CN102526238A

CN102526238A - Chinese medicinal composition for preventing and treating rheumatoid arthritis

Info

Publication number: CN102526238A
Application number: CN2012100290734A
Authority: CN
Inventors: 林娜; 孔祥英; 刘春芳
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2012-02-09
Filing date: 2012-02-09
Publication date: 2012-07-04

Abstract

The invention discloses a Chinese medicinal composition for preventing and treating rheumatoid arthritis. The Chinese medicinal composition is prepared from the following Chinese medicines in part by weight: 5 to 20 parts of blast-fried prepared common monkshood daughter root, 5 to 30 parts of cassia twig, 5 to 30 parts of largehead atractylodes rhizome and 5 to 20 parts of honey-fired tamariskoid spikemoss herb. The invention also provides a preparation containing the Chinese medicinal composition and a preparation method for the preparation. Experiments prove that the Chinese medicinal composition has a definite curative effect of treating rheumatoid arthritis.

Description

A kind of Chinese medicine composition of preventing and treating rheumatoid arthritis

Technical field

The present invention relates to medical invention field, be specifically related to a kind of Chinese medicine composition of preventing and treating rheumatoid arthritis and preparation method thereof.

Background technology

Rheumatoid arthritis (rheumatoid arthritis; RA) be a kind of cause of disease not bright be the autoimmune systemic disease of main chronic, general with the destruction of joint, more with person between twenty and fifty, send out the age well and be 20-45 year; Women's sickness rate is higher than the male, about 1: 3 of men and women's ratio.Its main pathology link comprises the synovial membrane inflammation hypertrophy, and pannus forms, and several aspects such as destruction of cartilage and osseous tissue.Clinical research finds that the outgrowth degree of RA intraarticular new vessels becomes positive correlation with the degree of patient's the state of an illness, synovial hyperplasia and cell infiltration, and the medicine that suppresses angiogenesis can be alleviated the state of an illness of RA.

The medicine of at present clinical treatment rheumatoid arthritis commonly used is mainly methotrexate, sulfasalazine, leflunomide and TNF alpha-2 antagonists.But existing medicine a lot of patients in clinical practice can not reach curative effect preferably, perhaps because side effect can't be adhered to long-term prescription.In addition, in developing country, some effective biological products use limited, and factor such as the part price is higher, have limited their extensive use.Therefore from Chinese medicine, develop the clear and definite anti-RA medicine of curative effect, will have great importance.

Summary of the invention

The purpose of this invention is to provide a kind of Chinese medicine composition of preventing and treating rheumatoid arthritis.

Another object of the present invention is the preparation that contains above-mentioned Chinese medicine composition.

Another object of the present invention is the method for the above-mentioned preparation of preparation.

The present invention has further comprised the application at field of medicaments of this Chinese medicine composition or its preparation.

A kind of Chinese medicine composition of preventing and treating rheumatoid arthritis provided by the invention, process by following parts by weight of Chinese traditional medicine: gun additioner 5-20 part, Ramulus Cinnamomi 5-30 part, Rhizoma Atractylodis Macrocephalae 5-30 part is processed Herba Selaginellae 5-20 part.

Preferably, this Chinese medicine composition is processed by following parts by weight of Chinese traditional medicine: gun additioner 5-15 part, and Ramulus Cinnamomi 10-20 part, Rhizoma Atractylodis Macrocephalae 6-20 part is processed Herba Selaginellae 5-15 part.

Further preferred, this Chinese medicine composition is processed by following parts by weight of Chinese traditional medicine: 10 parts of gun additioners, and 12 parts of Ramulus Cinnamomi, 15 parts of the Rhizoma Atractylodis Macrocephalaes are processed 9 parts of Herba Selaginellaes.

The present invention also provides the preparation that contains above-mentioned Chinese medicine composition, and said preparation is made up of Chinese medicine composition and pharmaceutically acceptable carrier or diluent.

Said pharmaceutically acceptable carrier or diluent are meant the pharmaceutical carrier that pharmaceutical field is conventional, are selected from filler, binding agent, disintegrating agent, lubricant, surfactant or the correctives one or more.

Wherein, said filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline Cellulose or glucose etc.;

Said binding agent is selected from cellulose derivative, alginate, gelatin or polyvinylpyrrolidone etc.;

Said disintegrating agent is selected from microcrystalline Cellulose, carboxymethyl starch sodium, polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose or cross-linking sodium carboxymethyl cellulose;

Said lubricant is selected from stearic acid, Polyethylene Glycol, calcium carbonate, sodium bicarbonate, micropowder silica gel, Pulvis Talci or magnesium stearate;

Said surfactant is selected from dodecylbenzene sodium sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, the fatty acid Pyrusussuriensis is smooth or Polysorbate (tween) etc.;

Said correctives is selected from aspartame, steviosin, Sucralose or saccharin sodium.

Chinese medicine composition provided by the invention can administered through oral, snuffing is gone into, the mode of rectum or parenteral is applied to the patient who needs this treatment.When wherein oral, can be made into conventional solid preparation such as tablet, granule, pill, drop pill, capsule or powder; Process liquid preparation such as water or oil-suspending agent or other liquid preparations such as syrup, oral liquid etc.; When being used for parenteral, can be made into solution, water or the oily suspensions etc. of injection.

Chinese medicine composition provided by the invention is preferably sheet, capsule, granule.

The present invention also provides a kind of method for preparing above-mentioned preparation, may further comprise the steps:

1) respectively with gun additioner, Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae, process Herba Selaginellae, pulverize, sieve, subsequent use;

2) take by weighing recipe quantity gun additioner, Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae, process Herba Selaginellae and pharmaceutically acceptable carrier according to the equivalent method mix homogeneously that progressively increases, process preparation according to the common process of preparation.

The present invention also provides other a kind of method for preparing above-mentioned preparation, may further comprise the steps:

Gun additioner was decocted first 30 minutes, added Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae then and processed Herba Selaginellae, and water decocts 2-3 time, adds the water that 5-10 doubly measures at every turn; (mixed medicine) each decocting time is 1-3 hour, filters merging filtrate; Being concentrated into relative density is 1.03-1.10, and drying is ground into fine powder; Sieve, with pharmaceutically acceptable carrier according to the equivalent method mix homogeneously that progressively increases, process preparation according to the common process of preparation.

With gun additioner, Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae with process Herba Selaginellae with alcohol reflux 2-3 time, add the ethanol of the 10-50% that 5-10 doubly measures at every turn, each extraction time is 1-3 hour; Filter, merging filtrate, it is 1.03-1.40 that filtrating is concentrated into relative density; Drying is ground into fine powder, sieves; With pharmaceutically acceptable carrier according to the equivalent method mix homogeneously that progressively increases, process preparation according to the common process of preparation.

The present invention also provides above-mentioned Chinese medicine composition or the application of its preparation in the medicine of preparation treatment rheumatoid arthritis.

Chinese medicine composition provided by the invention has the following advantages:

1, modern study shows that RA angiogenesis and traditional Chinese medical science appoplexy involving the collateral disorders of meridian become closely related.The diseased region of RA mainly is fascia, joint, and the essence of its " network is sick " is the synovial membrane blood vessel hyperplasia.The scorching academician of Wang Yong thinks that arthromyodynia (RA) is the representative disease of " new disease is gone into network ", and " poison decreases podomere channels, the high change in network road, channels is empty stagnates " be its main pathogenesis, and that advocates " divide the card opinion controls, cuts that poison is preapred for an unfavorable turn of events, unobstructed channels " discusses method of treatment then from " network ".Thus, " collateral dredging method " is the important rule of treatment RA.

The present invention is under the traditional Chinese medical science " network is sick " theoretical direction, discusses the representative prescription of controlling, determined curative effect to the RA syndrome of cold-dampness blocking collaterals.

2, Chinese medicine composition provided by the invention derives from Ramuli Cinnamomi and Aconiti Praeparatae Decoction, Atracty-lodis Macrocephalae and Aconiti Praeparatae Decoction sanction in " Medical Treasures of the Golden Chamber ", serves as main the composition with hot medicine (Radix Aconiti Lateralis Preparata, Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae, Herba Selaginellae), and the merit of " dehumidifying of dispeling cold, collateral dredging are opened numbness " to the RA syndrome of cold-dampness blocking collaterals, is arranged.Implemented the main pathogenesis that is directed against RA " channels is empty to stagnate for malicious damage podomere channels, the high change in network road ", and emphasical branch card opinion is controlled (dispeling cold), emphasical early treatment (section poison is preapred for an unfavorable turn of events), emphasical " reason, method " of correcting pathology (unobstructed channels) on treating.

Through experiment confirm, Chinese medicine composition provided by the invention is used to treat the rheumatoid arthritis determined curative effect.

3, the present invention starts with from " network is sick " and the relation of blood vessel hyperplasia; Adopt collagen-induced property arthritis mouse model similar and external RA synovioblast system, venous endothelial cell system with human RA characteristics of lesion; Observe the influence of the present invention, and inquired into the effect and the mechanism of its anti-RA synovial membrane angiogenesis RA synovial membrane blood vessel hyperplasia and regulatory pathway (VEGF/VEGF receptor pathway and Ang/Tie-2 truck) thereof.

Description of drawings

Fig. 1: to the influence (100 *) of mice matrigel embolus medium vessels formation;

Fig. 2: to the influence (100 *) of HUVEC chemotactic migration;

Fig. 3: the HUVEC tube chamber is formed the influence (100 *) of ability;

Fig. 4: to the influence (100 *) of RA-HFLS chemotactic migration;

Among the figure: WL 2,4,8,16,32 is the medicine of variable concentrations.

The specific embodiment

Following examples are used to explain the present invention, but should not be used for limiting scope of the present invention.

Embodiment 1: the granule of preventing and treating rheumatoid arthritis

1, raw material of Chinese medicine: gun additioner 10kg, Ramulus Cinnamomi 12kg, Rhizoma Atractylodis Macrocephalae 15kg, process Herba Selaginellae 9kg.

2, method for preparing:

1) with Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae, process the water logging bubble 30min of Herba Selaginellae with 8 times of its weight, subsequent use;

2) gun additioner is placed in the rustless steel decocting medicine pot, add the water of 8 times of its weight, soak 30min earlier, slow fire boiling 30min adds medicine and soak that step 1) prepares then, decocts 1.5h again, filters with four layers of filtered through gauze, collects filtrating;

The decocting that adds for the second time 6 times of medicinal residues boils 1h, with four layers of filtered through gauze, merges the decocting liquid of twice decoction, and it is 1.10 (30 ℃) that filtrating is concentrated into relative density, and is dry then, pulverizes, and promptly gets Chinese medicine extract.

Is 10: 10: 1 according to Chinese medicine extract and lactose, steviosin and essence according to weight ratio: 0.1 proportioning, according to the equivalent method mix homogeneously that progressively increases, packing promptly gets granule.

Embodiment 2: the capsule of preventing and treating rheumatoid arthritis

1, raw material of Chinese medicine: gun additioner 15kg, Ramulus Cinnamomi 20kg, Rhizoma Atractylodis Macrocephalae 10kg, process Herba Selaginellae 6kg.

2, method for preparing: with embodiment 1.

Embodiment 3: the injection of preventing and treating rheumatoid arthritis

2, method for preparing: 1) take by weighing medical material according to proportioning, add 8 times of amount 30% ethanol then, reflux, extract, 2 hours filters, and collects filtrating;

2) the step 1) residual medicine dreg was measured 30% alcohol reflux 1.5 hours with 5 times, filtered, and collected filtrating;

3) combining step 1), 2) filtrating, decompression recycling ethanol, it is that 1.03 concentrated solution is dry that water-bath (60 ℃) is concentrated into relative density, is ground into fine powder, promptly gets Chinese medicine extract.

4) use water for injection that the Chinese medicine extract dilution is 320mg/ml, 0.22um filters, and sterilization promptly gets injection.

Embodiment 4: the capsule of preventing and treating rheumatoid arthritis

1, raw material of Chinese medicine: gun additioner 5kg, Ramulus Cinnamomi 15kg, Rhizoma Atractylodis Macrocephalae 6kg, process Herba Selaginellae 15kg.

2, method for preparing:

1) take by weighing medical material according to proportioning, add 6 times of amount 50% ethanol then, reflux, extract, 2 hours filters, and collects filtrating;

2) the step 1) residual medicine dreg was measured 50% alcohol reflux 1.5 hours with 5 times, filtered, and collected filtrating;

3) combining step 1), 2) filtrating, decompression recycling ethanol, water-bath (60 ℃) is concentrated into the concentrated solution that relative density is 1.34 (60 ℃), drying is ground into fine powder, promptly gets Chinese medicine extract.

4) be 10: 1: 1 according to Chinese medicine extract and magnesium stearate, starch weight ratio, with Chinese medicine extract and magnesium stearate mix homogeneously, packing promptly gets capsule.

Embodiment 5: the capsule of preventing and treating rheumatoid arthritis

1, raw material of Chinese medicine: gun additioner 20kg, Ramulus Cinnamomi 30kg, Rhizoma Atractylodis Macrocephalae 30kg, process Herba Selaginellae 5kg.

2, method for preparing: take by weighing gun additioner, Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae respectively, process Herba Selaginellae, pulverize, cross 40 mesh sieves, subsequent use; Take by weighing the starch of 10kg then, the magnesium stearate of 3kg, with the Chinese medicine of pulverizing according to the equivalent method mix homogeneously that progressively increases, packing promptly gets capsule.

Experimental example 1: to the influence of CIA rat synovium of joint angiogenesis, vessel landmarks thing CD31 expression, vessel density

1, laboratory animal

Animal that this test is adopted is healthy, male DA rat (breathing out the Second Academy of medical university zoopery center), SPF level, body weight 170 ± 5g.Constant temperature, constant humidity were fed 3 days.Experimental session is freely drunk water, and raises solid feed, feeds the Experimental Animal Center in fundamental research institute of Chinese department of Chinese medicine institute.

2, experiment is divided into groups and method:

Modeling method that this test is adopted: by the 0.1mol/L glacial acetic acid solution being added cattle II Collagen Type VI (available from Chondrex company; Lot number: 080280); 4 ℃ are spent the night; (Chondrex company, lot number: 090070) fully emulsified, obtaining final concentration is the collagen emulsifying agent of 1mg/ml to add the equal-volume incomplete Freund's adjuvant again.Except that the normal control group, give rat root of the tail portion 2-3cm place intradermal injection collagen Emulsion (100 μ l/ only), injection was designated as the 0th day (D0) of immunity the same day, and the 7th day (D7) injects isodose collagen emulsifying agent, activate immunity once more.The normal control group is with equivalent normal saline control treatment.

Divide 8 groups at random with the DA rat, that is: normal group, model group; Test 1 low dose group (give embodiment the granule of 1 preparation, give 6.9g (according to crude drug amount meter, down together)) according to per kilogram of body weight; Test 1 high dose group and (give embodiment the granule of 1 preparation; Dosage is the 13.8g/kg body weight), test 2 groups of (give embodiment 2 capsule of preparation, dosage is the 10.4g/kg body weight), test 3 groups of (give embodiment the capsule of 4 preparations, dosage is the 10.4g/kg body weight), tests (give embodiment the capsule of 5 preparations for 4 groups; Dosage is the 10.4g/kg body weight); It irritates stomach according to rat administration volume according to the 10ml/kg body weight, and normal group and model group are irritated and obeyed isometric(al) distilled water every day 1 time, successive administration 35 days.

3, tissue is drawn materials

Last administration rat next day eye socket rear vein beard is got blood, 4 ℃ leave standstill 1h after, the centrifugal 15min of 3000rpm gets serum ,-20 ℃ of preservations detect in order to serology.

Use sterilized instrument, get left and right sides hind leg knee joint, ankle joint under the aseptic condition, peel off fur, put 4% paraformaldehyde solution and fix.

4, detect index

4.1 cardinal principle index

4.1.1 arthritis index: from immunity beginning in 8 days, carried out once clinical integration in every 2-3 days, every foot was made totally 16 minutes in 4 minutes.Standard is seen table 1: (ARTHRITIS&RHEUMATISM.1996; 39 (3): 515-521)

Table 1: arthritis index

4.1.2 arthritis incidence rate: take place to have at least a clinical integration of pawl >=2 minutes to be judged as arthritis.

4.2 joint iconography

Through micro-CT, estimate the joint injury situation.

4.3 joint pathology damage

Knee joint of all animals and ankle joint are fixed with 4% paraformaldehyde, 10%EDTA decalcification, conventional section, haematoxylin-Yihong (HE) dyeing.OLYMPUS observation by light microscope synovium of joint, cartilage and osteopathia reason change, and synovial membrane blood vessel hyperplasia situation.

4.3.1 inflammation joint (knee joint, ankle) pathology carries out calculus from four aspects such as inflammatory cell infiltration, pannus formation, cartilage destruction and bone erosions, standard is seen table 2.With reference to (Arthritits Rheum 1999,43:498-506)

Table 2: inflammation joint (knee joint, ankle) case calculus

Standards of grading	0 part	1 minute	2 minutes	3 minutes	4 minutes
						Inflammatory cell infiltration	No inflammatory cell	The sparse inflammatory cell that is dispersed in	The comparatively dense inflammatory cell	A large amount of inflammatory cells	--
Pannus forms	Do not have and form	A small amount of existence＜50%	Pannus area＞50%	The a large amount of existence	--
						Articular cartilage is destroyed situation	Do not have and destroy	The special mess shape destroys＜1/3	1/2 destroys	2/3 destroys	All destroy
The bone erosion degree	Do not have and corrode	A small amount of erosion＜50%	Corrode area＞50%	The a large amount of erosion	--

4.3.2 inflammation synovium of joint vascular counts: under 10 * 40 times of mirrors, choose the relatively significantly visual field of synovial membrane blood vessel hyperplasia, calculate the quantity of little blood vessel, medium vessels, trunk respectively, standard is seen table 3.With reference to (Arthritis Rheum.2003; 48 (9): 2660-9)

Table 3: inflammation synovium of joint vascular counts

5, statistical analysis

Adopt one factor analysis of variance.Experimental result is represented with mean ± standard deviation

.

6, experimental result

6.1 influence to CIA rat arthritis disease severity and sickness rate

The normal rat joint does not have red and swollen symptom, and CIA rat (being model group) redness and swelling of joints is obvious, red or swollen symptom after first immunisation the 11st day even more early begin to occur, and symptom increases the weight of gradually subsequently, 3-4 week peak, sickness rate reaches 100%.

Test 1 low dose group sx, test 1 high dose group, test time and reduction arthritis index and sickness rate that the 2-4 group all can significantly improve the appearance of CIA rat arthritis, each is organized and is not all seen obvious joint deformity.

6.2 influence to CIA rat joint iconography

The three-dimensional position of micro-CT rat ankle joint, longitudinal section figure show that the normal rat joint does not have destruction and is out of shape; CIA rat destruction of joint and distortion, joint are corroded obviously, the coarse injustice of articular surface; Test 1 high dose group, test 2-4 group articular surface is more coarse, the little destruction of ossa articularia light weight, distortion a little.

6.3 influence to CIA rat inflammation joint tissue pathological change

6.3.1 inflammation joint (knee joint, ankle) pathological change om observation

Clean in the normal rats knee joint cavity, articular cartilage surface is smooth, and cartilage matrix and chondrocyte do not have degeneration, and synovial tissue does not have hypertrophy.

The a large amount of hypertrophy of model group rat knee joint synovial tissue, pannus forms, and the cartilage degeneration necrosis strips off, and subchondral bone matter is destroyed, and intracavity has slough.

Test a small amount of hypertrophy of 1 low dose group knee joint synovial tissue, have pannus, cartilage is degeneration slightly, and subchondral bone matter is destroyed, and intracavity has the slough of few part.

Test 1 high dose group, test 2-4 group rat knee cartilage face is essentially smooth, the slight hypertrophy of synovial tissue, the slight degeneration of substrate.

6.3.2 the micro-score of inflammation joint (knee joint, ankle) pathology

Choose the knee joint and the ankle joint of model group and each treatment group, change from the RA pathomorphology and carry out integration like four aspects such as inflammatory cell infiltration, pannus formation, cartilage destruction and bone erosions.The result is following:

Inflammatory cell infiltration: with the model group ratio, test 1 low, high dose group, test 2-4 group can alleviate knee joint, ankle joint inflammatory cell infiltration degree in various degree.Pannus forms: with the model group ratio, test that 1 low, high dose group, test 2-4 group all can alleviate knee joint, the ankle joint pannus forms, (low be respectively P＜0.05) with high dose group, and become dose dependent.Cartilage destruction: with the model group ratio, test 1 low, high dose group, test 2-4 group all can alleviate knee joint, ankle joint cartilage destruction.Bone erosion: with the model group ratio, test 1 low, high dose group, test 2-4 group all can alleviate knee joint, ankle joint bone erosion.

6.4 influence to CIA rat inflammation synovium of joint angiogenesis

6.4.1 inflammation joint HE dyeing (knee joint, ankle) synovial membrane angiogenesis om observation

The normal control group: synovial cell's marshalling, no hypertrophy, vascular morphology is normal, does not see that obvious new vessels forms.

Model group: synovial cell's arrangement disorder, hypertrophy is obvious, and is the fine hair shape or mamillary stretches to the articular cavity depths.It is thus clear that tangible neovascularity hypertrophy, mostly new vessels is blood capillary, is made up of 1 to 2 layer of endotheliocyte, and no smooth muscle cell centers on.The also visible new vesselses that do not form luminal structure in a large number are point-like and are dispersed in and are distributed in the middle of the synovial membrane.Test 1 low dose group: synovial cell's arrangement disorder, hypertrophy is obvious.It is thus clear that a large amount of angiogenesiss and pannus are made up of 1-2 layer endotheliocyte, how rounded, smooth muscle layer is thinner.With model group relatively: test 1 high dose group and test 2-4 group all can suppress or slow down the synovial membrane blood vessel hyperplasia, the formation of minimizing pannus.

6.4.2 the expression and the vascular counts of inflammation synovium of joint medium vessels endothelium mark CD31 specific stain

CD31 is a platelet source endothelial cell adhesion molecule, is the vascular endothelial cell mark.Inflammation joint CD31 immunohistochemical staining experimental result shows that CD31 does not have expression basically in the normal rat synovial membrane blood vessel, the visible brown positive expression of CIA rat model synovium of joint medium vessels endotheliocyte and synovial cell.With the model group ratio, the positive expression of testing 1 low, high dose group, test 2-4 group significantly reduces.

The synovial membrane vessel density result demonstration that further obtains from the CD31 specific stain, after the success of CIA model, a large amount of hypertrophy of synovium of joint blood vessel, blood vessel hyperplasia is main with little blood vessel, tests 1 high dose group, tests the hypertrophy that the 2-4 group can suppress the little blood vessel of synovial membrane.

Experimental example 2: the effect of compositions to forming at body mice matrigel embolus medium vessels

1, laboratory animal: the C57BL/6 mice, male, available from Department Of Medicine, Peking University's animal center, 6 weeks are big or small.Animal credit number: SCXK (capital) 2006-0008 raises the Experimental Animal Center in fundamental research institute of Chinese department of Chinese medicine institute.

2, experiment is divided into groups and medication:

Experimental group is got an amount of embodiment 3, and the dilution of sterilization tri-distilled water is 32mg/ml, and 0.22um filters back 4 ℃ of preservations.Blank is the equal-volume normal saline.

19.5mg/ml matrigel (BD Company products) 500ul contains VEGF165 400ng/ml, heparin sodium 10U/ml adds the medicine for preparing, the abundant mixing of whirlpool vortex mixer.

With 75% alcohol disinfecting mice local skin, bottom right inguinal region subcutaneous injection pastille matrigel.

Behind the 12d, take off vertebra and put to death mice, cut off skin, peritoneum exposes embolus, and passivity is separated taking-up matrigel embolus, formalin fixed, FFPE, section 5um, HE dyeing.

3, experimental result: see Fig. 1, VEGF can promote the infiltration of endotheliocyte in the mice matrigel; The embodiment 3 of 32mg/ml dosage can suppress the infiltration of the inductive mice matrigel of VEGF medium vessels endotheliocyte.

Experimental example 3: to the influence of external human umbilical vein endothelial cell (HUVEC) propagation, migration, adhesion, tube chamber formation

1, cell culture

The cultivation of external HUVEC: the HUVEC routine is incubated at and contains 5%FBS, in the endotheliocyte culture medium of 1% endothelial cell growth additive (ECGS) and 1% penicillin/streptomycin solution (P/S), puts 37 ℃, 5%CO ₂, cultivate in the saturated humidity incubator.For guaranteeing that cell has kilter, experiment was all for 4 generations to 6 generations with cell.

The influence of 2, HUVEC being bred

1) getting the HUVEC use concentration in the 4th generation is after 0.25% trypsinization becomes single cell suspension, the centrifugal supernatant that goes, adjustment cell concentration to 3 * 10 ⁴/ ml is inoculated in 96 orifice plates, every hole 100 μ l.

2) behind the cultivation 24h, get the injection of embodiment 3, be added in the culture plate, make its final concentration be respectively 2mg/ml, 4mg/ml, 8mg/ml, 16mg/ml and 32mg/ml; The matched group orifice plate adds isodose solvent.

3) continue to cultivate 24h, every hole adds MTS 20 μ l.

4) behind the cultivation 4h, on ELIASA, survey absorbance (A) value, wavelength is 492nm.

5) establish 3 multiple holes, experiment repetition 3 times for every group.

3, adopt of the influence of the two chamber culture plate observation groups of Transwell compound to the HUVEC chemotactic migration

1) growth of HUVEC cell is merged to 80%, tranquillization 12h, and digestion, the serum-free medium re-suspended cell, last chamber adds 200 μ l pastille cell suspension, and making cell quantity is 1 * 106/hole.Following chamber adds the ECM that 500ul contains 5%FBS, and VEGF165 20ng/ml gets the injection of embodiment 3, is added in the culture plate, makes its final concentration be respectively 2mg/ml, 4mg/ml, 8mg/ml, 16mg/ml and 32mg/ml;

2) migration time: 4h;

3) cotton swab of moistening is wiped the cell that does not move on the film away;

4) the fixing 10min of methanol (37 ℃ of water-baths);

5) 0.1% violet staining 10min, the 650ul/ hole;

6) distilled water flushing 10min * 3 time

7) slot is flipped upside down, move to the cell film under with just putting optical microscope 200 * counting, at every turn the individual visual field at random of the experimental calculation 3-5 counting of taking pictures.

4, to the adherent influence of HUVEC

1) FN encapsulates 96 orifice plates: FN (1mg/ml) is configured to the working solution of 20mg/L, adds in 96 orifice plates, every hole 50 μ l absorb unnecessary liquid after 4 ℃ of overnight incubation, and every hole adds 1%BSA 100 μ l sealing again, in 37 ℃, 5%CO ₂Cultivate 60min in the incubator.

2) inoculum density 200 cell suspension, HUVEC 1 * 10 ⁶Individual/hole, get the injection of embodiment 3, be added in the culture plate, make its final concentration be respectively 2mg/ml, 4mg/ml, 8mg/ml, 16mg/ml and 32mg/ml; Every group 4 multiple hole;

3) hatch 1.5h, the PBS flushing is removed not attached cell 2 times

4) supernatant 100ul is abandoned in suction, adds MTS 20ul/ hole, hatches 4h for 37 ℃

5) 490nm surveys the A value, gets 4 hole meansigma methodss.

5, the HUVEC tube chamber is formed the influence of ability

1) matrigel is in the thawing of spending the night on ice of 4 ℃ of refrigerators; The rifle head that autoclaving is crossed and 4 ℃ of refrigerator pre-coolings of EP pipe.

2) matrigel (10mg/ml) encapsulates 96 orifice plates, remains on operation on ice, and the 45ul/ hole is placed 60min for 37 ℃ it is solidified.

3) digestion grows to the HUVEC that 70-80% merges, and centrifugal back endotheliocyte culture medium (5%FBS, ECGS) resuspended, inoculating cell 2 * 10 ⁴Individual/hole (cell suspension 160ul) gets the injection of embodiment 3, is added in the culture plate, makes its final concentration be respectively 2mg/ml, 4mg/ml, 8mg/ml, 16mg/ml and 32mg/ml; Matched group adds the equivalent culture medium.

4) 37 ℃, 5%CO ₂Hatch 16-18h in the incubator, phase contrast microscope observation of cell form is also taken pictures, with 5 selected at random low power field (50 *) the inferior division points in each hole of Image Pro Plus v7.0 analytical system counting;

6, to the influence of HUVEC angiogenesis associated receptor VEGFR2 and Tie2 expression

1) collection of specimens and preservation HUVEC are seeded in the T25 bottle, and when waiting to reach 80% fusion, set up following each group separately: normal group, embodiment group (give embodiment the injection of 3 preparations, make its final concentration be respectively 2mg/ml; 4mg/ml, 8mg/ml, 16mg/ml; 32mg/ml), 24h is handled in dosing, receives cell; Cracking, leach protein ,-80 ℃ are frozen.

2) the ELISA test kit detects the associated receptor expression.

Step is the same.

7, statistical procedures

Adopt SPSS 11.5 variance analysis statistical datas, P＜0.05 expression has statistical significance.

8, experimental result

8.1 the influence to HUVEC propagation: see table 4

Table 4: to the influence of HUVEC propagation

Group	Dosage (mg/mL)	The A value
			Normal group	-	1.35±0.09
WL	2	1.28±0.01
			WL	4	1.21±0.02
WL	8	1.22±0.06
			WL	16	1.18±0.05*
WL	32	1.11±0.04*

Compare * P＜0.05 with normal group.

As shown in table 4: WL group increases with concentration, and A value also is and reduces trend, and WL16, WL32 group A value significantly reduce (P＜0.05) than normal group, explains that WL16, WL32 organize all can show inhibition its propagation (P＜0.01).

8.2 the influence to the HUVEC chemotactic migration: see Fig. 2 and table 5

Table 5: to the influence of the inductive HUVEC migration of VEGF

Group	Dosage (mg/mL)	The migrating cell number
			Normal group	-	67.7±7.1
Model group	-	111.7±4.2 ^##
			WL	2	101.3±6.5
WL	4	98.0±9.5
			WL	8	84.3±9.9*
WL	16	60.7±5.7**
			WL	32	32.0±7.0**

Compare with normal group ^##P＜0.01; Compare * P＜0.05, * * P＜0.01 with model group;

Shown in Fig. 2 and table 5: model group obviously increases than normal group HUVEC transport number, explains that VEGF can significantly promote the migration (P＜0.05) of HUVEC; Increase with concentration, WL group HUVEC migration all significantly reduces (P＜0.05 or P＜0.01) than model group, explains that the WL8-32 group all can significantly suppress its migration, and presents tangible dose dependent.

8.3 to the adherent influence of HUVEC: see table 6

Table 8: to the adherent influence of HUVEC

Group	Dosage (mg/mL)	The A value
			BSA	-	0.31±0.03
Normal group	-	1.23±0.04
			Model group	-	1.32±0.03 ^#
WL	2	1.48±0.14
			WL	4	1.37±0.27
WL	8	1.74±0.18
			WL	16	1.24±0.02*
WL	32	1.20±0.05**

Compare with normal group ^#P＜0.05; Compare with model group ^*P＜0.05, ^*P＜0.01;

As shown in table 6: with the increase of concentration, WL group A value also has the trend of reduction, WL16, and 32 groups of A values than model group significantly reduce (P＜0.05 or P＜0.01), and WL16 is described, and 32 groups can significantly be reduced HUVEC and stick ability.

8.4 the HUVEC tube chamber is formed the influence of ability:

The down visible HUVEC of optical microscope moves on matrigel and creeps; At first form cell colony; Further stretch then, interconnection, converge and form the bar rope, acellular cavity appearance gap can appear between the HUVEC that converges; The bar Cable Structure can be cross-linked to each other and form the netted luminal structure of blood capillary appearance, forms situation through counting tube chamber branch point number reflection tube chamber.The result sees Fig. 3 and table 7.

Table 7: the inductive HUVEC tube chamber of VEGF is formed the influence of ability

Group	Dosage (mg/mL)	The intersecting blood vessels point
			Normal group	-	43.3±5.0
Model group	-	54.0±3.6 ^#
			WL	2	27.3±1.5**
WL	4	26.0±1.7**
			WL	8	25.9±2.0**
WL	16	19.3±2.3**
			WL	32	3.0±1.0**

Compare with normal group ^#P＜0.05; Compare with model group ^*P＜0.01;

Shown in Fig. 3 and table 7: model group is than compared with normal, tube chamber branch point showed increased (P＞0.05), and explaining that VEGF induces can significantly increase the tube chamber of HUVEC on Matrigel and form ability; Each group of WL all can significantly suppress its tube chamber and form (P＜0.01); Increase with concentration, the WL group of branches is counted out and is reduction trend, and the WL group is compared than model group and all significantly reduced (P＜0.01), explains that each group of WL all has the ability that its tube chamber forms that suppresses.

8.5 the influence to HUVEC angiogenesis associated receptor albumen VEGFR2 and Tie2 level: see table 8

Table 8:WL is to the influence of VEGFR2 and Tie 2 expression

Group	Dosage (mg/mL)	VEGFR2(ng/mg)	Tie?2(ng/mg)
				Normal group	-	2.44±0.26	4.28±0.06
WL	2	1.99±0.01	4.32±0.09
				WL	4	2.50±0.05	4.72±0.05*
WL	8	2.63±0.03**	5.68±0.06**
				WL	16	3.54±0.05*	6.83±0.08**
WL	32	3.94±0.04	6.54±0.05**

Compare with normal group ^*P＜0.05, ^*P＜0.01;

As shown in table 8: increase with dosage, the VEGFR2 level of WL group is in rising trend, but compares WL16 with normal group, and 32 groups expression significantly raises (P＜0.01), explains that WL can raise the VEGFR2 level with the concentration increase.

Increase with dosage, the Tie2 level of WL group is all in rising trend, except that the WL2 group, than all significantly risings (P＜0.05 or P＜0.01) of normal group.

Experimental example 4: to the effect of propagation, migration, adhesion and the angiogenesis correlation factor of the RA-HFLS of In vitro culture

1, cell culture

The cultivation of RA-HFLS: the RA-HFLS routine is incubated in the synovial cell's special culture media that contains 2mM (being 2mmol/L) glutamine, 100U/mL penicillin, 80U/mL streptomycin, 10%FBS and 10% somatomedin, puts 37 ℃, 5%CO ₂, cultivate in the saturated humidity incubator.For guaranteeing that cell has kilter, experiment was all for 4 generations to 8 generations with cell.

The influence of 2, the beta induced RA-HFLS of IL1-being bred

1) after the RA-HFLS that got for the 6th generation becomes single cell suspension with 0.25% trypsinization, the centrifugal supernatant that goes, adjustment cell concentration to 3 * 10 ⁴/ ml is inoculated in 96 orifice plates, every hole 100 μ l.

2) behind the cultivation 24h, IL1-β (10ng/mL) induces, and gets the injection of embodiment 3, adds the water for injection dilution and also is added in the culture plate, makes its final concentration be respectively 2mg/ml, 4mg/ml, 8mg/ml, 16mg/ml, 32mg/ml; And will remain orifice plate and add isodose solvent as matched group.

3) continue to cultivate 48h, every hole adds MTS 20 μ l.

5) establish 3 multiple holes, experiment repetition 3 times for every group.

3, adopt of the influence of the two chamber culture plate observation groups of Transwell compound to the RA-HFLS chemotactic migration

1) RA-HFLS cell growth is merged to 80%, tranquillization 24h, digestion, serum-free medium re-suspended cell, last chamber adding 200ul contain medicament extract (final concentration is 2mg/ml, 4mg/ml, 8mg/ml, 16mg/ml, 32mg/ml), cell suspension, 5 * 10 ⁴Individual/hole; Following chamber adds the normal cultured base that 500ul contains 10%FBS, rhVEGF165 50ng/ml.

2) migration time 7h

3) cotton swab of moistening is wiped the cell that does not move on the film away

4) the fixing 10min of methanol (37 ℃ of water-baths)

5) 0.1% violet staining 10min, the 650ul/ hole

6) distilled water flushing 10min * 3 time

4, to the adherent influence of RA-HFLS

2) inoculation 200ul RA-HFLS cell suspension, 3 * 10 ⁴Individual/hole, get the injection of embodiment 3, add the water for injection dilution and also be added in the culture plate, make its final concentration be respectively 2mg/ml, 4mg/ml, 8mg/ml, 16mg/ml, 32mg/ml; Every group 4 multiple hole

3) hatch 1.5h, the PBS flushing is removed not attached cell 2 times

5) 490nm surveys the A value, gets 4 hole meansigma methodss.

5, to the excretory angiogenesis relevant cell factor of RA-HFLS (TNF α, IL-17, VEGF, Ang1, Ang2) influence

1) collection of specimens and preservation RA-HFLS are seeded to 12 orifice plates, when waiting to reach 80% fusion, with the DMEM culture fluid tranquillization 24h that contains 0.1%FBS; Setting up following each group separately: blank control group normal group, the beta induced group model group of IL-1, IL-1 be beta induced+and the contrivance thing (gets the injection of embodiment 3; Be added in the culture plate, make its final concentration be respectively 2mg/ml, 4mg/ml, 8mg/ml, 16mg/ml, 32mg/ml) group, hatch 24h altogether with RA-HFLS; Receive supernatant ,-80 ℃ frozen.

2) the ELISA method detects the correlation factor expression.

Each reagent before use balance to room temperature.

Application of sample: establish blank well, gauge orifice, testing sample hole respectively.Except that blank well, Yu Kongfen adds standard solution or testing sample 100 μ l (medicine of experimental example 2), mixing gently, and 37 ℃, 60min discards liquid, dries.

Every hole adds detects solution 1100 μ l, 37 ℃, 60min.Wash plate 3 times, the every hole of 350 μ l/ dries.

Every hole adds detects solution 2100 μ l, and 37 ℃, 60min washes plate 5 times, dries.

Every in regular turn hole adds substrate solution 100 μ l, 37 ℃ of lucifuge colour developing 30min.

Every in regular turn hole adds stop bath 50 μ l, cessation reaction.Join appearance is measured each hole in regular turn under the 450nm wavelength optical density (OD value) with enzyme.

6, statistical procedures

7, experimental result

7.1 the influence to the beta induced RA-HFLS propagation of IL1-: see table 9

Table 9: to the influence of the beta induced RA-HFLS propagation of IL1-

Group	Dosage (mg/mL)	The A value
			Normal group	-	0.93±0.05
Model group	-	1.25±0.10 ^##
			WL	2	1.31±0.22
WL	4	1.29±0.11
			WL	8	1.22±0.06**
WL	16	0.81±0.04**
			WL	32	0.35±0.04**

Compare with normal group ^##P＜0.01; Compare with model group ^*P＜0.05, ^*P＜0.01;

As shown in table 9: model group A value obviously increases (P＜0.01) than normal group, explains that IL-1 β can obviously improve the proliferation activity of cell; Along with the increase of drug level, the A value reduces, and compares with model group, and WL respectively organizes the A value and is reduction trend, WL16, and 32 groups significantly reduce (P＜0.01) than model group A value, explain that WL16, WL32 all can significantly suppress its propagation.

7.2 the influence to the RA-HFLS chemotactic migration: see Fig. 4 and table 10

Table 10: to the influence of RA-HFLS chemotactic migration

Group	Dosage (mg/mL)	The migrating cell number
			Normal group	-	32.1±3.5
Model group	-	58.7±7.4 ^##
			WL	2	47.7±6.5
WL	4	44.3±5.1
			WL	8	41.8±3.0*
WL	16	30.3±2.5**
			WL	32	21.3±2.5**

Shown in Fig. 4 and table 10: model group RA-FLS transport number explains that than normal group showed increased (P＜0.01) VEGF can significantly promote its migration; The WL group increases with concentration, and the RA-FLS transport number also is reduction trend, and the WL8-32 group obviously reduces (P＜0.05 or P＜0.01) than model group migrating cell number average, explains that the WL8-32 group all can significantly suppress its migration, and significant dose dependent is arranged.

7.3 to the adherent influence of RA-HFLS:

Adopt one of extracellular matrix main component FN to encapsulate 96 well culture plates, investigate the influence that each medicine pair cell and extracellular matrix stick effect, the MTS method is surveyed A value reflection adherent cell number indirectly.

Table 11: to the adherent influence of RA-HFLS

Group	Dosage (mg/mL)	Adherence rate
			BSA	-	0.27±0.02
Normal group	-	1.60±0.13
			Model group	-	1.83±0.04 ^#
WL	2	1.81±0.06
			WL	4	1.78±0.09
WL	8	1.74±0.06
			WL	16	1.70±0.08*
WL	32	1.67±0.07**

Compare with normal group ^#P＜0.05, ^##P＜0.01; Compare with model group ^*P＜0.05, ^*P＜0.01

As shown in table 11, BSA negative control group and compared with normal A value significantly reduce, and explain that RA-FLS has the stronger ability of sticking (P＜0.01) to FN; Model group A value enlarges markedly than normal group, and beta induced stick (P＜0.05) that can significantly increase RA-FLS to FN of IL-1 is described; With the increase of concentration, WL group A value has the trend of reduction, WL16, and 32 groups significantly reduce (P＜0.05 or P＜0.01) than model group A value.

7.4 to the newborn correlation factor TNF of RA-HFLS supernatant medium vessels α, IL-17, VEGF, Ang1, Ang2) influence

7.4.1 the influence to TNF α, IL-17, VEGF level in the supernatant: see table 12

Table 12: to the influence of TNF α, VEGF and IL-17 content in the RA-HFLS supernatant

Group	Dosage (mg/mL)	TNFα(pg/mg)	VEGF(pg/mg)	IL-17(pg/mg)
					Normal group	-	28.7±4.4	50.9±2.6	50.4±2.4
Model group	-	68.8±5.1 ^##	85.4±12.9 ^##	85.3±9.1 ^##
					WL	2	67.8±6.8	79.5±9.8	79.3±6.9
WL	4	67.4±4.0	77.2±11.1	77.2±7.8
					WL	8	59.5±5.4	66.4±21.0	66.4±14.9
WL	16	48.3±5.9*	69.3±6.5	65.9±3.0*
					WL	32	40.2±1.7**	52.2±8.3*	52.2±5.9**

Compare with normal group ^#P＜0.05, ^##P＜0.01; Compare with model group ^*P＜0.05, ^*P＜0.01;

As shown in table 12: model group TNF alpha levels significantly raises (P＜0.01), explains that IL-1 β can significantly promote RA-FLS TNF secretion α; It is also on a declining curve that WL group increases the TNF alpha levels with dosage, and WL16,32 groups of TNF alpha levels significantly reduce (P＜0.05 or P＜0.01), explains that WL16,32 groups have significant inhibitory effect to RA-FLS TNF secretion α, and be significant concentration dependent;

Model group IL-17 level significantly raises (P＜0.01), and prompting IL-1 β can significantly raise the level of RA-FLS supernatant secretion IL-17; It is also on a declining curve that the WL group increases the IL-17 level with concentration, WL16, and 32 groups of IL-17 levels significantly reduce (P＜0.05 or P＜0.01), and prompting WL16,32 prescriptions have the effect that suppresses RA-FLS secretion IL-17;

Model group VEGF level significantly raises (P＜0.01) than normal group, and the beta induced VEGF level that can significantly increase in the RA-FLS supernatant of IL-1 is described; The WL group increases with concentration, and the VEGF level also is the trend of reduction, and WL32 group VEGF level significantly reduces (P＜0.01), and prompting WL32 group can significantly suppress the RA-FLS secretion of VEGF.

7.4.2 the influence to Ang1, Ang2 level in the supernatant: see table 13

Table 13: to the influence of Ang in the RA-HFLS supernatant 1 and Ang2

Compare #P＜0.05 with normal group; Compare * P＜0.05 with model group

As shown in table 13: the level of model group Ang1 significantly raises (P＜0.01) than normal group, explains that IL-1 β can promote RA-FLS secretion Ang1; The WL group increases with concentration, and the Ang1 level also is reduction trend, and WL8-32 group Ang1 level significantly reduces (P＜0.05 or P＜0.01); Prompting WL8～32 prescriptions can significantly suppress RA-FLS secretion Ang1.

Model group Ang2 level significantly increases (P＜0.05) than normal group, and prompting IL-1 β can promote RA-FLS secretion Ang2; The WL4-16 group increases with concentration, and the Ang2 level is on a declining curve, and comparing with model group does not all have marked difference, and WL32 group Ang2 level significantly reduces (P＜0.05); Prompting WL32 group can significantly suppress the expression of Ang2 in the RA-FLS supernatant.

Equally, all the other embodiment of the present invention and proportioning are carried out corresponding experiment, the result is similar with embodiment 3.

Though, used general explanation, the specific embodiment and test in the preceding text, the present invention has been done detailed description, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.

Claims

1. a Chinese medicine composition of preventing and treating rheumatoid arthritis is characterized in that, this Chinese medicine composition is processed by following parts by weight of Chinese traditional medicine: gun additioner 5-20 part, Ramulus Cinnamomi 5-30 part, Rhizoma Atractylodis Macrocephalae 5-30 part and process Herba Selaginellae 5-20 part.

2. Chinese medicine composition according to claim 1 is characterized in that, this Chinese medicine composition is processed by following parts by weight of Chinese traditional medicine: gun additioner 5-15 part, Ramulus Cinnamomi 10-20 part, Rhizoma Atractylodis Macrocephalae 6-20 part and process Herba Selaginellae 5-15 part.

3. Chinese medicine composition according to claim 1 and 2 is characterized in that, this Chinese medicine composition is processed by following parts by weight of Chinese traditional medicine: 10 parts of gun additioners, 12 parts of Ramulus Cinnamomi, 15 parts of the Rhizoma Atractylodis Macrocephalaes and process 9 parts of Herba Selaginellaes.

4. contain the preparation that right requires each described Chinese medicine composition of 1-3, it is characterized in that, said preparation is made up of Chinese medicine composition and pharmaceutically acceptable carrier or diluent.

5. preparation according to claim 4 is characterized in that, said preparation is tablet, granule, pill, drop pill, capsule, powder, syrup or oral liquid.

6. method for preparing claim 4 or 5 said preparations is characterized in that this method may further comprise the steps:

7. method for preparing claim 4 or 5 said preparations, it is characterized in that this method may further comprise the steps: gun additioner was decocted first 30 minutes; Add Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae then and process Herba Selaginellae, water decocts 2-3 time, adds the water that 5-10 doubly measures at every turn; The each decocting time of mixed Chinese medicine is 1-3 hour, filters merging filtrate; Being concentrated into relative density is 1.03-1.10, and drying is ground into fine powder; Sieve, with pharmaceutically acceptable carrier according to the equivalent method mix homogeneously that progressively increases, process preparation according to the common process of preparation.

8. method for preparing claim 4 or 5 said preparations is characterized in that this method may further comprise the steps: with gun additioner, Ramulus Cinnamomi, the Rhizoma Atractylodis Macrocephalae with process Herba Selaginellae with alcohol reflux 2-3 time; The ethanol that at every turn adds the 10-50% that 5-10 doubly measures, each extraction time is 1-3 hour, filters; Merging filtrate, it is 1.03-1.40 that filtrating is concentrated into relative density, drying; Be ground into fine powder; Sieve, with pharmaceutically acceptable carrier according to the equivalent method mix homogeneously that progressively increases, process preparation according to the common process of preparation.

9. each described Chinese medicine composition of claim 1-3 or claim 4 or the 5 described preparations application in the medicine of preparation treatment rheumatoid arthritis.