CN103417907A

CN103417907A - Applications of traditional Chinese medicine composition in preparation of drug used for inhibiting angiogenesis

Info

Publication number: CN103417907A
Application number: CN2012101662005A
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: 2013-12-04
Anticipated expiration: 2032-05-26
Also published as: CN103417907B

Abstract

The invention discloses applications of a traditional Chinese medicine composition in preparation of a drug used for inhibiting angiogenesis. The traditional Chinese medicine composition comprises traditional Chinese medicine ingredients such as radix astragali and ligustrum lucidum. The traditional Chinese medicine composition is capable of benefiting qi and nourishing yin, invigorating spleen and tonifying kidney, and removing stasis and relaxing vein; and the applications of the traditional Chinese medicine composition are right focused on pathomechanism. It is confirmed by experiments that the traditional Chinese medicine composition is capable of inhibiting angiogenesis effectively, and can be used for treating malignant tumor, rheumatoid arthritis, diabetic retinopathy and unstable arterial plaque in clinic. The traditional Chinese medicine composition is reasonable in compatibility of medicines, small in adverse drug reaction and taken by patients for a long term.

Description

The application of a kind of Chinese medicine composition in the medicine of preparation inhibition angiogenesis

Technical field

The present invention relates to a kind of new purposes of Chinese medicine composition, particularly, relate to the application of a kind of Chinese medicine composition in preparing the medicine of angiogenesis inhibiting, belong to the Chinese medicine application.

Background technology

Angiogenesis (being that new vessels forms) comprises that blood vessel occurs and angiogenesis; Blood vessel refers to by existing blood vessel to form new blood vessel by the mode of sprouting, and angiogenesis refers to that raising new vessels by angioblast/endothelial progenitor cells forms site and be divided into endotheliocyte.Angiogenic process is issued to poised state in the fine adjustment of the various factors that promote angiogenesis and inhibition angiogenesis under normal circumstances.Angiogenesis plays a significant role in the normal physiological processes such as human development, tissue repair, menstrual cycle of female.Simultaneously, the development of various diseases is also closely related with the blood vessel angiogenesis.

With normal blood vessels, compare, tumor vascular structure lacks integrity, there is larger gap between endotheliocyte, permeability is strong, the netted structure disturbance of blood vessel, a large amount of blood vessel cecum and arteriovenous shunt are arranged, extremely the causing of this structure ooze out increase and tissue between high pressure, also be easy to cancerous cell and shift simultaneously.The growth of tumor has two different stages, from avascular slow growth stage, changes into the fast breeding of the blood vessel stage is arranged.If there is no angiogenesis, the growth of primary tumo(u)r can not surpass 1 ~ 2 mm ³.Suppressing angiogenesis is the New Policy of antineoplaston.Generally, except when menstrual phase, inflammation or the wound, adult's vascular endothelial cell all remains static.VEGF (vascular endothelial growth factor, VEGF), basic fibroblast growth factor (bFGF) is important angiogenesis factor, Human Umbilical Vein Endothelial Cells has high degree of specificity, and the medicine that they are target of take is proved to be has few side effect.The process that cell surface receptor and extracellular factors can modulating vascular generate, this prompting medicine can not brought into play the effect of disturbing angiogenesis by Cell uptake.

Removed unconfined growth, the feature of tumor cell comprises adhesion, migration, invasion and attack, and this is also that tumor cell leaves primary tumor, transport the reason of sending out to body part or remote organ with body cavity by blood.Angiogenesis is also very important to the distal spread of tumor cell.The function of tumor cell and the transfer ability of tumor cell are closely related.Focal adhesion kinase (FAK) cell stick and move in play vital effect, focal adhesion kinase (FAK) inhibitor can effectively stop the transfer of tumor cell.

Phosphatidyl-inositol 3-kinase (PI3K) signal participates in the adjusting of the various kinds of cell functions such as propagation, differentiation, apoptosis and glucose transport.Discovered in recent years, the signal path that IA type PI3K and its downstream molecules protein kinase B (Akt) form and the genesis of human tumor are closely related.The propagation of this path regulate tumor cell and survival, its activity can not only cause malignant transformation of cells extremely, and to the migration of tumor cell, stick, the degraded of tumor-blood-vessel growth and extracellular matrix etc. is relevant, the ideas of cancer therapy that the PI3K-Akt signal path key molecule of take at present is target spot develops.Wortmannin (Wortmannin) is exactly wherein most typical representative, has entered the clinical research stage at present.The wortmannin structural formula is as follows:

CAS accession number: 19545-26-7.

The migration and adhesion of tumor cell plays an important role in tumor invasion shifts.PI3K can transmit the invasion and attack signal that integrin mediates, especially to integrin alpha ₂β ₁, α ₆β ₄And α _Vβ ₃The invasion and attack behavior be necessary, as integrin alpha in PI3K mediation carcinoma of prostate _Vβ ₃The invasion and attack characteristic driven, in breast carcinoma and ovarian cancer, crossing of Akt2 expressed and can be raised integrin beta by the IV collagen type ₁Thereby increase invasion and attack and the transfer of cell.

The etiology unknown of current rheumatoid arthritis, the rheumatoid arthritis main manifestations is progressive destruction of joint, finally cause dysfunction, in morphology, main manifestations is that synovial membrane propagation forms synovial tissue to fine hair shape projection in articular cavity, the synovial tissue of hypertrophy produces the multiple factor, have the effect that destroys cartilage, a large amount of vascular components is contained in the synovial tissue of hypertrophy.Data shows, angiogenesis plays an important role in the progress of this disease.For patient with rheumatoid arthritis, angiogenesis be one by Angiogensis with press down the complex process of angiogenesis medium regulation and control.

Diabetic renal papillary necrosis (diabetic retinopathy, DR) is a kind of diabetes chronic vascular complication that jeopardizes vision, no matter be insulin-dependent or non-insulin-depending type patient, DR finally affects nearly all diabetics.The characteristics of DR show as the retinal microvascular pathological changes progressively increased the weight of, concrete pathological manifestations has microcirculation disturbance, retina microangioma formation, blood-retina barrier destruction, blood capillary obturation, retinal nonperfusion district and new vessels formation etc., also may cause vitreous hemorrhage and detachment of retina, finally cause the infringement of visual function irreversibility.Pars papillaris is looked on optical fundus or new vessels appears in other positions, how with connective tissue proliferation, visual function is seriously damaged and causes that blind person is quite a few to be seen, therefore suppress new vessels, occurs it being the important step for the treatment of diabetic retinopathy (DR).

The unstable plaque rupture that causes of atherosclerosis (AS) speckle, and then acute coronary artery syndrome (ACS) occurs.The Recent study discovery, in speckle, angiogenesis can promote the development of atherosclerotic lesion, increases the unstability of speckle, therefore suppresses angiogenesis in speckle and may play key effect to stablizing vulnerable plaque.Therapeutic Angiogenesis can promote local vascular new life, improves myocardium compensatory capacity, finally reaches the purpose for the treatment of ischemic heart desease.

The present invention is the improvement invention of carrying out on the basis of No. 200410012347.4, at this, quotes in full the content of this patent document record.Above-mentioned patent discloses this Chinese medicine composition strengthening spleen, tonifying kidney, and the effect of dissolving blood stasis and detoxication, for adjuvant therapy of tumors.

Summary of the invention

The present invention relates to a kind of new purposes of Chinese medicine composition, particularly, relate to a kind of Chinese medicine composition and suppress the application in angiogenesis drug in preparation.

Chinese medicine of the present invention all can be concocted according to " national Chinese medicine processing standard " or " Chinese medicine voluminous dictionary ".Medicine of the present invention is to be made by the crude drug of following weight ratio:

Radix Astragali 120-360, Fructus Ligustri Lucidi 100-300, Radix Ginseng 30-95, Ganoderma 30-95, Rhizoma Curcumae 65-195, Rhizoma Atractylodis Macrocephalae 30-90, Herba Scutellariae Barbatae 65-195, Herb Gynostemmae Pentaphylli 120-360, Poria 30-95, Endothelium Corneum Gigeriae Galli 15-45, Herba Duchesneae Indicae 65-195, Herba Solani Lyrati 65-195, Herba Artemisiae Scopariae 65-195, Radix Cynanchi Paniculati 65-195, Eupolyphaga Seu Steleophaga 10-30, Herba Hedyotidis Diffusae 65-195.

Preferably, this Chinese medicine composition is made by the crude drug of following weight portion:

The Radix Astragali 120, Fructus Ligustri Lucidi 300, Radix Ginseng 30, Ganoderma 95, Rhizoma Curcumae 65, the Rhizoma Atractylodis Macrocephalae 90, Herba Scutellariae Barbatae 65, Herb Gynostemmae Pentaphylli 360, Poria 30, Endothelium Corneum Gigeriae Galli 45, Herba Duchesneae Indicae 65, Herba Solani Lyrati 195, Herba Artemisiae Scopariae 65, Radix Cynanchi Paniculati 195, Eupolyphaga Seu Steleophaga 10, Herba Hedyotidis Diffusae 195.

Or

The Radix Astragali 360, Fructus Ligustri Lucidi 100, Radix Ginseng 95, Ganoderma 30, Rhizoma Curcumae 195, the Rhizoma Atractylodis Macrocephalae 30, Herba Scutellariae Barbatae 195, Herb Gynostemmae Pentaphylli 120, Poria 95, Endothelium Corneum Gigeriae Galli 15, Herba Duchesneae Indicae 195, Herba Solani Lyrati 65, Herba Artemisiae Scopariae 195, Radix Cynanchi Paniculati 65, Eupolyphaga Seu Steleophaga 30, Herba Hedyotidis Diffusae 65.

Or

The Radix Astragali 250, Fructus Ligustri Lucidi 200, Radix Ginseng 65, Ganoderma 65, Rhizoma Curcumae 132, the Rhizoma Atractylodis Macrocephalae 64, Herba Scutellariae Barbatae 128, Herb Gynostemmae Pentaphylli 256, Poria 65, Endothelium Corneum Gigeriae Galli 30, Herba Duchesneae Indicae 128, Herba Solani Lyrati 128, Herba Artemisiae Scopariae 128, Radix Cynanchi Paniculati 128, Eupolyphaga Seu Steleophaga 20, Herba Hedyotidis Diffusae 128.

Preferably, in the raw materials used medicine of described Chinese medicine composition, the Rhizoma Atractylodis Macrocephalae is Rhizoma Atractylodis Macrocephalae (parched).

The present invention also provides the active component of described Chinese medicine composition to be made by following steps:

(1), according to the crude drug part by weight, take Chinese crude drug, clean;

(2), Fructus Ligustri Lucidi, people participate in 6-10 and doubly measure the 50-90% ethanol extraction 1-3 time, each 1-4 hour, merge extractive liquid,, filter, filtrate recycling ethanol is to without the alcohol flavor, filtrate and medicinal residues are standby;

(3), Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati add 4-8 times of water gaging and extract volatile oil, collects volatile oil, another device is collected, residue and aqueous solution are standby;

(4), Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli, Poria powder are broken into fine powder;

(5), the Radix Astragali, Ganoderma, Herba Hedyotidis Diffusae, Herba Scutellariae Barbatae, Herb Gynostemmae Pentaphylli, Herba Duchesneae Indicae, Herba Solani Lyrati, Herba Artemisiae Scopariae; with residue after the alcohol extraction of gained Radix Ginseng, Fructus Ligustri Lucidi in step (2); and after in step (3), gained Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati are carried oil, residue merges; add 7-10 times of water gaging; heating decocts 1-3 time; each 1-3 hour; merge decoction liquor; add the aqueous solution in gained Radix Ginseng, Fructus Ligustri Lucidi filtrate and step (3) in step (2); being concentrated into medicinal liquid relative density when surveying for 65 ℃ is 1.15-1.30; dry, pulverize, standby;

The dried cream powder of step (4) gained comminuted powder, step (3) gained volatile oil and step (5) gained forms the active component of this Chinese medicine composition jointly.

The dosage form of medicine of the present invention is capsule, tablet, powder, oral liquid, soft capsule, pill, tincture, syrup, suppository, gel, spray or injection.

The preparation method of capsule wherein is to be made by following steps:

(2), Radix Ginseng, Fructus Ligustri Lucidi, add 8 times of amount 70% alcohol reflux 2 times, 3 hours for the first time, 2 hours for the second time, merge extractive liquid,, reclaimed ethanol to without the alcohol flavor, filtrate and Radix Ginseng, Fructus Ligustri Lucidi residue are standby;

(3), Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati, united extraction volatile oil, carry the oil time and be no less than 8 hours, the another device of volatile oil is collected, residue and aqueous solution are standby;

(4), Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli two flavor animal drugs, washing, 60 ℃ of oven dry, merge with Poria, is ground into 100 order powder, standby after sterilizing;

(5), the Radix Astragali, Ganoderma, Herba Hedyotidis Diffusae, Herba Scutellariae Barbatae, Herb Gynostemmae Pentaphylli, Herba Duchesneae Indicae, Herba Solani Lyrati, Herba Artemisiae Scopariae, with residue after the alcohol extraction of gained Radix Ginseng, Fructus Ligustri Lucidi in step (2), and after in step (3), gained Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati are carried oil, residue merges, add 9 times of water gagings, heating decocts 2 times, each 2 hours, merge decoction liquor, add the aqueous solution in gained Radix Ginseng, Fructus Ligustri Lucidi alcohol extract and step (3) in step (2), be condensed into the clear paste of relative density 1.20-1.25, dry, pulverize, powder gets dry extract; Add suitable pharmaceutically acceptable adjuvant to granulate the gained dried cream powder;

(6), gained volatile oil in step (3) is sprayed in the fine powder of gained Poria, Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli in step (4), mix, with the granule of gained in step (5), mix, airtight half an hour, encapsulated and get final product.

Other dosage forms of medicine of the present invention are in proportion after weighting raw materials, adopt conventional preparation method preparation, for example, the preparation technology of Fan Biting " pharmacy of Chinese materia medica " (Shanghai Science Press 1997 December the 1st edition) record, make the acceptable regular dosage form of pharmaceutics.

For above-mentioned dosage form can be realized, need when these dosage forms of preparation, add the acceptable adjuvant of pharmacy, such as: filler, disintegrating agent, lubricant, suspending agent, binding agent, sweeting agent, correctives, antiseptic, substrate etc.Filler comprises: starch, pregelatinized Starch, lactose, mannitol, chitin, microcrystalline Cellulose, sucrose etc.; Disintegrating agent comprises: starch, pregelatinized Starch, microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linking sodium carboxymethyl cellulose etc.; Lubricant comprises: magnesium stearate, sodium lauryl sulphate, Pulvis Talci, silicon dioxide etc.; Suspending agent comprises: polyvinylpyrrolidone, microcrystalline Cellulose, sucrose, agar, hydroxypropyl emthylcellulose etc.; Binding agent comprises, starch slurry, polyvinylpyrrolidone, hydroxypropyl emthylcellulose etc.; Sweeting agent comprises: saccharin sodium, Aspartane, sucrose, cyclamate, enoxolone etc.; Correctives comprises: sweeting agent and various essence; Antiseptic comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzalkonium bromide, acetic acid chloroethene are determined, the Folium eucalypti globueli (Eucalyptus globulus Labill.) wet goods; Substrate comprises: PEG6000, PEG4000, insect wax etc.For making above-mentioned dosage form can realize pharmacy of Chinese materia medica, need add acceptable other adjuvant of pharmacy (Fan Biting " pharmacy of Chinese materia medica ", the adjuvant of each dosage form record in Shanghai Science Press December in 1997 the 1st edition) during these dosage forms in preparation.

Experiment confirms, pharmaceutical composition of the present invention has direct repression to angiogenesis and vascular endothelial cell and migration in vitro, there is direct repression for angiogenesis, and blood capillary, cellular matrix adhesion and cell migration are had to significant inhibitory action.Simultaneously, can suppress significantly the FAK phosphorylation, collaborative FAK inhibitor has the inhibition angiogenesis function.

The experiment confirmation, pharmaceutical composition of the present invention can be worked in coordination with the FAK inhibitor and be prevented neoplasm metastasis, can also work in coordination with PI3K inhibitor and AKT (serine/threonine kinase) inhibitor and prevent neoplasm metastasis.

Experiment is confirmation also, and this Chinese medicine composition is inhibition tumor cell directly, and can prevent cancer metastasis, particularly osteosarcoma, pulmonary carcinoma, human breast carcinoma, carcinoma of prostate, colon cancer or gastric cancer is had and prevents preferably the effect of shifting.

This Chinese medicine composition can effectively suppress the angiogenesis of rheumatoid arthritis, diabetic renal papillary necrosis and artery plaque, and effectively treats rheumatoid arthritis, diabetic renal papillary necrosis and the increase of prevention of arterial speckle and come off.

Compatibility of the present invention is reasonable, simple, is pure Chinese medicinal preparation, and untoward reaction is little, can supply patient's life-time service.

The accompanying drawing explanation

Fig. 1The effect that DME25 generates external HECV cell blood capillary tubule.The HECV cell between the basement membrane interlayer, with HECV (A) as a control group, FAK inhibitor intervention group (25nM) (B), DME25 intervention group (1:2000) (C), FAK inhibitor and DME25 Combination intervention group (D).Put microscopic examination tubule image (A-D), with Digital image analysis technique, quantize length (E).DME25 can significantly suppress that tubule forms and and the FAK inhibitor between have synergism.

Fig. 2DME25 is not good enough to the action effect of vitro endothelial cell growth.Process cell 72h with the DME25 of different diluted concentrations.Carry out cell counting by crystal violet.The HECV Growth of Cells is not had to obvious effect.

Fig. 3 A: DME25 dose-dependent inhibition HECV cellular matrix adheres to, and DME25 is diluted from 1:40 to 1:125,000 (A).Dilution is lower than 1:25, and 000 shows inhibitory action.B: the three-dimensional imaging of adhesion.Left side: blank.Right side: 1:1, the cell that the DME25 of 000 dilution processes.X-axis: frequency.Y-axis: resistance.Z axis: time.C and D: conventional method research cellular matrix adheres to.C: Gentian Violet dyeing adherent cell imaging.D: adherent cell number under every high power field.Compare * p<0.01 with the blank group; With independent DME and independent FAK inhibitor, compare, ^#P<0.01.

Fig. 4The dependency (B) of concentration dependence (A) and FAK approach between DME25 and cell adhesion.Shown in data from the Rb model.

Fig. 5.The effect of DME25 inhibitory action and FAK.DME25 can significantly reduce and stick, and it is enhanced while acting on the coupling of FAK inhibitor, migration path shown in figure (A) and threedimensional model (B).I: matched group; The ii:FAK inhibitor is intervened cell (25nM); Iii:DME25 intervenes cell (1:1000); IV: ii and iii Combination intervention.X-axis: frequency; Y-axis: resistance; Z axis: time.

Fig. 6The inhibitory action of DME25 on cell migration and the effect of FAK.DME25 can significantly reduce cell migration, and it is enhanced while acting on the coupling of FAK inhibitor, migration path shown in figure (A) and threedimensional model (B).I: matched group; The ii:FAK inhibitor is intervened cell (25nM); Iii:DME25 intervenes cell (1:1000); IV: ii and iii Combination intervention.X-axis: frequency; Y-axis: resistance; Z axis: time.The time point that arrow prompting electric injury in A figure is induced.

Fig. 7DME25 suppresses the phosphorylation of HECV endotheliocyte FAK and Paxillin.After serum starvation 2h, DME25(2 kind variable concentrations, 1:1000 and 1:5000), FAK inhibitor (25nM) or the Combination intervention HECV cell 60min of the two.Separate equal protein on the SDS-polyacrylamide gel electrophoresis after, with FAK and paxillin phosphorylation specific antibody, phosphorylation FAK and Paxillin are carried out to probe in detecting.

Fig. 8Focal adhesion kinase in the HECV cell (FAK) and pFAK immunofluorescence dyeing.20,000 HECV cells are added to respectively in blank culture medium, DME25 (1:1,000), FAK inhibitor and the two coupling.After 2 hours, cell is rinsed and fixes, after using respectively anti-FAK and anti-pFAK antibody treatment, total FAK(left side) and FAK phosphorylation (right side) by engrain.After irradiating by 100ms, the FAK imaging obtains, and after 400ms irradiates, the pFAK imaging obtains.

Fig. 9DME25 to MCF-7 (on), MG-63 (in) and A549 cells (under) substrate adheres to and to obtain impact.Concentration is higher than 1:125, and 000 all has inhibitory action.

Figure 10Wideband detects DME25 and suppresses cell adhesion.The 3D ideograph confirms the effect of extract to cell adhesion, and X-axis is frequency; Y-axis is resistance; Z axis is the time.

Figure 11A549 cell Rb mode data.

Figure 12DME25 suppresses the A549 cell migration.

Figure 13The PI3K/AKT path is induced the effect in the DU-145 cell adhesion at DME25.A: add the impact of Wortmannin on cell adhesion.B:AKT inhibition and the DME25 effect to cell adhesion.

Figure 14The impact of DME25 on A549 cell AKT (Ser 473) phosphorylation.A:West-blot detects AKT (Ser 473) phosphorylation; B:ImageJ. Bar graphical analysis pAKT/GAPDH band ratio.

The specific embodiment

Embodiment 1:

The crude drug formula is:

Radix Astragali 250g, Fructus Ligustri Lucidi 200g, Radix Ginseng 65g, Ganoderma 65g, Rhizoma Curcumae 132g, Rhizoma Atractylodis Macrocephalae (parched) 64g, Herba Scutellariae Barbatae 128g, Herb Gynostemmae Pentaphylli 256g, Poria 65g, Endothelium Corneum Gigeriae Galli 30g, Herba Duchesneae Indicae 128g, Herba Solani Lyrati 128g, Herba Artemisiae Scopariae 128g, Radix Cynanchi Paniculati 128g, Eupolyphaga Seu Steleophaga 20g, Herba Hedyotidis Diffusae 128g.

Preparation method is:

(3), Rhizoma Curcumae, Rhizoma Atractylodis Macrocephalae (parched), Radix Cynanchi Paniculati, united extraction volatile oil, carry the 8 hours time of oil, the another device of volatile oil is collected, residue and aqueous solution are standby;

(4), Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli, washing, 60 ℃ of oven dry, merge with Poria, is ground into 100 order powder, in 3KGY ⁶⁰Standby after the CO-r radiation sterilization;

(5), the Radix Astragali, Ganoderma, Herba Hedyotidis Diffusae, Herba Scutellariae Barbatae, Herb Gynostemmae Pentaphylli, Herba Duchesneae Indicae, Herba Solani Lyrati, Herba Artemisiae Scopariae; with residue after the alcohol extraction of gained Radix Ginseng, Fructus Ligustri Lucidi in step (2); and the residue that in step (3), gained Rhizoma Curcumae, Rhizoma Atractylodis Macrocephalae (parched), Radix Cynanchi Paniculati are carried after oil merges; add 9 times of water gagings; heating decocts 2 times; each 2 hours; merge decoction liquor; add the aqueous solution in gained Radix Ginseng, Fructus Ligustri Lucidi alcohol extract and step (3) in step (2); be condensed into the clear paste of relative density 1.25; dry, pulverize, standby;

(6), step (5) gained dried cream powder is added to starch 134 grams, use 85% alcohol granulation;

(7), by gained volatile oil 85% dissolve with ethanol in step (3); spray in the fine powder of gained Poria, Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli in step (4), mix, mix with the granule of gained in step (6); airtight half an hour, 1000 capsules of packing into and get final product.

Embodiment 2:

The crude drug formula is:

Radix Astragali 360g, Fructus Ligustri Lucidi 100g, Radix Ginseng 95g, Ganoderma 30g, Rhizoma Curcumae 195g, Rhizoma Atractylodis Macrocephalae (parched) 30g, Herba Scutellariae Barbatae 195g, Herb Gynostemmae Pentaphylli 120g, Poria 95g, Endothelium Corneum Gigeriae Galli 15g, Herba Duchesneae Indicae 195g, Herba Solani Lyrati 65g, Herba Artemisiae Scopariae 195g, Radix Cynanchi Paniculati 65g, Eupolyphaga Seu Steleophaga 30g, Herba Hedyotidis Diffusae 65g.

Preparation method is:

(2), Radix Ginseng, Fructus Ligustri Lucidi, add 6 times of amount 90% alcohol reflux 4 hours, extracting solution reclaims ethanol to without the alcohol flavor, filtrate and Radix Ginseng, Fructus Ligustri Lucidi residue are standby;

(3), Rhizoma Curcumae, Rhizoma Atractylodis Macrocephalae (parched), Radix Cynanchi Paniculati merge, and adds 4 times of water gagings and extract volatile oil, carries the 10 hours time of oil, the another device of volatile oil is collected, residue and aqueous solution are standby;

(5), the Radix Astragali, Ganoderma, Herba Hedyotidis Diffusae, Herba Scutellariae Barbatae, Herb Gynostemmae Pentaphylli, Herba Duchesneae Indicae, Herba Solani Lyrati, Herba Artemisiae Scopariae, with residue after the alcohol extraction of gained Radix Ginseng, Fructus Ligustri Lucidi in step (2), and the residue that in step (3), gained Rhizoma Curcumae, Rhizoma Atractylodis Macrocephalae (parched), Radix Cynanchi Paniculati are carried after oil merges, add 7 times of water gagings, heating decocts 3 times, 1 hour for the first time, 2 hours for the second time, 3 hours for the third time, merge decoction liquor, add the aqueous solution in gained Radix Ginseng, Fructus Ligustri Lucidi alcohol extract and step (3) in step (2), be condensed into the clear paste of relative density 1.15, dry, pulverize, standby;

(6), step (5) gained dried cream powder is added to starch 112 grams, use 78% alcohol granulation;

(7), by gained volatile oil 85% dissolve with ethanol in step (3); spray in the fine powder of gained Poria, Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli in step (4); mix, mix with the granule of gained in step (6), formulation method is made 1000 tablets of tablets routinely.

Embodiment 3:

The crude drug formula is:

Radix Astragali 120g, Fructus Ligustri Lucidi 300g, Radix Ginseng 30g, Ganoderma 95g, Rhizoma Curcumae 65g, Rhizoma Atractylodis Macrocephalae 90g, Herba Scutellariae Barbatae 65g, Herb Gynostemmae Pentaphylli 360g, Poria 30g, Endothelium Corneum Gigeriae Galli 45g, Herba Duchesneae Indicae 65g, Herba Solani Lyrati 195g, Herba Artemisiae Scopariae 65g, Radix Cynanchi Paniculati 195g, Eupolyphaga Seu Steleophaga 10g, Herba Hedyotidis Diffusae 195g.

Preparation method is:

(2), Radix Ginseng, Fructus Ligustri Lucidi, add 10 times of amount 50% alcohol reflux 2 times, 3 hours for the first time, 2 hours for the second time, merge extractive liquid,, reclaimed ethanol to without the alcohol flavor, filtrate and Radix Ginseng, Fructus Ligustri Lucidi residue are standby;

(3), Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati merge, and adds 10 times of water gagings to extract volatile oil, carries the 6 hours time of oil, the another device of volatile oil is collected, residue and aqueous solution are standby;

(5), the Radix Astragali, Ganoderma, Herba Hedyotidis Diffusae, Herba Scutellariae Barbatae, Herb Gynostemmae Pentaphylli, Herba Duchesneae Indicae, Herba Solani Lyrati, Herba Artemisiae Scopariae, with residue after the alcohol extraction of gained Radix Ginseng, Fructus Ligustri Lucidi in step (2), and the residue that in step (3), gained Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati are carried after oil merges, add 10 times of water gagings, heating decocts 3 hours, merge decoction liquor, add the aqueous solution in gained Radix Ginseng, Fructus Ligustri Lucidi alcohol extract and step (3) in step (2), be condensed into the clear paste of relative density 1.30, dry, pulverize, standby;

(6), by gained volatile oil 80% dissolve with ethanol in step (3), spray in the fine powder of gained Poria, Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli in step (4), mix, with the dried cream powder of gained in step (5), formulation method is made 1000 ball pills routinely.

At therapeutic effect, use the capsule (hereinafter referred to as medicine of the present invention) made by embodiment 1 for proved invention medicine, carried out following experimental study:

Experimental example 1 medicine of the present invention suppresses angiogenesis and medicine of the present invention and the synergistic experiment of FAK inhibitor

Materials and methods

Material

Human umbilical vein endothelial cells (HECV) is purchased from Interlab(Milan, Italy).These cells maintain (DMEM) (Sigma's aldrich, pul, many Saites, Britain) in the middle of cell culture medium, are aided with the hyclone (Sigma's aldrich) of penicillin, streptomycin and 10%.Cell culture is at 37 ℃, 5%CO ₂With under 95% humidity, carry out.Artificial basement membrane (reconstituted basement membrane) is purchased from Collaborative Research Products company (Bedford, Massachusetts, the U.S.).A kind of selective depressant focal adhesion kinase (FP573228) is purchased from Tocris company (Bristol, Britain).Paxillin comes from the transduction laboratory, and phosphorylation specific antibody (pFAK and pPaxillin) is purchased from santa-cruz biotech company (Santa Cruz, California, the U.S.).

Method

Tested medicine pretreatment

For convenience of experiment, carry out, medicine of the present invention (Shijiazhuang Yiling Pharmaceutical Co., Ltd's production) is done to following pretreatment: medicament capsule content 400mg of the present invention adds DMSO1ml, under room temperature, swiveling wheel is 20 rev/mins, and 12 hours, centrifugal 30 minutes of rotating speed 14000rpm, get supernatant, add balanced salt solution, filter, absorb at 405nm photometry degree, dilution extracting solution to luminosity absorbs 0.25, this is called DME25, and packing is preserved as standardized extract, standby.

The in-vitro cell growth method

The HECV cell with 3000, every hole cell kind on 96 orifice plates.Hatch an evening with in triplicate culture plate, 3 days and 5 days.After fully cultivating, plank is shifted out from incubator, the formaldehyde with 4% fixing with 5% violet staining.Slough crystal violet with 10% acetic acid subsequently, use the multi-functional microplate reader of Bio-Tek ELx800 (Bio-Tek instrument company, Vermont State, the U.S.) to detect the density of cell.

Electric fine cytoplasmic matrix impedance sensing (ECIS) is basic cell adhesion and migration experiment

ECIS-Z θ instrument (applying biological physics, New Jersey, the U.S.) is used to cell adhesion and mobility's (damage check) detects research.Cell model has adopted ECIS RbA modeling software, by manufacturer, is provided.Current producer adopts the 96W1E array.ECIS is that the gold film electrode by being placed in the culture dish surface is measured the interaction between cell and its substrate adhered to.The array surface that contains aminothiopropionic acid solution with post processing, complete culture medium culturing 1 hour for this array.Add the cell of equal number in each culture hole.In the cell adhesion test, its adhesiveness is at once followed the tracks of after cell is added to array.For the cell migration test, cellular array reached and converges after 3 hours.Cell monolayer is hindered 20 seconds with the electric current electricity of 2000MA.The impedance of cellular layer and resistance record 20 hours.Signal transduction inhibitor detects, and in the test plate hole, inhibitor separately all is among them.Adhesion and migration are to copy to use ECIS RbA cell modeling software.

External tube chamber forms experiment

External blood capillary tube chamber forms assessment and uses basement membrane endotheliocyte tubule to form test.Under serum-free medium, the basement membrane of 250 μ g is planted on 96 orifice plates, and is placed in incubator at least 40 minutes and makes it gel.After this, 35000HECV is inoculated into and contains and do not contain the basal layer of MDE25 or FAK inhibitor and cultivate 4-5 hour, and tube chamber in the training period forms and records and take with high power microscope.Tube chamber girth total in each visual field quantizes with Image J software.

Immunofluorescence dyeing (IFC)

HECV cell kind in 16 vestibule formula microscope slides (LAB-TEK Fisher, Britain), 20000/hole, overnight incubation (25).Absorb culture fluid, 4% formalin fixed cell 20 min, BBS incubated at room 20 min, then drip 0.1 % Triton X-100 BBS solution, cell perforation 5 min.Adopt MenaPath Autowash buffer confining liquid (A. Menarini Diagnostics, Britain) to hatch 20 min, with the blocking-up non-specific binding, every 5 milliliters of confining liquids are containing 2 of horse serums (Sigma, Britain).After buffer rinse 2 times, adopt specific antibody labelled protein paxillin, p-Paxilli, FAK, p-FAK (Santa-Cruz, the U.S.).Primary antibodie is with rear incubated cell 1 h of 1:100 dilution, and buffer rinse 3 times, remove remaining primary antibodie.Drip the anti-Mus two anti-(Insight Biotechnology, Britain) of FITC labelling, microscope slide is placed on shaking table, lucifuge is hatched 1 h.Finally, microscope slide rinse three times, remove and resist in conjunction with two, after Fluor-save mounting (Calbiochem-Novabiochem, Britain), adopts Olympus BX51 fluorescence microscope to observe under 100 times of object lens.

Gel electrophoresis and immune protein

Cell is at 25 cm ³Grow to fusion in tissue culture flasks, collecting cell, add the HCMF buffer (to contain 1% Triton X-100,2 mM CaCl2,100 μ g/ml Phenylmethanesulfonyl fluorides, 1 mg/ml leupeptin, 1 mg/ml aprotinin and, 10 mM sodium orthovanadates), be placed in rotor wheel instrument cell lysis 1 h, the centrifugal removal insoluble matter of 13,000g.The gained sample adopts Bio-Rad DC protein reagent box (Bio-Rad, the U.S.) to carry out protein quantification.

Protein sample is fully separated, adopt Hybond-C Extra nitrocellulose filter (Amersham Biosciences, Britain) transferring film, after 10% milk sealing, measure the expression of specific protein.Adopt respectively FAK and the paxillin (27) of anti-pFAK antibody and anti-pPaxillin antibody labeling phosphorylation.Adopt GAPDH specific antibody (Santa-Cruz, the U.S.) to measure GAPDH and express, with total protein level and the concordance of assess sample.Protein band adopts SWDED substrate chemiluminescence system to be developed the color (Perbio Science, Britain), adopts the UVIProChem imaging system to be detected (UVItec, Britain).

Result

Medicine of the present invention can suppress the formation of blood capillary sample tubule but not affect endothelial cell growth

External tubule forms experimental result and shows, with matched group comparison DME25, can significantly shorten little length of tube (P=0.046) (seeing that Fig. 1 dilutes by 1:1000).This concentration does not produce the growth inhibited effect to tubule, HECV is not produced to cytotoxicity yet simultaneously.The not significant impact of the growth of (see figure 2) 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 of HECV cell, significantly inhibitory action concentration is at 1:5000 or lower (Fig. 3 A, Fig. 4).Use threedimensional model can see that the inhibitory action of DME25 is through repeated experiment checking (Fig. 3 B).Conventional cellular matrix adheres in model, and DME25 significantly suppresses cell adhesion (Fig. 3 C-D).

Medicine of the present invention can reduce endothelial cell migration

With cellular matrix, stick similarly, cell migration can be suppressed by DME25 equally, and cell migration can further be suppressed with the coupling of FAK inhibitor the time.(Fig. 5).

Medicine of the present invention and FAK inhibitor form and exist synergism at endothelial cell adhesion, migration and tubule

The FAK inhibitor is to HECV cytosis remarkable (Fig. 3 C, 3D, 4B, 5).In figure, can find out, during with the DME25 coupling, inhibitory action is worked in coordination with enhancing.

The FAK inhibitor shows as inhibitory action but does not have remarkable statistical significance (p=0.14) (Fig. 1 E) the formation of tubule.But DME25 and the coupling of FAK inhibitor and matched group, alone FAK inhibitor group and alone DME25 group compare tubule, generation has significant inhibitory action.（P=0.006，P=0.041，P=0.011）

Medicine of the present invention suppresses endotheliocyte FAK phosphorylation

We further infer the effect of DME25 to HECV cell FAK and paxillin protein active, and namely the tyrosine phosphorylation of these albumen, use the tyrosine phosphorylation specific antibody.As shown in Figure 7, DME25 suppresses the FAK phosphorylation and produced larger inhibitory action with the coupling of FAK inhibitor the time.Alone DME25 or FAK inhibitor and the two coupling can both remarkable effect in the phosphorylation of paxillin albumen.Visible by immunofluorescence method, in blank cell, local sticking point FAK is by (the Fig. 8 that significantly dyes, the position that left side is pointed out by arrow), after adding DME25, FAK inhibitor and the two coupling are compared with blank group, although the degree of dyeing does not change, and has shown less cell adhesion speckle (Fig. 8, left side).What is interesting is that the FAK of part FAK phosphorylation is by pFAK phosphorylation antibody engrain.(right side extends the time exposed) as shown in Figure 8, the local adhesion plaque of visible blank cell place is dyeed by pFAK, and DME25 and FAK inhibitor cause pFAK dyeing to reduce, however the cell of the two coupling is not dyeed by pFAK fully.

Conclusion

The angiogenesis inhibitor treatment, as Arastin, be used as the new show risen for the treatment of of solid tumor, and just in some tumor types, showing its clinical value.Some traditional antitumoral compounds also are found in the effect of anti-angiogenic rebirth aspect.Chinese medicine composition of the present invention, as a kind of compound medicine of Chinese medicine, has shown its advantage aspect the oncotherapy clinical efficacy, comprise these two kinds of hepatocarcinoma, gastric cancer in China the tumor disease in high incidence.Also verified its some immanoprotection actions in chemotherapy process.

This experiment confirms, the inhibitory action that Chinese medicine composition of the present invention sticks and moves for tumor cell.These two kinds of cytological effects have important function in the angiogenesis of endotheliocyte.

The tubule that can be suppressed significantly the extracorporeal blood vessel endotheliocyte by the visible DME25 of experimental result forms.Further research finds that the dependent inhibition cellular matrix of this extract doses sticks and cell migration.These Chinese medicine compositions of proved invention as a result have the effect that tubule forms that suppresses.

The result of this experiment confirms it is to use low dose of FAK inhibitor blocking-up FAK can significantly improve the effect of Chinese medicine composition of the present invention, and extract itself also has inhibitory action to the FAK activity simultaneously, namely to the inhibitory action of FAK tyrosine phosphorylation.The FAK approach cellular matrix stick with cell adhesion in effect be better than extracellular matrix.In the interaction with substrate, cell utilizes the film integrin to be combined with substrate and to trigger in a series of cells and activates, one of them crucial approach is exactly the activation of focal adhesion kinase (FAK), and FAK can activate the interaction of integrin and cytoskeleton system conversely.This has just formed the important component part in matrix adhesion and cell migration process next.FAK is considered to the signal of interest path in angiogenesis.The FAK inhibitor, shown antineoplastic action in the clinical experiment for pulmonary carcinoma and breast carcinoma in early days.

In general, one of them the important function approach that can say Chinese medicine composition curative effect of the present invention is by suppressing the effect of FAK approach in angiogenesis.In a word, Chinese medicine composition of the present invention generates and to have good effect for extracorporeal blood vessel, can reduce that cellular matrix sticks and cell migration, and this is because it acts on focal adhesion kinase (FAK) approach.

Experimental example 2 medicine of the present invention for the inhibitory action of tumor-blood-vessel growth, for the inhibitory action of the transfer of tumor cell and with the synergistic experiment of Pi3K inhibitor

Materials and methods

Material

MCF-7, MCF-7 and MDA MB-231; PC-3 PC-3 and DU-145; SGC-7901, HGC27 and AGS; CCL188, RKO and and HRT18; All from animal cell culture storehouse, ECACC(Europe, Salisbury, Britain).Human osteosarcoma MG-63 and human lung cancer cell line A549 are purchased from ATCC (Unite States Standard cell bank).These cells are stored in DMEM (Sigma-Aldrich, Poole Dorset, the England) culture medium of adding penicillin, streptomycin and 10% hyclone.Cell culture condition 37 ^oC, 5% CO ₂With 95% humidity.

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

Method

Tested medicine pretreatment

Body outer cell proliferation detects

Cell proliferation adopts body outer cell proliferation analytical method (11,12).Cell is inoculated in 96 orifice plates to 3000 cells/well.After three blocks of plates are distinguished overnight incubation, 3d, 5d, 4% (v/v) formaldehyde is fixed, 0.5% (w/v) violet staining.The purple spectrophotometer that adopts of 10% acetic acid extractive crystallization detects absorbance (Bio-Tek ELx800 reads plate device, Vermont, USA).

The cellular matrix electric impedance sensor detects cell adhesion and migration

ECIS-Zq (Applied Biophysics Inc, NJ, US) detects (13,14) for the motor capacity of cell adhesion volume.Adopt ECIS RbA three-dimensional software building cytological map.96W1E ECIS is for this research.Measure cell and substrate interphase interaction by the gold-foil electrode that is placed in the culture plate surface.1h, with after the serine solution pretreatment of 10Mm, is hatched by full culture medium in the analyser surface in cultivation.The equivalent cell adds each culture hole.In cell adhesion detects, cell adds spike immediately after analyser.In cell migration detects, require to merge after cell 3h.The cell monolayer electrical impedance is damaged at 2000iA.Impedance and resistance are recorded to 20h immediately.The employing signal transduction inhibitor is analyzed.Adhere to and move and adopt ECIS RbA three-dimensional software to carry out the axonometric chart structure.

Western blot analysis

Cell is at 25cm ³To merging, adopt subsequently and (contain 1% Triton X-100,2 mM CaCl in culture bottle ₂, 100 μ g/ml Phenylmethanesulfonyl fluorides, 1 mg/ml leupeptin, 1 mg/ml aprotinin and, the former sodium vanadate of 10 mM) the HCMF buffer salt blows and beats and the 1h cell lysis that vibrates, the centrifugal precipitation of removing of 13,000g.Protein content in sample adopts Bio-Rad DC test kit (Bio-Rad laboratories, California, USA) to detect.

Equal protein adopts the analysis of SDS-PAGE glue.Separated backward Hybond-C Extra nitrocellulose filter (Amersham Biosciences UK Ltd, Bucks, UK) transfer printing, 10% milk sealing, differential protein detects expresses.Phosphorylation AKT (threonine) expresses and adopts anti-pAkt antibody (Santa Cruz Biotechnology, Inc., California, USA) to detect.In addition, GAPDH expresses and also adopts specific antibody (Santa Cruz Biotechnology, Inc., California, USA) to detect.Primary antibodie adds the anti-mouse antibodies (Sigma, Dorset, UK) of two resist-connection peroxidase after connecting.Adopt Supersignal West Dura Extended Duration substrate chemiluminescence system (Perbio Science UK Ltd, Cramlington, UK) and UVIProChem imaging system (UVItec Ltd, Cambridge, UK) check protein expression.

Result

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

Use high flux ECIS to detect the cell line of DME25 to different tumor types, comprise human breast carcinoma, carcinoma of prostate, pulmonary carcinoma, colon cancer, gastric cancer and osteosarcoma cell.YZXJ concentration is from 1:40 to 1:600,000.Concentration is higher than 1:125, and 000 pair of cell adhesion has the inhibition effect.More sensitive cell comprises MCF-7, and MG-63 and A539, the results are shown in Figure 9.At present model, adopting wide range to detect also can confirm its inhibition effect, the results are shown in Figure 10.Use the Rb method to take the A549 cell as example (Figure 11) diagram quantitative analysis results.Parameter alpha means the average distance between basement membrane and electrode (substrate).Therefore, it can clearly illustrate that DME25 is to existing dose-dependent inhibition between tumor cell and substrate.

Chinese medicine composition of the present invention suppresses cell migration

After having detected the effect to tumor cell adhesion, carry out monolayer and merge the tumor cell electric injury.Adopting ECIS to detect the Tumors cell injury recovers.DME25 is to tumor cell as a result, pulmonary carcinoma and colon cancer, and migration table reveals concentration dependent and suppresses.Figure 12 means the impact of DME25 on the A549 cell migration.Drug level is at 1:125, and 000 can see the inhibition effect, similar with the cell adhesion experiment.

Chinese medicine composition of the present invention is faint on the impact of human tumor cells propagation

Further studied the impact of the external on cell proliferation of DME25.After DME25 (1:1000 and 1:25,000) processing 72h (the results are shown in Table 1), in wider concentration range, DME25 has no significant effect tumor cell proliferation.

Table 1. DME25 is for the growth inhibited exercising result of in vitro cancerous cell

Cell type	Matched group (490nm absorbance)	1:1000 (490nm absorbance)	1:25,000 (490nm absorbance)
				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

The impact of PI3K/AKT mediation Chinese medicine composition of the present invention on cell adhesion

For confirming the signal path of DNE25 to cell adhesion, for PI3K/AKT and AKT, detect the inhibitor effect.Add separately Wortmannin (PI3K inhibitor) or suppress prostate gland cancer cell (DU-145) with the DME25 coupling to adhere to, compare with matched group and alone DME25, adhere to all and reduce.Coupling is more alone also shows stronger inhibitory action.AKT is the downstream passages inhibitor of PI3K, and its inhibitor and DME25 coupling can improve the inhibitory action to cell adhesion.Alone inhibition is shown in Figure 13 a little less than suppressing to cell adhesion.Figure 14 means the impact of DME25 on lung cancer cell line A549 AKT (Ser 473) phosphorylation.High concentration (1:1000) DME25 reduces AKT phosphorylation (pAKT) level.Add AKT inhibitor and DME25 can work in coordination with inhibition to pAKT, especially with low concentration DME25(1:5000) coupling.

Conclusion

Chinese medicine composition of the present invention not only has antitumous effect, and directly adhesion and the migration of inhibition tumor cell, prevents neoplasm metastasis, and with the synergism of chemotherapeutics.

The effect of experimental example 3 Drug therapy rheumatoid arthritis of the present invention reaches the inhibitory action to the rheumatoid arthritis angiogenesis

1 material

1.1 animal SD rat, male and female half and half, body weight is 180-200g, is purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..Licence numbering SCXK(capital) 2006-0009.

1.2 medical material and main agents medicament capsule of the present invention (according to the method preparation of embodiment 1, Shijiazhuang Yiling Pharmaceutical Co., Ltd); Incomplete Freund's adjuvant (IFA) and solubility in acid II Collagen Type VI (collagen II, 3806) are the sigma product; Bacillus calmette-guerin vaccine is for being purchased from Nat'l Pharmaceutical & Biological Products Control Institute.

Method

2.1 the preparation of complete Freund's adjuvant (Freund, s Complete adjuvant CFA): add bacillus calmette-guerin vaccine at incomplete Freund's adjuvant (IFA), making it final concentration is 7.5mg/ml, repeatedly lashes into emulsifying agent.

2.2 the preparation of rat Juvenile rheumatoid arthritis model: cattle II Collagen Type VI is dissolved in the glacial acetic acid of 0.1M, and 4 ℃ are spent the night.Then with CFA, grind evenly, make and contain the Emulsion that the II Collagen Type VI is 2mg/ml, above-mentioned Emulsion, with 150 μ l/ only left foot and root of the tail section subcutaneous multiple spot injection model group and each administration group immune rat (every rat is equivalent to inject the II Collagen Type VI of 150 μ g), is injected to Emulsion 150 μ l booster immunizations of the same race in the 10th day every rat root of the tail section.Normal group is left intact.

2.3 the grouping of laboratory animal and administration: the SD rat is divided into 5 groups at random.Be Normal group, give the equal-volume solvent; The Juvenile rheumatoid arthritis model group, give the equal-volume solvent; The large, medium and small dosage group of medicine of the present invention, prepare model and start gavage next day and give medicine of the present invention, and dosage is respectively 1.56,0.78,0.39 gkg-1; Each is organized the administration volume and is the 1ml/100g body weight, successive administration 40 days.

2.4 the impact of medicine of the present invention on Juvenile rheumatoid arthritis rat arthritis index: after administration finishes, rat adopts the joint point system to be marked.Ear: tuberosity and rubescent symptom (without 0 minute, having 1 minute); Nose: red, swelling (without 0 minute, having 1 minute); Tail: tuberosity (without 0 minute, having 1 minute); Fore paw: the inflammation at least one joint (without 0 minute, having 1 minute); Right back pawl: swelling (without 0 minute, slight 1 minute, moderate 2 minutes, severe 3 minutes).The average of every group of rat is arthritis index.

2.5 the impact of medicine of the present invention on the toes volume of collagen-induced rats with arthritis: measure the right back whole toe volume of every rat with the toes capacity measurer, observe the variation of respectively organizing rat foot volume.

2.6 pathological change: put to death rat, get the right side ankle joint, formalin is fixed, specimens paraffin embedding slices, HE colouring method.

2.7 results of statistical analysis with

± sMean, adopt SPSS 11.5 softwares to carry out ANOVA analyzing and processing and Dunnett check.

Result

3.1 the impact of medicine of the present invention on Juvenile rheumatoid arthritis rat arthritis index: result shows, approximately within 14 days, start II Collagen Type VI rat model and each administration group rat after first immunisation and start to occur ear redness and redness and swelling of joints, the metapedes toe joint occurs spreading to front foot and afterbody after redness, and be on the rise, obvious tuberosity appears in some Mus afterbody.With Normal group relatively, model group arthritis index obviously raise (P<0.01); With model group, compare, medicine of the present invention can obviously reduce arthritis index, and middle and high dosage group has statistical significance (P<0.05, P<0.01).In Table 1.

3.2 the impact of medicine of the present invention on the toes volume of collagen-induced rats with arthritis: with Normal group relatively, model group toes volume obviously raise (P<0.01); With model group, compare, medicine of the present invention can obviously reduce the toes volume, and middle and high dosage group has statistical significance (P<0.05, P<0.01).In Table 2.

Table 2 medicine of the present invention on collagen-induced rat arthritis impact (

± s)

Group	N	Arthritis index	The toes volume
				Matched 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 dosage group	11	4.14±1.57 ³⁾	2.11 ±0.42 ³⁾
				Long-pending high dose group	11	4.02±0.73 ⁴⁾	2.06 ±0.28 ⁴⁾

Annotate: with matched group, compare, ¹⁾ P<0.05, ²⁾ P<0.01; With model group, compare, ³⁾ P<0.05, ⁴⁾ P<0.01.

3.3 pathological change:

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

Collagen model group joint connective tissue and the visible inflammatory cell infiltration of muscular tissue, neutrophilic granulocyte, mononuclear cell in the visible synovial tissue of articular cartilage, especially lymphocytic infiltration, blood vessel increase, tunica intima distortion hypertrophy, tube chamber narrows down or blocks, under the synovial cell, cellulose oozes out in a large number, the collagen fiber deposition.

The big or middle dosage group of medicine of the present invention has a better role to the tissue injury of Juvenile rheumatoid arthritis.

Conclusion

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

Discuss

Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind ofly to take that to involve joint on every side be main multisystem inflammatory autoimmune disease.The principal character of RA is arthritis reaction, synovial membrane angiogenesis, and then causes chronic synovial hyperplasia, further affects cartilage and sclerotin, causes the disease that joint deformity and afunction are basic pathological changes.II Collagen Type VI inductivity arthritis (collagen induced arthritis, CIA) animal model be generally acknowledge at present for studying the ideal animals model of RA pathogenesis and exploitation treatment RA new drug, this model is by II Collagen Type VI (Collagen II, CII) induce, CII is the main component of cartilage, is the autoantigen of rheumatoid arthritis morbidity.Can find II Collagen Type VI antibody in clinical Patients With Rheumatoid Arthritis serum and synovial fluid.Because CIA model pathogenesis more approaches mankind RA, thereby it is the first-selected model of Recent study rheumatoid arthritis.

Our laboratory observation ear redness and redness and swelling of joints occur to the rat model group, and the metapedes toe joint occurs spreading to front foot and afterbody after redness, and obvious tuberosity appears in some Mus afterbody.Pathology also shows joint connective tissue and the visible inflammatory cell infiltration of muscular tissue, and the visible synovial tissue of articular cartilage lymphocytic infiltration, blood vessel increase, tunica intima distortion hypertrophy, collagen fiber deposition.Medicine of the present invention can obviously improve general symptom and pathological change.

Model group arthritis index and toes volume obviously raise, medicine of the present invention can significantly reduce arthritis index and toes volume, improve the focus blood vessel and increase, the pathological change of tunica intima distortion hypertrophy, the rat arthritis that proved invention medicine is induced the II Collagen Type VI has good preventive and therapeutic effect.

The effect of experimental example 4 drug treatment of diabetic retinopathy of the present invention reaches the inhibitory action to the diabetic renal papillary necrosis angiogenesis

1 material

1.1 animal KK/Upj-Ay mice, 30～40 g; The C57BL/6 mice, 25～30 g, be the SPF level, male, in 12 week age, is purchased from Beijing China Fukang biotech inc.

1.2 medical material and main agents medicament capsule of the present invention (according to the method preparation of embodiment 1, Shijiazhuang Yiling Pharmaceutical Co., Ltd); Endothelial cell growth factor (ECGF) (VEGF) primary antibodie (Santa Cruz company).

2 methods

2.1 the animal grouping is divided into model group, the high, medium and low dosage group of medicine of the present invention with 40 KK/Upj-Ay mices of administration by fasting blood sugar (FBG), separately establishing the C57BL/6 mice is matched group, every group of 10 animals.Adopt gastric infusion, the high, medium and low dosage group of medicine of the present invention gives respectively 1.56,0.78,0.39 gkg ^-1Medicine of the present invention, matched group and model group give isopyknic distilled water, once a day, continuous 3 months.

2.2 morphological change

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

2.2.2 eyeball of mouse, get eyeball, is fixed in 48 h in 10% formalin, separates retina, 37 ℃ of about 3h of incubator digestion vibration of 3% tryptic digestive juice are put in the tap water rinsing, and only the retinal blood pipe network of remaining layer of transparent, carry out HE-PAS dyeing.

2.2.3 eyeball of mouse, be placed in glutaraldehyde and fix, the electron microscopic observation ultrastructure.

2.3 Western blot method detects the expression of vegf protein and gets retinal tissue, the RIPA cracking process extracts total protein, protein sample is carried out to 12% SDS-PAGE gel electrophoresis, 4 ℃ of constant voltage electrotransfers are to the PVDF film, 5% defatted milk powder sealing 1h, after add the primary antibodie (VEGF of 1:200 dilution, the GAPDH internal reference of 1:1000 dilution), 4 ℃ of overnight incubation, TBST washes film, two anti-(the diluted concentration 1:1000) that add horseradish peroxidase-labeled, the luminous colour developing in ECL darkroom, gel imaging system photographing scanning gray value is analyzed, gray value ratio with destination protein gray value and GAPDH is used for statistical analysis.

2.4 results of statistical analysis with ± sMean, adopt SPSS 11.5 softwares to carry out ANOVA analyzing and processing and Dunnett check.

3 results

3.1 HE dyeing: each confluent monolayer cells of matched group retina is well arranged, and cellularity is tight; The obvious attenuation of model group layer of retina,pigment epithelium, the cell arrangement disorder, medicine of the present invention can improve above-mentioned variation.

3.2 HE-PAS dyeing: normal mouse retinal capillary distribution rule, move towards more straight, the caliber even thickness is consistent; Model group retinal capillary net arrangement disorder, move towards irregular, and many blood capillaries are turned round and are polymerized to clump, part telangiectasis, tube chamber thickness inequality; Medicine of the present invention can obviously improve above-mentioned variation, the most obvious with high agent group.

3.3 Electronic Speculum result: the control group mice retina, the inside and outside joint intersection of photoreceptor cell, mitochondrial crista is clear, and cell arrangement is neat, and nucleus and organelle are normal; Vacuolar degeneration, appear in the model group retina, acromere membranous disc structural fuzzy, arrangement disorder, swelling and degeneration.The swelling of inside and outside stratum nucleare intracellular plastochondria, photoreceptor cell film rupture, karyopycnosis, peripheral visible cell chip.Medicine group acromere membranous disc of the present invention gap dwindles than model group, and the arrangement depth of parallelism is slightly good, and interior outer nuclear layer cell mitochondrial vacuolar degeneration reduces to some extent, and high dose group is the most obvious.

3.4 retina vegf expression: Western blot result show vegf protein express in the matched group normal retina, express lower, the model group vegf protein express apparently higher than matched group ( P<0.01), after pharmaceutical intervention of the present invention, vegf protein is expressed significantly lower than model group, and middle and high dosage group have statistical discrepancy ( P<0.05, P<0.01).In Table 3.

Table 3 medicine of the present invention on the impact of KK Mouse Retina vegf expression (n=3,

± s)

Group	VEGF
		Matched group	0.25±0.03
Model group	0.78±0.15 ²⁾
		Medicine of the present invention hangs down the agent group	0.57±0.11
Agent group in medicine of the present invention	0.44±0.07 ³⁾
		The high agent group of medicine of the present invention	0.31±0.05 ⁴⁾

4 conclusions

Medicine of the present invention has certain preventive and therapeutic effect to the diabetic renal papillary necrosis mice, and vegf protein is expressed significantly and reduced, and can effectively suppress angiogenesis.

5 discuss

Clinical middle type ii diabetes accounts for more than 90% of diabetes colony, and general Study person all selects type ii diabetes (DM) animal model.The KK mice of selecting in this experiment is spontaneous DM model, has been widely used in the basic research of DM.Report that the KK mice was raised retina after 3 months and obvious blood capillary and neuropathy occurred age in 8-12 week, this research has also been observed similar result by preliminary experiment, and therefore in this research, we have selected 8-12 KK mice in age in week to raise 3 months as diabetic renal papillary necrosis (DR) model.Our light microscopic and Electronic Speculum result have also shown the obvious attenuation of model group Mouse Retina pigment epithelium layer, cell arrangement disorder; The capillary network arrangement disorder, move towards irregular; Vacuolar degeneration appears, acromere membranous disc structural fuzzy, and arrangement disorder, the pathology damage such as swelling and degeneration, prompting diabetic renal papillary necrosis model forms.

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

In the normal eyes tissue, retinal pigment epithelium, bovine retinal capillary pericytes, endotheliocyte and M ü ller cell all can produce the VEGF of reduced levels, in order to maintain stabilization of vascular and retina normal development, but under the sugared condition of height, VEGF is released, cause a series of reaction, comprise the retinal vessel seepage, stimulate retina endothelial cell proliferation and migration, promote new vessels formation etc.This experiment has also confirmed that the expression of model group DR Mouse Retina vegf protein significantly raises.Give the Drug therapy of the present invention of various dose after 3 months, vegf expression all has reduction in various degree, and proved invention medicine can suppress angiogenesis by reducing vegf expression, thereby plays the amphiblestroid effect of protection diabetic mice.

Experimental example 5 medicine of the present invention reaches the inhibitory action to the artery plaque angiogenesis for the Stabilization of artery plaque

1 material

1.1 the animal large ear rabbit, regular grade, hero, 2.0 ~ 2.4 kg, buy in KeYu animal cultivation center, Beijing, licence numbering: the SCXK(capital) 2007-0003.

1.2 medical material and main agents medicament capsule of the present invention (according to the method preparation of embodiment 1, Shijiazhuang Yiling Pharmaceutical Co., Ltd); Oil red 0 is purchased from U.S. Sigma company; Sirius red is purchased from U.S. Sigma company.

Method

2.1 research method: 30 male large ear rabbits, application balloon injured ventral aorta+high cholesterol diet (l% cholesterol, every feed for nursing 120-140g/d) feed the method for 8 weeks, set up steady dynamic pulse atherosclerosis (As) Patchy model, be divided at random: medication therapy groups of the present invention (gives 0.39 gkg-1 medicament capsule of the present invention every day, medicine group of the present invention), full diet is fed spontaneous regression group (matched group), continues high fat and feeds matched group (model group).Every group of 10 rabbits.Model group and medicine of the present invention continue High-fat diet after setting up mould, respectively single cage is fed, automatic water-drinking, give Chinese speckle crab snake venom and histamine after 12 weeks and carry out medicine triggering 0.15mg/kg peritoneum hemostasis, auricular vein injection histamine 0.02mg/kg after 30min, put to death front 24 h of animal, twice medicine triggering of 48 h, to impel speckle, experimental breaking occurs, put to death after triggering.

2.1 the mensuration of intravascular ultrasonic imaging inspection (IVUS): medicine carries out the inspection of ventral aorta intravascular ultrasound after triggering respectively.After 3% pentobarbital sodium anesthesia, the experimental rabbit dorsal position is fixed on laboratory table, concrete operations are as follows: conventional application 4F lancet puncture left femoral artery, under 0.014 inch guide wire and convergent divergent channel are auxiliary, insert 5F sheath pipe, fixing, vein gives heparin 100u/kg anticoagulant immediately, along guide wire under the guiding of body surface Vascular Ultrasonography, insert the intravascular ultrasound probes conduit, by stenotic lesion, to the blood vessel far-end, then slowly withdraw probe conduit (0.5mm/s), labelling speckle far-end, near-end image, video recording is for off-line analysis and file.Measure the incidence rate of three treated animal plaque ruptures, measure simultaneously

(1) the outer elastic membrane area (external elastic membrance area, EEMA) of blood vessel: refer to the area that the outer elastic membrane of blood vessel comprises, comprise lumen of vessels area and plaque area sum.

(2) tube chamber area (lumen area, LA): refer to the area that tunica intima comprises.

(3) plaque area (plaque area, PA): outside blood vessel, elastic membrane area and tube chamber area is poor.

(4) the narrow percentage rate of tube chamber area (lumen area stenosis, LAS%): the ratio of the outer elastic membrane area of plaque area and blood vessel.

2.2 specific stain: utilize oil red O stain to observe speckle inner lipid, collagen relative amount.

2.3 results of statistical analysis with

Result

3.1 intravascular ultrasonic imaging inspection (IVUS):

With model group relatively, medicine group experimental rabbit EEMA of the present invention, PA, LAS% descend obviously ( P<0.01), (table 4).The rupture rate of the three treated animal ventral aorta specklees that record: medicine group of the present invention: 0%; Matched group: 0%; Model group: 33%.

The comparison of table 4 treatment experimental rabbit ventral aorta IVUS measured value after 12 weeks

Index	Medicine group of the present invention (n=9)	Matched group (n=9)	Model group (n=9)
				LA(mm ²)	6.59±1.28	7.27±1.74	7.93±2.26
EEMA(mm ²)	9.44±1.88**	12.82±2.34	15.23±2.39
				PA(mm ²)	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

Annotate: with model group, compare * * P<0.01

3.2 pathological change:

The positive percentage ratio of specific stain display model group oil red O stain, apparently higher than matched group, compares with model group, and the positive percentage ratio of medicine group of the present invention significantly reduces.

Conclusion

This experimental applications balloon injured ventral aorta+high cholesterol diet has been set up Corn Bract Decotion, check and show that medication therapy groups of the present invention can significantly reduce EEMA, PA, LAS% by IVUS, and the rupture rate of reduction speckle, thereby the effect of the vulnerable plaque of playing stably, microexamination can obviously reduce blood vessel quantity in speckle, suppresses angiogenesis.

Claims

1. a Chinese medicine composition suppresses the application in the medicine of angiogenesis in preparation, it is characterized in that the crude drug of following weight ratio is made:

Radix Astragali 120-360, Fructus Ligustri Lucidi 100-300, Radix Ginseng 30-95, Ganoderma 30-95, Rhizoma Curcumae 65-195, Rhizoma Atractylodis Macrocephalae 30-90, Herba Scutellariae Barbatae 65-195, Herb Gynostemmae Pentaphylli 120-360, Poria 30-95, Endothelium Corneum Gigeriae Galli 15-45, Herba Duchesneae Indicae 65-195, Herba Solani Lyrati 65-195, Herba Artemisiae Scopariae 65-195 Radix Cynanchi Paniculati 65-195 Eupolyphaga Seu Steleophaga 10-30 Herba Hedyotidis Diffusae 65-195.

2. application according to claim 1 is characterized in that being made by the crude drug of following weight portion:

3. application according to claim 1 is characterized in that being made by the crude drug of following weight portion:

4. application according to claim 1 is characterized in that being made by the crude drug of following weight portion:

5. according to the described application of claim 1-4 any one, it is characterized in that the active component of described Chinese medicine composition is made by following steps:

6. according to the described application of claim 1-4 any one, it is characterized in that described pharmaceutical dosage form is capsule, tablet, powder, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection.

7. application according to claim 6, the preparation method that it is characterized in that described medicine capsule is to be made by following steps:

(2), Radix Ginseng, Fructus Ligustri Lucidi, add 8 times of amount 70% alcohol reflux 2 times, 3 hours for the first time, 2 hours for the second time, merge extractive liquid,, reclaimed ethanol to without the alcohol flavor, filtrate and Radix Ginseng Fructus Ligustri Lucidi residue are standby;

(3), Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati, united extraction volatile oil, carry oil time 6-12 hour, the another device of volatile oil is collected, residue and aqueous solution are standby;

(5), the Radix Astragali, Ganoderma, Herba Hedyotidis Diffusae, Herba Scutellariae Barbatae, Herb Gynostemmae Pentaphylli, Herba Duchesneae Indicae, Herba Solani Lyrati, Herba Artemisiae Scopariae, with residue after the alcohol extraction of gained Radix Ginseng, Fructus Ligustri Lucidi in step (2), and after in step (3), gained Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati are carried oil, residue merges, add 9 times of water gagings, heating decocts 2 times, each 2 hours, merge decoction liquor, add gained Radix Ginseng, Fructus Ligustri Lucidi alcohol extract and step (3) obtained aqueous solution in step (2), be condensed into the clear paste of relative density 1.20-1.25, dry, pulverize, powder gets dry extract; Add suitable pharmaceutically acceptable adjuvant to granulate the gained dried cream powder;

8. according to the described application of claim 1-4 any one, it is characterized in that the application of described Chinese medicine composition in suppressing the cancer angiogenesis drug.

9. application according to claim 8, is characterized in that described Chinese medicine composition prevents the application in the medicine of cancer metastasis in preparation.

10. application according to claim 8, is characterized in that described Chinese medicine composition prevents the application in the medicine of cancer metastasis at preparation associating focal adhesion kinase inhibitor.

11. application according to claim 8, is characterized in that described Chinese medicine composition prevents the application in the medicine of cancer metastasis at preparation associating inhibitors of phosphatidylinositol3 3-kinase.

12. application according to claim 11, is characterized in that described inhibitors of phosphatidylinositol3 3-kinase is wortmannin.

13. application according to claim 8, is characterized in that described Chinese medicine composition prevents the application in the medicine of cancer metastasis at preparation associating AKT inhibitor.

14., according to the described application of any one in claim 9-13, it is characterized in that described cancer is osteosarcoma, pulmonary carcinoma, human breast carcinoma, carcinoma of prostate, colon cancer or gastric cancer.

15., according to the described application of claim 1-4 any one, it is characterized in that the application of described Chinese medicine composition in suppressing the rheumatoid arthritis angiogenesis drug.

16., according to the described application of claim 1-4 any one, it is characterized in that the application of described Chinese medicine composition in suppressing the diabetic renal papillary necrosis angiogenesis drug.

17., according to the described application of claim 1-4 any one, it is characterized in that the application of described Chinese medicine composition in suppressing the artery plaque angiogenesis drug.