CN103830662A - Application of traditional Chinese medicine composition in preparation of drug for inhibiting rheumatoid arthritis angiogenesis - Google Patents

Application of traditional Chinese medicine composition in preparation of drug for inhibiting rheumatoid arthritis angiogenesis Download PDF

Info

Publication number
CN103830662A
CN103830662A CN201410014362.6A CN201410014362A CN103830662A CN 103830662 A CN103830662 A CN 103830662A CN 201410014362 A CN201410014362 A CN 201410014362A CN 103830662 A CN103830662 A CN 103830662A
Authority
CN
China
Prior art keywords
herba
gained
chinese medicine
medicine composition
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201410014362.6A
Other languages
Chinese (zh)
Other versions
CN103830662B (en
Inventor
魏聪
王宏涛
张会欣
常丽萍
肖维刚
安军永
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shijiazhuang Yiling Pharmaceutical Co Ltd
Original Assignee
Shijiazhuang Yiling Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shijiazhuang Yiling Pharmaceutical Co Ltd filed Critical Shijiazhuang Yiling Pharmaceutical Co Ltd
Priority to CN201410014362.6A priority Critical patent/CN103830662B/en
Priority claimed from CN201210166200.5A external-priority patent/CN103417907B/en
Publication of CN103830662A publication Critical patent/CN103830662A/en
Application granted granted Critical
Publication of CN103830662B publication Critical patent/CN103830662B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Medicines Containing Plant Substances (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention discloses an application of a traditional Chinese medicine composition in preparation of drugs for inhibiting rheumatoid arthritis angiogenesis. The traditional Chinese medicine composition comprises traditional Chinese medicines such as radix astragali, ligustrum lucidum and the like. The traditional Chinese medicine composition has the effects of tonifying qi and nourishing yin, invigorating spleen and tonifying kidney, dissipating binds and dredging collaterals, and focuses on the pathogenesis; experiments confirm that the traditional Chinese medicine composition of the invention can effectively inhibit angiogenesis, and can be clinically used for rheumatoid arthritis. The traditional Chinese medicine composition of the invention is reasonable in combination, has less adverse drug reaction, and is suitable for long-term use by patients.

Description

The application of a kind of Chinese medicine composition in the medicine of preparation inhibition rheumatoid arthritis angiogenesis
Written or printed documents application for a patent for invention is divisional application, and original bill information is as follows:
Application number: 201210166200.5
The applying date: on May 26th, 2012
Denomination of invention: 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 the medicine of preparing angiogenesis inhibiting, belong to 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.Meanwhile, the development of various diseases is also closely related with blood vessel angiogenesis.
Compared with normal blood vessels, tumor vascular structure lacks integrity, between endotheliocyte, there is larger gap, permeability is strong, the netted structure disturbance of blood vessel, have a large amount of blood vessel cecum and arteriovenous shunt, extremely the causing of this structure ooze out increase and tissue between high pressure, be also easy to cancerous cell and shift simultaneously.The growth of tumor has two different stages, changes into and has the fast breeding of the blood vessel stage from avascular slow growth stage.If there is no angiogenesis, the growth of primary tumo(u)r can not exceed 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 take them as target 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 generation of human tumor development 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, develop take PI3K-Akt signal path key molecule as the ideas of cancer therapy of target spot at present.Wortmannin (Wortmannin) is exactly wherein most typical representative, has entered at present the clinical research stage.Wortmannin structural formula is as follows:
Figure 731838DEST_PATH_IMAGE001
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 3invasion and attack behavior be necessary, as integrin alpha in PI3K mediation carcinoma of prostate vβ 3the invasion and attack characteristic driving, in breast carcinoma and ovarian cancer, crossing of Akt2 expressed and can be raised integrin beta by IV collagen type 1thereby increase invasion and attack and the transfer of cell.
The etiology unknown of rheumatoid arthritis at present, 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 feature of DR shows as the retinal microvascular pathological changes progressively increasing 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.There is new vessels depending on pars papillaris or other positions in optical fundus, how with connective tissue proliferation, visual function be seriously damaged and cause that blind person is quite a few to be seen, occurring it being the important step for the treatment of diabetic retinopathy (DR) therefore suppress new vessels.
The unstable plaque rupture that causes of atherosclerosis (AS) speckle, and then there is acute coronary artery syndrome (ACS).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 object for the treatment of ischemic heart desease.
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 up of 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 up of 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 up of following steps:
(1), take Chinese crude drug according to crude drug part by weight, clean;
(2), Fructus Ligustri Lucidi, people participate in 6-10 and doubly measure 50-90% ethanol extraction 1-3 time, each 1-4 hour, merge extractive liquid,, filters, filtrate recycling ethanol is to without alcohol taste, filtrate and medicinal residues are for subsequent use;
(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 for subsequent use;
(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 in the time surveying for 65 ℃ is 1.15-1.30; dry, pulverize, for subsequent use;
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 wherein preparation method of capsule is to be made up of following steps:
(1), take Chinese crude drug according to crude drug part by weight, clean;
(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 alcohol taste, filtrate and Radix Ginseng, Fructus Ligustri Lucidi residue are for subsequent use;
(3), Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati, united extraction volatile oil, carries the oil time and is no less than 8 hours, the another device of volatile oil is collected, residue and aqueous solution are for subsequent use;
(4), Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli two taste animal drugs, washing, 60 ℃ of oven dry, merge with Poria, are ground into 100 order powder, for subsequent use 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 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, mix with the granule of gained in step (5), 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 that Fan Biting " pharmacy of Chinese materia medica " (Shanghai Science Press 1997 December the 1st edition) records, makes the acceptable regular dosage form of pharmaceutics.
For above-mentioned dosage form can be realized, need in the time of these dosage forms of preparation, add the acceptable adjuvant of pharmacy, for example: 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, 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 that in Shanghai Science Press December in 1997 the 1st edition, each dosage form is recorded) when 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.Meanwhile, can suppress significantly FAK phosphorylation, collaborative FAK inhibitor has inhibition angiogenesis function.
Experiment confirmation, pharmaceutical composition of the present invention can be worked in coordination with 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 the effect of the transfer of preventing preferably.
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.
Accompanying drawing explanation
fig. 1the effect that DME25 generates external HECV cell blood capillary tubule.HECV cell between 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), quantize length (E) with Digital image analysis technique.DME25 can significantly suppress tubule form and and FAK inhibitor between there is synergism.
fig. 2dME25 is not good enough to the action effect of vitro endothelial cell growth.With the DME25 processing cell 72h of different diluted concentrations.Carry out cell counting by crystal violet.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.With the comparison of blank group, * p<0.01; With independent DME and FAK inhibitor comparison separately, #p<0.01.
fig. 4the dependency (B) of concentration dependence (A) and FAK approach between DME25 and cell adhesion.Shown in data from 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; 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; 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 of the arrow prompting electric injury induction in A figure.
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.In SDS-polyacrylamide gel electrophoresis, separate after equal protein, phosphorylation FAK and Paxillin are carried out to probe in detecting with FAK and paxillin phosphorylation specific antibody.
fig. 8focal adhesion kinase in 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, and uses respectively after anti-FAK and anti-pFAK antibody treatment, total FAK(left side) and FAK phosphorylation (right side) by engrain.After irradiating by 100ms, FAK imaging obtains, and after 400ms irradiates, pFAK imaging obtains.
fig. 9dME25 to MCF-7 (on), MG-63 (in) and A549 cells (under) substrate adhere to obtain impact.Concentration is higher than 1:125, and 000 all has inhibitory action.
figure 10wideband detects DME25 and suppresses cell adhesion.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 A549 cell migration.
figure 13the effect of PI3K/AKT path in DME25 induction DU-145 cell adhesion.A: add the impact of Wortmannin on cell adhesion.B:AKT suppresses and the effect of DME25 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:
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:
(1), take Chinese crude drug according to crude drug part by weight, clean;
(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 alcohol taste, filtrate and Radix Ginseng, Fructus Ligustri Lucidi residue are for subsequent use;
(3), Rhizoma Curcumae, Rhizoma Atractylodis Macrocephalae (parched), Radix Cynanchi Paniculati, united extraction volatile oil, carries the 8 hours time of oil, the another device of volatile oil is collected, residue and aqueous solution are for subsequent use;
(4), Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli, washing, 60 ℃ of oven dry, merge with Poria, are ground into 100 order powder, in 3KGY 60for subsequent use after 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, for subsequent use;
(6), step (5) gained dried cream powder is added to 134 grams of starch, 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, pack 1000 capsules into and get final product.
embodiment 2:
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:
(1), take Chinese crude drug according to crude drug part by weight, clean;
(2), Radix Ginseng, Fructus Ligustri Lucidi, add 6 times of amount 90% alcohol reflux 4 hours, extracting solution reclaims ethanol to without alcohol taste, filtrate and Radix Ginseng, Fructus Ligustri Lucidi residue are for subsequent use;
(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 for subsequent use;
(4), Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli, washing, 60 ℃ of oven dry, merge with Poria, are ground into 100 order powder, in 3KGY 60for subsequent use after 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 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, for subsequent use;
(6), step (5) gained dried cream powder is added to 112 grams of starch, 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:
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:
(1), take Chinese crude drug according to crude drug part by weight, clean;
(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 alcohol taste, filtrate and Radix Ginseng, Fructus Ligustri Lucidi residue are for subsequent use;
(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 for subsequent use;
(4), Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli, washing, 60 ℃ of oven dry, merge with Poria, are ground into 100 order powder, in 3KGY 60for subsequent use after 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, 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, for subsequent use;
(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.
For proved invention medicine is at therapeutic effect, use the capsule (hereinafter referred to as medicine of the present invention) making by embodiment 1, carry 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 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
Carry out for convenience of experiment, 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, 20 revs/min of swiveling wheels under room temperature, 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 subpackage is preserved as standardized extract, for subsequent use.
in-vitro cell growth method
HECV cell with 3000, every hole cell kind on 96 orifice plates.Hatch an evening, 3 days and 5 days with in triplicate culture plate.After fully cultivating, plank is shifted out from incubator, 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, is provided by manufacturer.Current producer adopts 96W1E array.ECIS is that the gold film electrode by being placed in culture dish surface is measured the interaction between cell and its substrate adhering to.The array surface that contains aminothiopropionic acid solution with post processing, complete culture medium culturing 1 hour for this array.In each culture hole, add the cell of equal number.In cell adhesion test, its adhesiveness is at once followed the tracks of after cell is added to array.For 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 test plate hole, inhibitor separately is all 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 formation is in the training period recorded and taken 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, to block 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, removes remaining primary antibodie.The anti-Mus two anti-(Insight Biotechnology, Britain) that drips FITC labelling, is placed in microscope slide on shaking table, and lucifuge is hatched 1 h.Finally, microscope slide rinse three times, removes and does not resist in conjunction with two, after Fluor-save mounting (Calbiochem-Novabiochem, Britain), adopts Olympus BX51 fluorescence microscope in 100 times of thing Microscopic observations.
gel electrophoresis and immune protein
Cell is at 25 cm 3in tissue culture flasks, grow to fusion, collecting cell, add 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.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 develop the color (Perbio Science, Britain), adopts UVIProChem imaging system to detect (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, can significantly shorten little length of tube (P=0.046) (seeing that Fig. 1 dilutes by 1:1000) with matched group comparison DME25.This concentration does not produce 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 the inhibitory action process repeated experiment checking (Fig. 3 B) of DME25.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
Stick similarly with cellular matrix, cell migration can be suppressed by DME25 equally, and cell migration can further be suppressed when with the coupling of FAK inhibitor.(Fig. 5).
medicine of the present invention and FAK inhibitor form and exist synergism at endothelial cell adhesion, migration and tubule
FAK inhibitor is to significantly (Fig. 3 C, 3D, 4B, 5) of HECV cytosis.In figure, can find out, during with DME25 coupling, inhibitory action is worked in coordination with enhancing.
FAK inhibitor shows as inhibitory action to the formation of tubule but does not have remarkable statistical significance (p=0.14) (Fig. 1 E).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 the namely tyrosine phosphorylation of these albumen uses tyrosine phosphorylation specific antibody.As shown in Figure 7, DME25 suppresses FAK phosphorylation and when with the coupling of FAK inhibitor, has produced larger inhibitory action.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), add after 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 exposing) 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, but the cell of the two coupling is not dyeed by pFAK completely.
conclusion
Angiogenesis inhibitor treatment, as Arastin, is used as the new show rising for the treatment of of solid tumor, and just in some tumor types, is showing its clinical value.Some traditional antitumoral compounds are also 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 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 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 result proved invention Chinese medicine compositions 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 FAK activity, the namely inhibitory action to FAK tyrosine phosphorylation simultaneously.FAK approach cellular matrix stick with cell adhesion in effect be better than extracellular matrix.With the interaction of substrate in, cell utilizes 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.FAK inhibitor, has 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 has 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
Carry out for convenience of experiment, 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, 20 revs/min of swiveling wheels under room temperature, 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 subpackage is preserved as standardized extract, for subsequent use.
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).
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 culture plate surface.Analyser surface is with after the serine solution pretreatment of 10Mm, hatches 1h by full culture medium in cultivation.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.Cell monolayer electrical impedance is damaged at 2000iA.Impedance and resistance are recorded to 20h immediately.Employing signal transduction inhibitor is analyzed.Adhere to and move and adopt ECIS RbA three-dimensional software to carry out axonometric chart structure.
western blot analysis
Cell is at 25cm 3in culture bottle, to merging, adopt subsequently and (contain 1% Triton X-100,2 mM CaCl 2, 100 μ g/ml Phenylmethanesulfonyl fluorides, 1 mg/ml leupeptin, 1 mg/ml aprotinin and, the former sodium vanadate of 10 mM) 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.Separation completes backward Hybond-C Extra nitrocellulose filter (Amersham Biosciences UK Ltd, Bucks, UK) transfer printing, 10% milk sealing, and 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 inhibition effect.More sensitive cell comprises MCF-7, and MG-63 and A539, the results are shown in Figure 9.Adopting wide range to detect at present model also can confirm its inhibition effect, the results are shown in Figure 10.(Figure 11) illustrates quantitative analysis results as an example of A549 cell example to use Rb method.Parameter alpha represents 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
Detect after the effect of tumor cell adhesion, carried out monolayer and merge tumor cell electric injury.Adopting ECIS to detect Tumors cell injury recovers.Result DME25 is to tumor cell, pulmonary carcinoma and colon cancer, and migration table reveals concentration dependent and suppresses.Figure 12 represents the impact of DME25 on A549 cell migration.Drug level is at 1:125, and 000 can see inhibition effect, similar with cell adhesion experiment.
chinese medicine composition of the present invention is faint on the impact of human tumor cells propagation
Further study 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, detect inhibitor effect for PI3K/AKT and AKT.Add separately Wortmannin (PI3K inhibitor) or suppress prostate gland cancer cell (DU-145) with DME25 coupling to adhere to, with matched group and alone DME25 comparison, 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 represents 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 and the inhibitory action to rheumatoid arthritis angiogenesis
1 material
1.1 animal SD rats, 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 materials 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 acid-soluble II Collagen Type VI (collagen II, 3806) are sigma product; Bacillus calmette-guerin vaccine is for being purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
method
The preparation of 2.1 complete Freund's adjuvants (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.
The preparation of 2.2 rat Juvenile rheumatoid arthritis models: cattle II Collagen Type VI is dissolved in the glacial acetic acid of 0.1M, and 4 ℃ are spent the night.Then grind evenly with CFA, make and contain the Emulsion that II Collagen Type VI is 2mg/ml, above-mentioned Emulsion, with only left foot and root of the tail portion 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) of 150 μ l/, is injected to Emulsion 150 μ l booster immunizations of the same race in the 10th day every rat root of the tail portion.Normal group is left intact.
The grouping of 2.3 laboratory animals and administration: SD rat is divided into 5 groups at random.Be Normal group, give equal-volume solvent; Juvenile rheumatoid arthritis model group, gives equal-volume solvent; The large, medium and small dosage group of medicine of the present invention, prepares model and starts gavage next day and give medicine of the present invention, and dosage is respectively 1.56,0.78,0.39 gkg-1; Each group administration volume is 1ml/100g body weight, successive administration 40 days.
2.4 impacts of medicine of the present invention on Juvenile rheumatoid arthritis rat arthritis index: administration finishes rear rat and adopts joint point system to mark.Ear: tuberosity and rubescent symptom (without 0 point, having 1 point); Nose: red, swelling (without 0 point, having 1 point); Tail: tuberosity (without 0 point, having 1 point); Fore paw: the inflammation (without 0 point, having 1 point) at least one joint; Right back pawl: swelling (without 0 point, slight 1 point, 2 points of moderates, 3 points of severes).The average of every group of rat is arthritis index.
The impact of the toes volume of 2.5 medicines of the present invention on collagen-induced rats with arthritis: measure the right back whole toe volume of every rat with toes capacity measurer, observe the variation of each group of rat foot volume.
2.6 pathological change: put to death rat, get right side ankle joint, formalin is fixed, specimens paraffin embedding slices, HE colouring method.
2.7 results of statistical analysis with
Figure DEST_PATH_IMAGE003
± srepresent, adopt SPSS 11.5 softwares to carry out ANOVA analyzing and processing and Dunnett check.
result
3.1 impacts of medicine of the present invention on Juvenile rheumatoid arthritis rat arthritis index: result shows, within after first immunisation approximately 14 days, start II Collagen Type VI rat model and each administration group rat and start to occur ear redness and redness and swelling of joints, metapedes toe joint occurs spreading to front foot and afterbody after redness, and be on the rise, there is obvious tuberosity in some Mus afterbody.With Normal group comparison, model group arthritis index obviously raise (P<0.01); With model group comparison, 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.
The impact of the toes volume of 3.2 medicines of the present invention on collagen-induced rats with arthritis: with Normal group comparison, model group toes volume obviously raise (P<0.01); With model group comparison, medicine of the present invention can obviously reduce 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 (
Figure 445717DEST_PATH_IMAGE003
± s)
Group N Arthritis index 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)
Note: with matched group comparison, 1) p<0.05, 2) p<0.01; With model group comparison, 3) p<0.05, 4) p<0.01.
3.3 pathological changes:
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 or blocks, under synovial cell, cellulose oozes out in a large number, 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
Medicine of the present invention has good preventive and therapeutic effect to the rat arthritis of II Collagen Type VI induction.
discuss
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of to involve the multisystem inflammatory autoimmune disease of joint as leading around.The principal character of RA is arthritis reaction, synovial membrane angiogenesis, and then causes chronic synovial hyperplasia, further affects cartilage and sclerotin, and causing joint deformity and afunction is the disease of basic pathological changes.II Collagen Type VI inductivity arthritis (collagen induced arthritis, CIA) animal model is the ideal animals model for the treatment of RA new drug for studying RA pathogenesis and exploitation of generally acknowledging at present, this model is by II Collagen Type VI (Collagen II, CII) induction, CII is the main component of cartilage, is the autoantigen of rheumatoid arthritis morbidity.In clinical Patients With Rheumatoid Arthritis serum and synovial fluid, can find II Collagen Type VI antibody.Because CIA model pathogenesis more approaches mankind RA, because of but the first-selected model of Recent study rheumatoid arthritis.
There is ear redness and redness and swelling of joints to rat model group in our laboratory observation, 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 focus blood vessel and increase, the pathological change of tunica intima distortion hypertrophy, proved invention medicine has good preventive and therapeutic effect to the rat arthritis of II Collagen Type VI induction.
the effect of experimental example 4 drug treatment of diabetic retinopathy of the present invention and the inhibitory action to diabetic renal papillary necrosis angiogenesis
1 material
1.1 animal KK/Upj-Ay mices, 30～40 g; C57BL/6 mice, 25～30 g, are SPF level, male, in 12 week age, are purchased from Fukang biotech inc of China, Beijing.
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 animal groupings are 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), and separately establishing 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 after administration finishes, get eyeball of mouse, be placed in formaldehyde and fix, HE dyeing;
2.2.2 eyeball of mouse, gets eyeball, is fixed on 48 h in 10% formalin, separates retina, and 37 ℃ of about 3h of incubator digestion vibration of 3% tryptic digestive juice are put in tap water rinsing, and only the retinal blood pipe network of remaining layer of transparent, carries out HE-PAS dyeing.
2.2.3 eyeball of mouse, is placed in glutaraldehyde and fixes, electron microscopic observation ultrastructure.
2.3 Western blot methods detect the expression of vegf protein and get retinal tissue, RIPA cracking process extracts total protein, protein sample is carried out to 12% SDS-PAGE gel electrophoresis, 4 ℃ of constant voltage electrotransfers are to 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, add two anti-(diluted concentration 1:1000) of 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
Figure 881246DEST_PATH_IMAGE003
± srepresent, adopt SPSS 11.5 softwares to carry out ANOVA analyzing and processing and Dunnett check.
3 results
3.1 HE dyeing: the 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, 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, caliber even thickness is consistent; Model group retinal capillary net arrangement disorder, moves 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 results: 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; , there is vacuolar degeneration in 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, periphery visible cell chip.Medicine group acromere membranous disc of the present invention gap dwindles compared with 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 expressions: Western blot result show vegf protein express in matched group normal retina express lower, 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.
The impact of table 3 medicine of the present invention on KK Mouse Retina vegf expression (n=3,
Figure 807614DEST_PATH_IMAGE003
± s)
Group VEGF
Matched group 0.25±0.03
Model group 0.78±0.15 2)
Low dose of group of medicine of the present invention 0.57±0.11
Agent group in medicine of the present invention 0.44±0.07 3)
The high agent group of medicine of the present invention 0.31±0.05 4)
Note: with matched group comparison, 1) p<0.05, 2) p<0.01; With model group comparison, 3) p<0.05, 4) p<0.01.
4 conclusions
?medicine of the present invention has certain preventive and therapeutic effect to 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 the 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 KK mice was raised retina after 3 months and occurred obvious blood capillary and neuropathy 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; Capillary network arrangement disorder, moves towards irregular; There is vacuolar degeneration, acromere membranous disc structural fuzzy, 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 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 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, by reducing vegf expression, suppress angiogenesis, thereby plays the amphiblestroid effect of protection diabetic mice.
experimental example 5 medicine of the present invention is for the Stabilization of artery plaque and the inhibitory action to artery plaque angiogenesis
1 material
1.1 animal large ear rabbits, regular grade, hero, 2.0 ~ 2.4 kg, buy in KeYu animal cultivation center, Beijing, licence numbering: SCXK(capital) 2007-0003.
1.2 medical materials 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 Sigma company of the U.S.; Sirius red is purchased from Sigma company of the U.S..
method
2.1 research methoies: 30 male large ear rabbits, application balloon injured ventral aorta+high cholesterol diet (l% cholesterol, every feed for nursing 120-140g/d) the nursing method of 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, after 12 weeks, give Chinese speckle crab snake venom and histamine 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 that experimental breaking occurs, after triggering, put to death.
The mensuration of 2.1 intravascular ultrasonic imaging inspections (IVUS): medicine carries out the inspection of ventral aorta intravascular ultrasound after triggering respectively.After 3% pentobarbital sodium anesthesia, experimental rabbit dorsal position is fixed in laboratory table, concrete operations are as follows: conventional application 4F lancet puncture left femoral artery, under 0.014 inch of 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 intravascular ultrasound probes conduit, to blood vessel far-end, then slowly withdraw probe conduit (0.5mm/s) by stenotic lesion, 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%): plaque area and the blood vessel ratio of elastic membrane area outward.
2.2 specific stains: utilize oil red O stain to observe speckle inner lipid, collagen relative amount.
2.3 results of statistical analysis with
Figure 28511DEST_PATH_IMAGE003
± srepresent, adopt SPSS 11.5 softwares to carry out ANOVA analyzing and processing and Dunnett check.
result
3.1 intravascular ultrasonic imaging inspections (IVUS):
With model group comparison, medicine group experimental rabbit EEMA of the present invention, PA, LAS% decline 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%.
Table 4 is treated the comparison of 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
Note: with relatively * * of model group p<0.01
3.2 pathological changes:
The positive percentage ratio of specific stain display model group oil red O stain is apparently higher than matched group, and with model group comparison, 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 reduce the rupture rate of speckle, thereby the effect of the vulnerable plaque of playing stably, microexamination can obviously reduce blood vessel quantity in speckle, suppresses angiogenesis.

Claims (6)

1. Chinese medicine composition suppresses the application in the medicine of rheumatoid arthritis angiogenesis in preparation, it is characterized in that described Chinese medicine composition is made up of the crude drug of following parts by weight: 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 described Chinese medicine composition is made up of the crude drug of following parts by weight:
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.
3. application according to claim 1, is characterized in that described Chinese medicine composition is made up of the crude drug of following parts by weight:
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.
4. application according to claim 1, is characterized in that described Chinese medicine composition makes 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 by the crude drug of following parts by weight
According to the application described in claim 1-4 any one, the preparation formulation that it is characterized in that described Chinese medicine composition is capsule, tablet, powder, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection.
5. according to the application described in claim 1-4 any one, it is characterized in that the active component of described Chinese medicine composition is made up of following steps:
(1), take Chinese crude drug according to crude drug part by weight, clean;
(2), Fructus Ligustri Lucidi, people participate in 6-10 and doubly measure 50-90% ethanol extraction 1-3 time, each 1-4 hour, merge extractive liquid,, filters, filtrate recycling ethanol is to without alcohol taste, filtrate and medicinal residues are for subsequent use;
(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 for subsequent use;
(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 in the time surveying for 65 ℃ is 1.15-1.30; dry, pulverize, for subsequent use;
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.
6. application according to claim 5, is characterized in that the capsule of described Chinese medicine composition is made up of following steps:
(1), take Chinese crude drug according to crude drug part by weight, clean;
(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 alcohol taste, filtrate and Radix Ginseng, Fructus Ligustri Lucidi residue are for subsequent use;
(3), Rhizoma Curcumae, the Rhizoma Atractylodis Macrocephalae, Radix Cynanchi Paniculati, united extraction volatile oil, carries the oil time and is no less than 8 hours, the another device of volatile oil is collected, residue and aqueous solution are for subsequent use;
(4), Eupolyphaga Seu Steleophaga, Endothelium Corneum Gigeriae Galli two taste animal drugs, washing, 60 ℃ of oven dry, merge with Poria, are ground into 100 order powder, for subsequent use 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 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, mix with the granule of gained in step (5), airtight half an hour, encapsulated and get final product.
CN201410014362.6A 2012-05-26 2012-05-26 A kind of application of Chinese medicine composition in treatment medicine for treating rheumatoid arthritis is prepared Active CN103830662B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410014362.6A CN103830662B (en) 2012-05-26 2012-05-26 A kind of application of Chinese medicine composition in treatment medicine for treating rheumatoid arthritis is prepared

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410014362.6A CN103830662B (en) 2012-05-26 2012-05-26 A kind of application of Chinese medicine composition in treatment medicine for treating rheumatoid arthritis is prepared
CN201210166200.5A CN103417907B (en) 2012-05-26 2012-05-26 A kind of application of Chinese medicine composition in the medicine for suppressing angiogenesis is prepared

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN201210166200.5A Division CN103417907B (en) 2012-05-26 2012-05-26 A kind of application of Chinese medicine composition in the medicine for suppressing angiogenesis is prepared

Publications (2)

Publication Number Publication Date
CN103830662A true CN103830662A (en) 2014-06-04
CN103830662B CN103830662B (en) 2017-09-26

Family

ID=50794764

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410014362.6A Active CN103830662B (en) 2012-05-26 2012-05-26 A kind of application of Chinese medicine composition in treatment medicine for treating rheumatoid arthritis is prepared

Country Status (1)

Country Link
CN (1) CN103830662B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107375821A (en) * 2016-05-17 2017-11-24 石家庄以岭药业股份有限公司 A kind of Chinese medicine composition is preparing the application in protecting vascular endothelial cell medicine
CN114470137A (en) * 2022-03-01 2022-05-13 湖南省中医药研究院 Traditional Chinese medicine composition for treating lung cancer and preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101590200A (en) * 2008-05-29 2009-12-02 河北以岭医药研究院有限公司 The application of a kind of Chinese medicine composition in the medicine of preparation treatment tumor
CN102210836A (en) * 2010-04-08 2011-10-12 河北以岭医药研究院有限公司 Application of Chinese medicinal composition in preparation of medicine for treating stomach cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101590200A (en) * 2008-05-29 2009-12-02 河北以岭医药研究院有限公司 The application of a kind of Chinese medicine composition in the medicine of preparation treatment tumor
CN102210836A (en) * 2010-04-08 2011-10-12 河北以岭医药研究院有限公司 Application of Chinese medicinal composition in preparation of medicine for treating stomach cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
周清华等: "《肺癌新理论与新技术进展》", 31 December 2003 *
樊代明: "《肿瘤研究前沿（第11卷）》", 31 December 2011 *
樊代明: "《肿瘤研究前沿（第9卷）》", 31 December 2009 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107375821A (en) * 2016-05-17 2017-11-24 石家庄以岭药业股份有限公司 A kind of Chinese medicine composition is preparing the application in protecting vascular endothelial cell medicine
CN114470137A (en) * 2022-03-01 2022-05-13 湖南省中医药研究院 Traditional Chinese medicine composition for treating lung cancer and preparation method and application thereof

Also Published As

Publication number Publication date
CN103830662B (en) 2017-09-26

Similar Documents

Publication Publication Date Title
CN100382798C (en) Pharmaceutical composition containing caffeoylquinic acids
CN1931269B (en) Rutin containing medicine composition
CN103845418B (en) A kind of Chinese medicine composition for the treatment of retinal hemorrhage and uses thereof
CN103405582B (en) Traditional Chinese medicine composition for improving joint gall of rheumatoid arthritis
CN103417907B (en) A kind of application of Chinese medicine composition in the medicine for suppressing angiogenesis is prepared
CN103386078A (en) Application of traditional Chinese medicinal composition in preparation of medicines for treating colon cancer
CN106176773A (en) Berberine hydrochloride and stachyose composition compositions and application
WO2014176934A1 (en) Use of traditional chinese medicine composition in preparation of drugs for treating or preventing angiogenesis related diseases
CN105287812A (en) Medicine composition for treating irritable bowel syndromes and application of medicine composition
CN103830662A (en) Application of traditional Chinese medicine composition in preparation of drug for inhibiting rheumatoid arthritis angiogenesis
CN105412410A (en) Traditional Chinese medicine preparation for preventing and treating radiographic contrast nephropathy
CN101254186A (en) Medicament use of myricetin
CN103735712A (en) Preparation method of Chinese medicinal composition and Chinese medicinal composition prepared by using preparation method
CN105267867A (en) Application of traditional Chinese medicine to preparation of medicine for eliminating heart toxic and side effects of antitumor medicine or preparation of medicine with synergic antitumor effect with adriamycin
CN105982887A (en) Application of arctigenin in preparing medicine for treating blood hyperviscosity
CN110292607B (en) Traditional Chinese medicine composition for treating hypertension complicated with left ventricular hypertrophy and preparation method thereof
CN104013928B (en) A kind of medicine for treating fibroid, endometriosis and preparation method thereof
CN104095843B (en) Arctigenin is preparing the application in treating digestive tract ulcer disease medicament
KR20220138397A (en) Chinese medicine composition and its method and application
CN106420697A (en) Polymethoxylated flavone, composition and application of polymethoxylated flavone preparation in preventing or treating diabetes
CN108653401A (en) A kind of compound medicinal formulation and preparation method thereof for treating ulcerative colitis
CN105663152A (en) Application of triacetyl-3-hydroxyphenyl adenosine in preparing medicines for improving insulin resistance and related diseases
CN103520270A (en) Application of astragalus-salviae miltiorrhizae qi-benefiting dropping pills in resistance to anti-cancer medicine cardiotoxicity
WO2023025228A1 (en) Traditional chinese medicine composition, and preparation method therefor and use thereof
CN103917544A (en) Phased dosing of clopidogrel

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant