step (5), is added into 112 grams of starch, 78% alcohol granulation is used；

(7), gained volatile oil in step (3) dissolve with 85% ethanol, spray into step (4) it is middle obtained by Poria cocos, ground bettle, In the fine powder of the membrane of a chicken's gizzard, mix, with step（6）The particle of middle gained is mixed, and routinely 1000 tablets are made in formulation method.

Embodiment 3：

Raw material medicine composition is：

Radix Astragali 120g, fruit of glossy privet 300g, ginseng 30g, ganoderma lucidum 95g, curcuma zedoary 65g, bighead atractylodes rhizome 90g, Sculellaria barbata 65g, gynostemma pentaphylla 360g, Poria cocos 30g, the membrane of a chicken's gizzard 45g, mock-strawberry 65g, bittersweet 195g, oriental wormwood 65g, paniculate swallowwort 195g, ground bettle 10g, oldenlandia Careless 195g.

Preparation method is：

(2), ginseng, the fruit of glossy privet, plus 10 times of 50% alcohol refluxs of amount are extracted 2 times, 3 hours for the first time, second 2 hours, are closed And extract solution, ethanol is reclaimed to without alcohol taste, and filtrate and ginseng, fruit of glossy privet residue are standby；

(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort merge, and add 10 times of amount water and extract volatile oil, carry oily 6 hours time, volatile oil Another device is collected, and residue and the aqueous solution are standby；

(5), the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with gained in step (2) Gained curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort propose the merging of the residue after oil in residue after ginseng, the alcohol extracting of the fruit of glossy privet, and step (3), plus 10 Amount water, heating decoction 3 hours again, merges gained ginseng, fruit of glossy privet alcohol extract and step in decoction liquor, addition step (2)（3）In The aqueous solution, be condensed into the clear cream of relative density 1.30, dry, pulverize, it is standby；

(6), gained volatile oil in step (3) dissolve with 80% ethanol, spray into step (4) it is middle obtained by Poria cocos, ground bettle, In the fine powder of the membrane of a chicken's gizzard, mix, with step（5）The dried cream powder of middle gained, routinely formulation method 1000 ball pills are made.

To confirm medicine of the present invention in therapeutic effect, with the capsule as made from embodiment 1（Medicine hereinafter referred to as of the present invention）, Tests below research is carried out：

The experiment that the Drug inhibition angiogenesis of the present invention of experimental example 1 and medicine of the present invention act synergistically with Fak inhibitor

Materials and methods

Material

Human umbilical vein endothelial cells (HECV) are purchased from Interlab（Milan, Italy）.These cells maintain cell Among culture medium（DMEM）（Sigma-Aldrich, pul, many Saites, Britain）, it is aided with penicillin, streptomysin and 10% tire Cow's serum（Sigma-Aldrich）.Cell culture is in 37 DEG C, 5%CO₂Carried out with 95% humidity.Artificial basement membrane（Restructuring Basilar memebrane）Purchased from Collaborative Research Products companies（Bedford, Massachusetts, the U.S.）.One Plant selective depressant focal adhesion kinase（FP573228）Purchased from Tocris companies（Bristol, Britain）.Paxillin Come from transduction experiment room, phospho-specific antibody（PFAK and pPaxillin）It is public purchased from santa-cruz biotechnologys Department（Santa Cruz, California, the U.S.）.

Method

Test medicine is pre-processed

Carried out for convenience of experiment, by medicine of the present invention（Shijiazhuang Yiling Pharmaceutical Co., Ltd produces）Make following pre- place Reason：Medicament capsule content 400mg of the present invention, adds 20 revs/min of rotating wheel, 12 hours, rotating speed under DMSO1ml, normal temperature 14000rpm is centrifuged 30 minutes, takes supernatant, adds balanced salt solution, and filtration surveys photometric absorbance in 405nm, dilutes extract solution To photometric absorbance 0.25, referred to herein as DME25, packing preserves the extract as standardization, standby.

In-vitro cell growth method

HECV cells are with every 3000 cell kinds in hole on 96 orifice plates.Be incubated an evening with triplicate culture plate, 3 days and 5 days.After fully culture, plank is removed from incubator, the violet staining with 5% is fixed with 4% formaldehyde.Then Crystal violet is sloughed with 10% acetic acid, Bio-Tek ELx800 multi-function microplate readers are used（Bio-Tek instrument companies, Vermont State, the U.S.）Detect the density of cell.

Electronic cellular matrix impedance sensing（ECIS）Based on cell adherence and migration test

ECIS-Z θ instrument（Using biophysics, New Jersey, the U.S.）It is used for cell adherence and motility（Damage inspection Survey）Detection research.Cell model employs ECIS RbA modeling softwares, is provided by manufacturer.Current producer uses 96W1E times Row.ECIS is that the interaction between cell and its substrate adhered to is measured by being placed in the gold film electrode on culture dish surface. With array surface of the post processing containing NAC solution, the array complete medium culture 1 hour.In each culture hole Add the cell of identical quantity.In cell adhesion experiment, its adhesiveness is tracked at once after cell is added into array.For Cell migration assay, cellular array reaches after 3 hours to be converged.Cell monolayer is hindered 20 seconds with 2000MA electric current electricity.Cellular layer Impedance and resistance record 20 hours.Signal transduction inhibitor detects that respective inhibitor is all among them in experiment plate hole. Adhesion and migration are to copy to use ECIS RbA cell modeling softwares.

External segment dislocation experiment

External capilary segment dislocation is assessed to be tested using basilar memebrane capillary formation.In serum free medium Under, 250 μ g basilar memebrane is planted on 96 orifice plates, and be placed in incubator at least 40 minutes and be allowed to gel.After this, 35000HECV is inoculated into the basalis containing and without MDE25 or Fak inhibitor and cultivated 4-5 hours, pipe in the training period Chamber formation high-power microscope is recorded and shot.Total tube chamber girth is quantified with Image J softwares in each visual field.

Immunofluorescence dyeing（IFC）

HECV cells kind (LAB-TEK Fisher, Britain) in 16 hole chamber slides, is incubated overnight by 20000/hole （25）.Nutrient solution is absorbed, 4% formalin fixes the min of cell 20, and BBS is incubated at room temperature 20 min, then 0.1 % is added dropwise Triton X-100 BBS solution, the min of cell perforation 5.Using MenaPath Autowash buffer confining liquids（A. Menarini Diagnostics, Britain）20 min are incubated, to block non-specific binding, every 5 milliliters of confining liquid blood containing horse (Sigma, Britain) 2 drips clearly.After buffer solution rinse 2 times, using specific antibody labelled protein paxillin, p-Paxilli, FAK, p-FAK (Santa-Cruz, the U.S.).Primary antibody is with 1:The h of incubated cell 1 after 100 dilutions, buffer solution rinse 3 times is removed residual Remaining primary antibody.The anti-mouse secondary antibody (Insight Biotechnology, Britain) of FITC marks is added dropwise, slide is placed in shaking table On, lucifuge is incubated 1 h.Finally, slide rinse three times, remove uncombined secondary antibody, Fluor-save mountings (Calbiochem- Novabiochem, Britain) after, using Olympus BX51 fluorescence microscopes in 100 times of thing Microscopic observations.

Gel electrophoresis and immune protein

Cell is in 25 cm³Fusion is grown in tissue culture flasks, cell is collected, HCMF buffer solutions are added（Containing 1% Triton X-100,2 mM CaCl2,100 μ g/ml phenylmethylsulfonyl fluorides, 1 mg/ml leupeptins, 1 mg/ml suppression eggs White BPTI and, 10 mM sodium orthovanadates）, the h of cell lysis 1 in rotor wheel instrument is placed in, 13,000g centrifugations remove insoluble Thing.Gained sample carries out protein quantification using Bio-Rad DC protein reagents box (Bio-Rad, the U.S.).

Protein sample is sufficiently separated, using Hybond-C Extra nitrocellulose filters (Amersham Biosciences, Britain) transferring film, after the closing of 10% milk, determine the expression of specific protein.Be respectively adopted anti-pFAK antibody and The FAK and paxillin (27) of anti-pPaxillin antibody labelings phosphorylation.Using GAPDH specific antibodies (Santa- Cruz, the U.S.) GAPDH expression is determined, to evaluate the total protein levels and uniformity of sample.Protein band uses SWDED substrates Chemiluminescence system is developed the color (Perbio Science, Britain), is detected using UVIProChem imaging systems (UVItec, Britain).

As a result

Medicine of the present invention can suppress the formation of capilary sample tubule but not influence endothelial cell growth

External tubule formation test result indicates that, DME25 is compared with control group can significantly shorten small length of tube（P= 0.046）（See Fig. 1 by 1:1000 dilutions）.This concentration does not produce growth inhibition effect to tubule, while also not produced to HECV Raw cytotoxicity.（See Fig. 2）The no significant impact of growth of DME25 Human Umbilical Vein Endothelial Cells in quite wide concentration range.

Cellular matrix is sticked inhibited

DME25 shows as the inhibitory action of concentration dependent to sticking for HECV cells, it will be apparent that inhibitory action concentration exists 1:5000 or lower（Fig. 3 A, Fig. 4）.With threedimensional model it can be seen that DME25 inhibitory action is tested by repeated experiment Card（Fig. 3 B）.In conventional Cell-matrix adhesion model, DME25 significantly inhibits cell adhesion（Fig. 3 C-D）.

Medicine of the present invention can reduce endothelial cell migration

Stick with cellular matrix similar, cell migration can equally be suppressed by DME25, and be combined with Fak inhibitor When cell migration can be suppressed more.（Fig. 5）.

Medicine of the present invention and Fak inhibitor have synergy in the formation of endothelial cell adhesion, migration and tubule

Fak inhibitor is notable to HECV cytosiies（Fig. 3 C, 3D, 4B, 5）.It can be seen from the figure that, when being combined with DME25, Inhibitory action is strengthened by collaboration.

Formation of the Fak inhibitor to tubule shows as inhibitory action but without notable statistical significance（p=0.14）（Figure 1E）.But DME25 and Fak inhibitor are combined compared with control group, alone Fak inhibitor group and alone DME25 groups to tubule Generation has significant inhibitory action.（P=0.006, P=0.041, P=0.011）

Drug inhibition endothelial cell FAK phosphorylations of the present invention

We further infer that effects of the DME25 to HECV cell FAK and paxillin protein actives, that is, these eggs White tyrosine phosphorylation, with tyrosine phosphorylation specific antibody.As shown in Figure 7, DME25 suppresses FAK phosphorylations simultaneously And bigger inhibitory action is generated when being combined with Fak inhibitor.Alone DME25 or Fak inhibitor and the two combination are all Can remarkable effect in the phosphorylation of paxillin albumen.It is visible by immunofluorescence method, local adhesion point in blanc cell FAK is significantly dyed（Fig. 8, the position that left side is pointed out by arrow）, add after DME25, Fak inhibitor and the two combination and sky White group is compared, although the degree of dyeing does not change, but has shown less cell adherence spot（Fig. 8, left side）.What is interesting is portion The FAK of FAK phosphorylations is divided to be contaminated deeply by pFAK phospho-ABs.As shown in Figure 8（Right side, extends the time of exposure）, it is seen that blank Dyed at cell local adhesion spot by pFAK, DME25 and Fak inhibitor cause pFAK dyeing reductions, however, associated with the two Cell is not dyed by pFAK completely.

Conclusion

Antiangiogenesis therapy, such as Arastin, by the new show risen as treatment of solid tumor, and are just swelling at some Its clinical value is shown in knurl type.Some traditional antitumoral compounds be also found in anti-angiogenic rebirth in terms of work With.Chinese medicine composition of the present invention has shown it in tumor therapy clinical curative effect side as a kind of compound medicine of traditional Chinese medicine The advantage in face, including liver cancer, stomach cancer both China be in high incidence tumor disease.Also have confirmed that it in chemotherapy During some immanoprotection actions.

This is it is experimentally confirmed that Chinese medicine composition of the present invention is for tumor cell adhesion and the inhibitory action of migration.Both Cytological effect plays an important roll in the angiogenesis of endothelial cell.

Formed by the visible DME25 of the experimental result tubules that can significantly suppress extracorporeal blood vessel endothelial cell.Further grind Study carefully and find that the suppression cellular matrix of the extract doses dependence sticks and cell migration.These results confirm Chinese medicine group of the present invention Compound has the effect for suppressing tubule formation.

The result of this experiment confirms it is to block FAK to significantly improve Chinese medicine of the present invention with low dose of Fak inhibitor The effect of composition, while extract also has inhibitory action, that is, the suppression to FAK tyrosine phosphorylations to FAK activity in itself Make and use.FAK approach sticks in cellular matrix and is better than extracellular matrix with the effect in cell adhesion.In the phase interaction with matrix In, cell is combined with matrix using film integrin and triggers a series of intracellular activations, and one of crucial approach is just It is focal adhesion kinase（FAK）Activation, FAK can activate the interaction of integrin and cytoskeleton system in turn.This is just Form matrix adhesion and the important component in following cell migration process.During FAK is considered as angiogenesis Signal of interest path.Fak inhibitor, antineoplastic work is shown in clinical trial of the early stage for lung cancer and breast cancer With.

In general, it may be said that one of important function approach of Chinese medicine composition curative effect of the present invention is by suppressing Effect of the FAK approach in angiogenesis.In a word, Chinese medicine composition of the present invention has work well for extracorporeal blood vessel generation With can reduce cellular matrix and stick and cell migration, this is due to that it acts on focal adhesion kinase（FAK）Approach.

Inhibitory action of the medicine of the present invention for Tumor Angiongesis of experimental example 2, the suppression of the transfer for tumour cell Effect and the experiment acted synergistically with Pi3K inhibitor

Material and method

Material

Human breast cancer cell line, MCF-7 and MDA MB-231；Human prostate cancer cell line PC-3 and DU-145；People Gastric carcinoma cell lines, HGC27 and AGS；Human colon cancer cell line, RKO and and HRT18；It is all from ECACC（European animal is thin Born of the same parents' incubator, Salisbury, Britain）.Human osteosarcoma MG-63 and human lung cancer cell line A549 is purchased from ATCC (Unite States Standard cells Storehouse).These cells are stored in DMEM (Sigma-Aldrich, the Poole of addition penicillin, streptomysin and 10% hyclone Dorset, England) in culture medium.Cell culture condition 37^oC, 5% CO₂With 95% humidity.

ROCK inhibitor (Y27632) is purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz Biotechnologies, Inc., CA, US) jnk inhibitors II (SP60015), ERK inhibitor II (FR180204), JAK-3 inhibitor ({ 4- (4 '-Hydroxyphenyl) amino-6,7- of Jak-3 inhibitor 1 dimethoxyquinazoline;WHI-P131 }), and PLC-g (U73122) is purchased from Calbiochem (Merck Chemicals Ltd, Nottingham, England, UK) cMet kinase inhibitor come from Pfizer bases Matter glue (artificial substrate's film) purchased from Collaborative Research Products (Bedford, Massachusetts, USA) the anti-human GAPDH and anti-phopho-Akt antibody of is purchased from Santa-Cruz Biotechnologies.

Method

Test medicine is pre-processed

Cell in vitro propagation detection

Cell propagation is using cell in vitro proliferation assay (11,12).Cell is inoculated in 96 orifice plates to 3000 thin Born of the same parents/hole.4% (v/v) formaldehyde is fixed after three pieces of plate difference overnight incubations, 3d, 5d, 0.5% (w/v) violet staining.10% acetic acid Extractive crystallization purple detects absorbance using spectrophotometer（Bio-Tek ELx800 plate reader, Vermont, USA）.

Cellular matrix electric impedance sensor detects cell adherence and migration

ECIS-Zq (Applied Biophysics Inc, NJ, US) has been used for the detection of cell adherence volume locomitivity (13,14).Using ECIS RbA three-dimensional software building cytological maps.96W1E ECIS are studied for this.By being placed in culture Gold-foil electrode measurement cell and the matrix interphase interaction of plate surface.Analyzer surface is pre-processed with 10Mm serine solution Afterwards, 1h is incubated in culture with full culture medium.Equal cell adds each culture hole.In cell adherence detection, cell is added Spike immediately after analyzer.In cell migration detection, it is desirable to merged after cell 3h.Cell monolayer electrical impedance is carried out in 2000iA Damage.Impedance and resistance are recorded to 20h immediately.Analyzed using signal transduction inhibitor.Adhesion and migration are stood using ECIS RbA Body software carries out stereogram structure.

Western blot analysis

Cell is in 25cm³To merging in blake bottle, then use（Containing 1% Triton X-100,2 mM CaCl₂, 100 μ g/ml phenylmethylsulfonyl fluorides, 1 mg/ml leupeptins, 1 mg/ml Aprotinins and 10 mM original sodium vanadates）HCMF buffer salts Blow and beat and vibrate 1h cell lysis, precipitation is removed in 13,000g centrifugations.Protein content in sample uses Bio-Rad DC kits (Bio-Rad laboratories, California, USA) is detected.

Equal protein is analyzed using SDS-PAGE glue.Separation completes backward Hybond-C Extra nitrocellulose filters (Amersham Biosciences UK Ltd, Bucks, UK) is transferred, the closing of 10% milk, differential protein detection expression.Phosphorus The anti-pAkt antibody of acidifying AKT (threonine) expression uses (Santa Cruz Biotechnology, Inc., California, USA) detection.In addition, GAPDH expression also using specific antibody (Santa Cruz Biotechnology, Inc., California, USA) detection.The anti-mouse antibody of secondary antibody-connection peroxidase is added after primary antibody connection (Sigma, Dorset, UK).Using Supersignal West Dura Extended Duration substrate chemiluminescences system Unite (Perbio Science UK Ltd, Cramlington, UK) and UVIProChem imaging systems (UVItec Ltd, Cambridge, UK) check protein expression.

As a result

The effect that Chinese medicine composition of the present invention is adhered to human tumor cells

Detect DME25 to the cell line of different tumor types using high flux ECIS, including human breast carcinoma, prostate cancer, Lung cancer, colon cancer, stomach cancer and osteosarcoma cell.YZXJ concentration is from 1:40 to 1:600,000.Concentration is higher than 1:125,000 pairs Cell adherence has inhibition effect.More sensitive cell includes MCF-7, MG-63 and A539, as a result sees Fig. 9.In present mould Type may also confirm that its inhibition is acted on using wide range detection, as a result see Figure 10.Using Rb methods by taking A549 cells as an example（Figure 11） Illustrate quantitative analysis results.Parameter alpha represents basilar memebrane and electrode（Matrix）Between average distance.Therefore, it can be clearly Illustrate DME25 to there is dose-dependent inhibition between tumour cell and matrix.

Chinese medicine composition of the present invention suppresses cell migration

It has detected after the effect to tumor cell adhesion, carry out single layer of confluent tumour cell electric injury.Examined using ECIS Survey surrounding tumor cells injury recovery.As a result DME25 is to tumour cell, lung cancer and colon cancer, and migration shows concentration dependent Suppress.Figure 12 represents influences of the DME25 to A549 cell migrations.Drug concentration is 1:125,000 can be seen inhibition effect, It is similar with cell adhesion experiments.

Chinese medicine composition of the present invention is faint to human tumor cells proliferative effect

It further study the influence of DME25 vitro on cell propagation.DME25 (1:1000 and 1:25,000) handle After 72h（It the results are shown in Table 1）, in wider concentration range, DME25 has no significant effect to tumor cell proliferation.

Growth inhibition effect results of the DME25 of table 1. in vitro cancer cell

Cell type	Control group (490nm absorbances)	1:1000 (490nm absorbances)	1:25,000 (490nm absorbances)
				RKO, colorectal cancer cells	0.82±0.0.31	0.94±0.0.15	1.01±0.1
PC-3, prostate gland cancer cell	0.39±0.11	0.37±0.08	0.35±0.05
				MCF-7, breast cancer cell	1.39±0.34	1.52±0.41	1.32±0.54
HGC27, stomach cancer cell	0.72±0.1	0.95±0.31	0.97±0.1
				A549, lung carcinoma cell	0.24±0.08	0.21±0.07	0.24±0.03
Osteosarcoma cell	0.52±0.02	0.49±0.03	0.54±0.02

PI3K/AKT mediates influence of the Chinese medicine composition of the present invention to cell adherence

To confirm signal paths of the DNE25 to cell adhesion, make for PI3K/AKT and AKT detection inhibitor With.It is individually added into Wortmannin (PI3K inhibitor) or suppresses prostate gland cancer cell (DU-145) with DME25 combinations and glues It is attached, compared with control group and alone DME25, adhesion is reduced.Combination is more alone also to show stronger inhibitory action.AKT is PI3K downstream passages inhibitor, its inhibitor can improve the inhibitory action to cell adherence with DME25 combinations.Alone suppression pair Cell adherence suppresses weaker, sees Figure 13.Figure 14 represents shadows of the DME25 to lung cancer cell line A549 AKT (Ser 473) phosphorylation Ring.High concentration（1:1000）DME25 reduces AKT phosphorylations (pAKT) level.Adding AKT inhibitor and DME25 can assist to pAKT With suppressing, especially with low concentration DME25（1:5000）Combination.

Conclusion

Chinese medicine composition of the present invention not only has antitumous effect, and can directly suppress the adhesion of tumour cell and move Move, prevent metastases, and with the synergy of chemotherapeutics.

The effect of the drug therapy rheumatoid arthritis of the present invention of experimental example 3 and to rheumatoid arthritis angiogenesis Inhibitory action

1 material

1.1 animal SD rats, male and female half and half, body weight is 180-200g, and being purchased from Beijing magnificent experimental animal technology of dimension tonneau has Limit company.Licensing numbering SCXK（Capital）2006-0009.

1.2 medicinal materials and main agents medicament capsule of the present invention（Prepared according to the method for embodiment 1, Shijiazhuang Yi Ling medicine companies Limited company）；Incomplete Freund's adjuvant（IFA）And acid-soluble II Collagen Type VIs（Collagen II, 3806）It is sigma Product；BCG vaccine is to be purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute.

Method

2.1 complete Freund's adjuvant（Freund,s Complete adjuvantCFA）Preparation：In incomplete Freund Agent（IFA）BCG vaccine is added, final concentration of 7.5mg/ml is allowed to, emulsifying agent is lashed into repeatedly.

The preparation of 2.2 anti-rat collagen arthritis models：Ox II Collagen Type VIs are dissolved in 0.1M glacial acetic acid, 4 DEG C overnight. Then it is uniform with CFA grindings, the emulsion that Collagen Type VI containing II is 2mg/ml is made, by above-mentioned emulsion with 150 μ l/ left foots and root of the tail The subcutaneous multiple spot injection model group in portion and each administration group immune rat（II Collagen Type VI of the every rat equivalent to 150 μ g of injection）, in The μ l booster immunizations of emulsion 150 of the same race are injected in every rat root of the tail portion within 10th day.Normal group is without any processing.

The packet and administration of 2.3 experimental animals：SD rats are randomly divided into 5 groups.That is Normal group, to isometric solvent； Juvenile rheumatoid arthritis model group, to isometric solvent；The large, medium and small dosage group of medicine of the present invention, prepare model next day start gavage to Medicine of the present invention is given, dosage is respectively 1.56,0.78,0.39 gkg-1；Each group administration volume is 1ml/100g body weight, even Continuous administration 40 days.

Influence of 2.4 medicines of the present invention to Juvenile rheumatoid arthritis rat arthritis index：Administration terminates rear rat and uses joint Point system is scored.Ear：Tubercle and redness（Without 0 point, there is 1 point）；Nose：Red, swelling（Without 0 point, there is 1 point）；Tail：Tubercle （Without 0 point, there is 1 point）；Fore paw：The inflammation at least one joint（Without 0 point, there is 1 point）；Right rear solid end：Swelling（Without 0 point, slight 1 point, 2 points of moderate, 3 points of severe）.The average of every group of rat is arthritis index.

Influence of 2.5 medicines of the present invention to the toes volume of Collagen-Induced Arthritis rat：Use toes capacity measurer Measure the right metapedes toes volume of every rat, the change of the sufficient volume of observation each group rat.

2.6 pathological change：Rat is put to death, right side ankle-joint is taken, formalin is fixed, specimens paraffin embedding slices, HE dyeing sides Method.

2.7 results of statistical analysis with±sRepresent, using the softwares of SPSS 11.5 carry out ANOVA analyzing and processing and Dunnett is examined.

As a result

Influence of 3.1 medicines of the present invention to Juvenile rheumatoid arthritis rat arthritis index：As a result show, about 14 after first immunisation It starts II Collagen Type VIs rat model and each administration group rat starts ear redness and redness and swelling of joints occur, and metapedes toe joint occurs red Front foot and afterbody are spread to after swollen, and is on the rise, obvious tubercle occur in some rat-tail portions.Compared with Normal group, mould Type group arthritis index is significantly raised（P<0.01）；Compared with model group, medicine of the present invention can substantially reduce arthritis index, Middle and high dosage group is statistically significant（P<0.05, P<0.01）.It is shown in Table 1.

Influence of 3.2 medicines of the present invention to the toes volume of Collagen-Induced Arthritis rat：Compared with Normal group, Model group toes volume is significantly raised（P<0.01）；Compared with model group, medicine of the present invention can substantially reduce toes volume, in, High dose group is statistically significant（P<0.05, P<0.01）.It is shown in Table 2.

Influence (± s) of the medicine of the present invention of table 2 to collagen-induced rat arthritis

Group	N	Arthritis index	Toes volume
				Control group	9	0±0	1.71 ±0.08
Model group	11	5.82±0.84^2）	2.63 ±0.51^2）
				Low dose group	10	4.99±1.21	2.33±0.41
Middle dose group	11	4.14±1.57³⁾	2.11 ±0.42³⁾
				Product high dose group	11	4.02±0.73⁴⁾	2.06 ±0.28⁴⁾

Note：Compared with control group,¹⁾ P<0.05,²⁾ P<0.01；Compared with model group,³⁾ P<0.05,⁴⁾ P<0.01。

3.3 pathological change：

The visible 1-2 layers of synovial cell of normal group articular cartilage, articular surface is smooth, without exudate in articular cavity.

Collagen model group joint connective tissue and the visible inflammatory cell infiltration of musculature, the visible synovial tissue of articular cartilage Middle neutrophil leucocyte, monocyte, especially lymphocytic infiltration, increased vascularization, endangium deformation hyperplasia, tube chamber narrow or Cellulose largely oozes out under obstruction, synovial cell, Collagen fiber deposition.

Medicine of the present invention is big, tissue damage of the middle dose group to Juvenile rheumatoid arthritis has a better role.

Conclusion

The rat arthritis that medicine of the present invention is induced II Collagen Type VIs have good preventive and therapeutic effect.

Discuss

Rheumatoid arthritis（Rheumatoid arthritis, RA）It is a kind of to involve the multisystem based on periarticular Inflammatory autoimmune disease.RA's is mainly characterized by arthritis reaction, synovial membrane angiogenesis, and then causes chronic synovial membrane to increase Raw, further influence cartilage and sclerotin, cause joint deformity and function to lose the disease for basic pathological changes.II Collagen Type VIs are lured The property led arthritis（Collagen induced arthritis, CIA）Animal model is generally acknowledged at present for studying RA morbidities The ideal animals model of mechanism and exploitation treatment RA new drugs, the model is by II Collagen Type VIs（Collagen II, CII）Induction, CII It is the main component of cartilage, is the autoantigen of rheumatoid arthritis morbidity.Clinical Patients With Rheumatoid Arthritis serum and synovial membrane II Collagen Type VI antibody can be found in liquid.Because CIA models pathogenesis is closer to mankind RA, thus it is Recent study rheumatoid The arthritic preferred model of property.

There is ear redness and redness and swelling of joints to rat model group in our Germicidal efficacy, and metapedes toe joint occurs climing after redness Prolong to front foot and afterbody, obvious tubercle occur in some rat-tail portions.Pathology displays that joint connective tissue and musculature are visible Inflammatory cell infiltration, the visible synovial tissue's lymphocytic infiltration of articular cartilage, increased vascularization, endangium deformation hyperplasia, collagen Fiber is deposited.Medicine of the present invention can be obviously improved general symptom and pathological change.

Model group arthritis index and toes volume are significantly raised, and medicine of the present invention can significantly reduce arthritis index and foot Toe volume, improves focus increased vascularization, and endangium deforms the pathological change of hyperplasia, it was demonstrated that medicine of the present invention is lured II Collagen Type VIs The rat arthritis led have good preventive and therapeutic effect.

The effect of the drug treatment of diabetic PVR of the present invention of experimental example 4 and to diabetic retinopathy blood vessel give birth to Into inhibitory action

1 material

1.1 animal KK/Upj-Ay mouse, 30~40 g；C57BL/6 mouse, 25~30 g are SPF grades, male, 12 Week old, is purchased from Beijing HFK Bio-Technology Co., Ltd..

1.2 medicinal materials and main agents medicament capsule of the present invention（Prepared according to the method for embodiment 1, Shijiazhuang Yi Ling medicines Industry limited company）；Endothelial growth factors（VEGF）Primary antibody（Santa Cruz companies）.

2 methods

40 KK/Upj-Ay mouse of 2.1 animal packets and administration press fasting blood sugar（FBG）It is divided into model group, the present invention The high, medium and low dosage group of medicine, separately sets C57BL/6 mouse as control group, every group of 10 animals.Using gastric infusion, medicine of the present invention The high, medium and low dosage group of thing gives 1.56,0.78,0.39 gkg respectively^-1Medicine of the present invention, control group and model group are given Isometric distilled water, once a day, continuous 3 months.

2.2 morphological change

2.2.1 after administration terminates, eyeball of mouse is taken, formaldehyde is placed in and fixes, HE dyeing；

2.2.2 eyeball of mouse, takes eyeball, is fixed on 48 h in 10% formalin, separates retina, and tap water rinse is put Enter digestion vibration about 3h in 3% tryptic digestive juice, 37 DEG C of incubators, be only left the retinal blood pipe network of layer of transparent, carry out HE- PAS is dyed.

2.2.3 eyeball of mouse, is placed in glutaraldehyde and fixes, electron microscopic observation ultra microstructure.

The expression of 2.3Western blot methods detection vegf protein takes retinal tissue, and RIPA cracking process extracts total egg In vain, by the PAGE gel electrophoresis of protein sample progress 12%, on 4 DEG C of constant pressure electrotransfers to PVDF films, 5% skimmed milk power envelope 1h is closed, primary antibody is added afterwards（1:The VEGF of 200 dilutions, 1:The GAPDH internal references of 1000 dilutions）, 4 DEG C are incubated overnight, and TBST is washed Film, adds the secondary antibody (diluted concentration 1 of horseradish peroxidase-labeled:1000), the luminous colour developing in ECL darkrooms, gel imaging system Photographing scanning gray value is analyzed, and is used for statistical analysis with destination protein gray value and GAPDH gray value ratio.

2.4 results of statistical analysis with±sRepresent, using the softwares of SPSS 11.5 carry out ANOVA analyzing and processing and Dunnett is examined.

3 results

3.1 HE are dyed：Each confluent monolayer cells of control group retina are well arranged, and eucaryotic cell structure is close；Model group retinal pigment Epithelial layer is substantially thinning, and cell arrangement is disorderly, and medicine of the present invention can improve above-mentioned change.

3.2 HE-PAS are dyed：Normal mouse retina blood capillary distribution rule, moves towards more straight, caliber even thickness one Cause；Model group retina blood capillary net arrangement disorder, moves towards irregular, and many capillaries, which are turned round, is polymerized to clump, part capillary Blood vessel dilatation, tube chamber thickness are uneven；Medicine of the present invention can be obviously improved above-mentioned change, the most obvious with high agent group.

3.3 Electronic Speculum results：Control group mice retina, the inside and outside section intersection of photoreceptor cell, mitochondrial cristae is clear, Cell arrangement is neat, and nucleus and organelle are normal；, there is vacuolar degeneration, acromere membranous disc structural fuzzy, row in model group retina Row are disorderly, swelling and degeneration.Inside and outside nuclear layer cell mitochondrial swelling, photoreceptor cell film rupture, karyopycnosis, periphery is visible Cell debris.Medicine group acromere membranous disc gap of the present invention reduces compared with model group, and the arrangement depth of parallelism is slightly good, interior photoreceptor cell nuclei line grain Body vacuolar degeneration has been reduced, and high dose group is the most obvious.

3.4 retina vegf expressions：Western blot results show vegf protein expression in the normal view of control group Express relatively low in film, model group vegf protein is expressed apparently higher than control group（P<0.01）, VEGF eggs after pharmaceutical intervention of the present invention White expression is substantially less than model group, and middle and high dosage group has statistical discrepancy（P<0.05,P<0.01）.It is shown in Table 3.

Influence (n=3, ± s) of the medicine of the present invention of table 3 to KK Mouse Retina vegf expressions

Group	VEGF
		Control group	0.25±0.03
Model group	0.78±0.15²⁾
		Low dose of group of medicine of the present invention	0.57±0.11
Agent group in medicine of the present invention	0.44±0.07³⁾
		The high agent group of medicine of the present invention	0.31±0.05⁴⁾

4 conclusions

Medicine of the present invention has certain preventive and therapeutic effect, the notable drop of vegf protein expression to diabetic retinopathy mouse It is low, it can effectively suppress angiogenesis.

5 discuss

Type ii diabetes account for more than the 90% of diabetes community in clinic, and general Study person selects type ii diabetes（DM） Animal model.The KK mouse selected in this experiment are spontaneity DM models, have been widely used for DM basic research.It has been reported that 8-12 week old KK Mouse feeders after 3 months retina occur in that obvious capillary and DPN, this research passes through pre- reality Test and also observed similar result, therefore we have selected 8-12 week old KK Mouse feeders 3 months as sugar in this research The sick PVR of urine（DR）Model.Our light microscopic and Electronic Speculum result also show model group Mouse Retina pigment epithelial layer Substantially thinning, cell arrangement is disorderly；Capillary network arrangement disorder, moves towards irregular；There is vacuolar degeneration, acromere membranous disc knot Structure is obscured, arrangement disorder, the pathology damage such as swelling and degeneration, points out diabetic retinopathy model to be formed.

Light microscopic and Electronic Speculum result show that medicine of the present invention can improve retina pathology damage, confirm from morphology simultaneously Medicine of the present invention can delay the appearance of diabetic retinal tissue in rat lesion, and preventive and therapeutic effect is truly had to DR.

In normal eye tissue, retinal pigment epithelium, bovine retinal capillary pericytes, endothelial cell and M ü ller Cell can produce the VEGF of reduced levels, to maintain vascular stability and retina normal development, but under the conditions of high sugar, VEGF is released, and causes a series of reaction, including retinal blood vessels leak, stimulation retinal endothelial cell to breed and migrate, Promote new vessels formation etc..This experiment also demonstrate that the notable rise of model group DR Mouse Retinas vegf protein expression.Give The drug therapy of the present invention of various dose is after 3 months, and vegf expression has different degrees of reduction, it was demonstrated that medicine of the present invention can lead to Reduction vegf expression is crossed, suppresses angiogenesis, so as to play a part of protecting diabetic mice retina.

Stabilization of the medicine of the present invention of experimental example 5 for artery plaque and the suppression to artery plaque angiogenesis are made With

1 material

1.1 animal large ear rabbits, regular grade is male, 2.0 ~ 2.4 kg, buys in Beijing KeYu animal-breeding center, license Card numbering：SCXK（Capital）2007-0003.

1.2 medicinal materials and main agents medicament capsule of the present invention（Prepared according to the method for embodiment 1, Shijiazhuang Yi Ling medicines Industry limited company）；Oil red 0 is purchased from Sigma Co., USA；Sirius red is purchased from Sigma Co., USA.

Method

2.1 research method：30 male large ear rabbits, using balloon injured abdominal aorta+high cholesterol diet (l% courages Sterol, every feed for nursing 120-140g/d) method of 8 weeks is fed, set up stably atherosclerosis（As）Patchy model, It is randomly divided into：Medication therapy groups (giving 0.39 gkg-1 medicament capsules of the present invention, medicine group of the present invention daily) of the present invention are general Logical diet spontaneous regression group（Control group）, continue high fat diet control group (model group).Every group of 10 rabbits.Model group and Continue High-fat diet after medicine group modeling of the present invention, single cage is fed respectively, and automatic water-drinking gives Chinese spot after 12 weeks Crab snake venom and histamine injected under medicine triggering 0.15mg/kg peritonaeums, auricular vein injection histamine 0.02mg/kg after 30min, Putting to death 24 h, 48 h before animal, medicine is triggered twice, to promote patch to occur experimental rupture, is put to death after triggering.

2.1 intravascular ultrasonic imaging inspections（IVUS）Measure：Abdominal aorta to be carried out after medicine triggering intravascular super respectively Acoustic inspection.After the anesthesia of 3% yellow Jackets, experimental rabbit dorsal position is fixed on experimental bench, concrete operations are as follows：Conventional application 4F lancet puncture left femoral arteries, under 0.014 inch of guide wire and expansion pipe auxiliary, insert 5F sheaths, fixed, immediately Vein gives heparin 100u/kg anti-freezings, along guide wire under the guiding of peripheral vascular ultrasound, and insertion intravascular ultrasound probes are led Pipe, by stenotic lesion to blood vessel distal end, then slowly withdraws probe catheter（0.5mm/s）, mark patch distal end, proximal end view Picture, video recording supplies off-line analysis and archive.The incidence of three groups of animal plaque ruptures is measured, is determined simultaneously

（1）The outer elastic force membrane area of blood vessel（External elastic membrance area, EEMA）：Refer to the outer bullet of blood vessel The area that power film is included, including blood vessel cavity area and plaque area sum.

（2）Lumen Area（Lumen area, LA）：Refer to the area that endangium is included.

（3）Plaque area（Plaque area, PA）：The difference of the outer elastic force membrane area of blood vessel and Lumen Area.

（4）Lumen Area percent stenosis（Lumen area stenosis, LAS%）：Plaque area and the outer elastic force of blood vessel The ratio between membrane area.

2.2 specific stain：Utilize oil red O stain observation patch inner lipid, collagen relative amount.

2.3 results of statistical analysis with±sRepresent, using the softwares of SPSS 11.5 carry out ANOVA analyzing and processing and Dunnett is examined.

As a result

3.1 intravascular ultrasonic imaging inspections（IVUS）：

Compared with model group, medicine group experimental rabbit EEMA, PA, LAS% of the present invention decline obvious（P<0.01）,（Table 4）.Survey The rupture rate of the three groups of animal abdominal aorta patches obtained：Medicine group of the present invention：0%；Control group：0% ；Model group：33%.

The comparison of experimental rabbit abdominal aorta IVUS measured values after table 4 is treated 12 weeks

Index	Medicine group (n=9) of the present invention	Control group (n=9)	Model group (n=9)
				LA(mm²)	6.59±1.28	7.27±1.74	7.93±2.26
EEMA(mm²)	9.44±1.88**	12.82±2.34	15.23±2.39
				PA(mm²)	2.55±1.04**	5.19±2.24	5.99±2.17
LAS(%)	25.76±2.11**	39.15±2.94	42.83±3.62

Note：Compared * * with model groupP<0.01

3.2 pathological change：

Specific stain display model group oil red O stain percent positive is compared, this hair apparently higher than control group with model group Bright medicine group percent positive is significantly reduced.

Conclusion

This experimental applications balloon injured abdominal aorta+high cholesterol diet establishes Corn Bract Decotion, leads to Cross IVUS and check and show that medication therapy groups of the present invention can significantly reduce EEMA, PA, LAS%, and reduce the rupture rate of patch, so that Play stably the effect of vulnerable plaque, microexamination can obviously reduce blood vessel number in patch, suppress angiogenesis.

Claims

1. a kind of application of Chinese medicine composition in treatment medicine for treating rheumatoid arthritis is prepared, it is characterised in that the Chinese medicine group Compound is made up of the bulk drug of following parts by weight：Radix Astragali 120-360, fruit of glossy privet 100-300, ginseng 30-95, ganoderma lucidum 30- 95th, curcuma zedoary 65-195, bighead atractylodes rhizome 30-90, Sculellaria barbata 65-195, gynostemma pentaphylla 120-360, Poria cocos 30-95, the membrane of a chicken's gizzard 15-45, mock-strawberry 65-195, bittersweet 65-195, oriental wormwood 65-195, paniculate swallowwort 65-195, ground bettle 10-30, oldenlandia diffusa 65-195.

2. application according to claim 1, it is characterised in that the Chinese medicine composition by following parts by weight bulk drug It is made：

The Radix Astragali 120, the fruit of glossy privet 300, ginseng 30, ganoderma lucidum 95, curcuma zedoary 65, the bighead atractylodes rhizome 90, Sculellaria barbata 65, gynostemma pentaphylla 360, Poria cocos 30, The membrane of a chicken's gizzard 45, mock-strawberry 65, bittersweet 195, oriental wormwood 65, paniculate swallowwort 195, ground bettle 10, oldenlandia diffusa 195.

3. application according to claim 1, it is characterised in that the Chinese medicine composition by following parts by weight bulk drug It is made：

The Radix Astragali 360, the fruit of glossy privet 100, ginseng 95, ganoderma lucidum 30, curcuma zedoary 195, the bighead atractylodes rhizome 30, Sculellaria barbata 195, gynostemma pentaphylla 120, Poria cocos 95th, the membrane of a chicken's gizzard 15, mock-strawberry 195, bittersweet 65, oriental wormwood 195, paniculate swallowwort 65, ground bettle 30, oldenlandia diffusa 65.

4. application according to claim 1, it is characterised in that the Chinese medicine composition by following parts by weight bulk drug The Radix Astragali 250, the fruit of glossy privet 200, ginseng 65, ganoderma lucidum 65, curcuma zedoary 132, the bighead atractylodes rhizome 64, Sculellaria barbata 128, gynostemma pentaphylla 256, Poria cocos is made 65th, the membrane of a chicken's gizzard 30, mock-strawberry 128, bittersweet 128, oriental wormwood 128, paniculate swallowwort 128, ground bettle 20, oldenlandia diffusa 128.

5. the application according to claim any one of 1-4, it is characterised in that the preparation formulation of the Chinese medicine composition is glue Wafer, tablet, powder, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection.

6. the application according to claim any one of 1-4, it is characterised in that the active component of the Chinese medicine composition by with Lower step is made：

(2), the fruit of glossy privet, people participate in 6-10 times and measure 50-90% ethanol extraction 1-3 times, each 1-4 hours, merge extract solution, filter, Filtrate recycling ethanol is to without alcohol taste, and filtrate and the dregs of a decoction are standby；

(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort add 4-8 times to measure water and extract volatile oil, collect volatile oil, another device is collected, residue and water-soluble Liquid is standby;

(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2) Gained curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort carry residue after oil and merged in residue after ginseng, the alcohol extracting of the fruit of glossy privet, and step (3), plus 7-10 times Measure water, heating is decocted 1-3 time, each 1-3 hour, merge decoction liquor, add step (2) it is middle obtained by ginseng, fruit of glossy privet filtrate and Step（3）In the aqueous solution, be concentrated into decoction 65 DEG C survey when relative density be 1.15-1.30, dry, pulverize, it is standby;

Step（4）Gained comminuted powder, step（3）Gained volatile oil and step（5）The dried cream powder of gained collectively forms the Chinese medicine group The active component of compound.

7. application according to claim 5, it is characterised in that the capsule of the Chinese medicine composition is made up of following steps：

(2), ginseng, the fruit of glossy privet, plus 8 times of 70% alcohol refluxs of amount are extracted 2 times, and 3 hours for the first time, second 2 hours, merging was carried Liquid is taken, ethanol is reclaimed to without alcohol taste, filtrate and ginseng, fruit of glossy privet residue are standby;

(3), curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort, merge and extract volatile oil, carry the oily time no less than 8 hours, and the another device of volatile oil is collected, residual Slag and the aqueous solution are standby;

(4), ground bettle, the taste animal drugs of the membrane of a chicken's gizzard two, washing, 60 DEG C of drying merge with Poria cocos, 100 mesh powder are ground into, after sterilizing It is standby;

(5) income earner in, the Radix Astragali, ganoderma lucidum, oldenlandia diffusa, Sculellaria barbata, gynostemma pentaphylla, mock-strawberry, bittersweet, oriental wormwood, with step (2) Gained curcuma zedoary, the bighead atractylodes rhizome, paniculate swallowwort carry residue after oil and merged in residue after ginseng, the alcohol extracting of the fruit of glossy privet, and step (3), plus 9 times of amounts Water, heating is decocted 2 times, 2 hours every time, merges decoction liquor, adds gained ginseng, fruit of glossy privet alcohol extract and step in step (2) （3）In the aqueous solution, be condensed into relative density 1.20-1.25 clear cream, dry, pulverize, get dry extract powder；Gained dried cream powder is added Enter appropriate pharmaceutically acceptable auxiliary material granulation；

(6), gained volatile oil in step (3) is sprayed into the middle gained Poria cocos of step (4), ground bettle, the fine powder of the membrane of a chicken's gizzard, mixed It is even, with step（5）The particle of middle gained is mixed, closed half an hour, encapsulated to produce.