CN1292786C - Method for preparing Chinese medicine composition for treating ischemic brain apoplexy - Google Patents

Abstract

The present invention discloses a method for preparing Chinese medicine compositions for treating ischemic brain apoplexy, which has the following steps: (1) supercritical CO2 water extracted angelica is taken; (2) angelica dregs, astragalus root, scrophularia root, honeysuckle flowers, dendrobium are licorice root are mixed and extracted by ethanol to obtain alcohol extract; (3) the alcohol extract is decompressed to recover alcohol, concentrated, centrifuged, washed by water and precipitated to obtain supernatant; the supernatant is adsorbed through macroporous resin and arranged on a column or a circulation sample, and is eluted by alcohol to obtain elutent; (4) the eluent is decompressed to recover alcohol, concentrated, dried and pulverized into dry extract powder; (5) the components of the step (1) and the step (4) are combined to obtain a Chinese medicine composition. The present invention has the advantages that the method overcomes the defect that alcohol is extracted by originally technical water and is difficult to reserve the volatile ingredients of angelica; main active ingredients are enriched through the separation of macroporous resin. The present invention also has the advantages of high therapeutic effect, low dosage consumption, good solubility of intermediate extract, low hygroscopicity and convenient preparation.

Description

A kind of preparation method for the treatment of the Chinese medicine composition of ischemia apoplexy

One, technical field

The present invention relates to the preparation method of Chinese medicine composition, specifically relate to a kind of preparation method for the treatment of the Chinese medicine composition of ischemia apoplexy.

Two, background technology

Apoplexy is a kind of commonly encountered diseases and frequently-occurring disease, and apoplexy generally is divided into hemorrhagic cerebral apoplexy and ischemia apoplexy again, and the application relates to the latter's treatment Chinese medicine composition.

Utilize the traditional Chinese medical herbal treatment ischemia apoplexy that a lot of examples have been arranged in the prior art, the oral Chinese patent medicine that is applied to ischemia apoplexy at present has TONGMAI KELI, NAOLUOTONG JIAONANG etc.TONGSAIMAI PIAN is former to be a kind of prescriptions of Chinese medicine for the treatment of thromboangiitis obliterans, full side is made up of eight flavor Chinese medicines such as the Radix Astragali, Radix Codonopsis, Herba Dendrobii, Radix Scrophulariae, Flos Lonicerae, Radix Achyranthis Bidentatae, Radix Angelicae Sinensis, Radix Glycyrrhizae, has effects such as training fills blood, replenishing YIN and removing heat, blood circulation promoting and blood stasis dispelling, dredge the meridian passage.This prescription is that units such as Nanjing University of Traditional Chinese Medicine and the Jiangsu Province academy of traditional Chinese medicine, Jiangsu TCM Hospital were in beginning in 1962, method with the combination of Chinese and Western medicine, determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs, successively sum up through three times, based on " soup of looking around ", the clinical and obtained scientific achievement of basic research through more than two decades.Nineteen eighty-two, " TONGSAIMAI PIAN treatment thromboangiitis obliterans " is by provincial-level appraisal; November 8 in the same year, the approval of Nikkei Jiangsu Province Department of Public Health was formally produced by Jiangsu Nanxing Pharmaceutical Co., Ltd; Be listed in national Chinese medicine two classes protection kind in April, 1994.Principle according to traditional Chinese medical science treating different diseases with the same therapeutic principle, TONGSAIMAI PIAN has further been widened clinical application range again in process of clinical application, can be used for having deficient qi and blood, deficiency of YIN-blood, the multiple disease of blood stasis disease is as diseases such as the insomnia of coronary heart disease, angina pectoris, cerebral arteriosclerosis, forgetful, dementias; Also can be used for the rehabilitation of chronic hepatitis, liver cirrhosis, fatty liver, the auxiliary treatment of diseases such as hypertension, hyperlipidemia; Also can be used for particularly cerebral hemorrhage hemiplegia etc. of apoplexy and apoplexy sequela.

TONGSAIMAI PIAN is the good Chinese patent medicine of treatment thrombotic disease, clinical verification is 97.8% to the thromboangiitis obliterans total effective rate, and obvious effective rate is 95.6%, and to cerebral thrombosis and sequela thereof, Arteriosclerosis obliterans, venous thrombosis and diabetic gangrene all have satisfied curative effect.Secular clinical practice shows that TONGSAIMAI PIAN is the good Chinese patent medicine of treatment cardiovascular and cerebrovascular disease, and oral safety is convenient.But main active site group is indeterminate for this tablet, taking dose (a time 4～5,3 times on the one) bigger than normal, and cause the less stable of tablet greatly owing to full extractum sheet hygroscopicity.Therefore, in order further to illustrate the main active site group of TONGSAIMAI PIAN treatment ischemia apoplexy, develop that effective part group is comparatively clear and definite, taking dose is less relatively, preparation stability new product of Chinese medicine preferably, we have carried out following research.

Three, summary of the invention

1. goal of the invention

The object of the present invention is to provide a kind of preparation method for the treatment of the Chinese medicine composition of ischemia apoplexy.

2. technical scheme

The inventor is in order further to explore the mechanism of action of TONGSAIMAI PIAN prescribed treatment ischemia apoplexy, on the basis of the composition of analyzing former prescription and compatibility relationship thereof, and the pharmacological research of bound drug, proposed the closely-related Radix Astragali of the ischemia apoplexy mechanism of action, Radix Angelicae Sinensis, Radix Scrophulariae, Flos Lonicerae is the basic composition medicine, to remaining Radix Codonopsis, Radix Achyranthis Bidentatae, Herba Dendrobii, the Radix Glycyrrhizae four Chinese medicine carries out the plus-minus test of the quadrature side of tearing open, and verify with the relevant pharmacodynamics test of the treatment ischemia apoplexy mechanism of action, estimate the reasonability of prescription, and the raw material of effective prescription of acquisition treatment ischemia apoplexy is the Radix Astragali, Radix Angelicae Sinensis, Radix Scrophulariae, Flos Lonicerae, Herba Dendrobii and glycyrrhizic liuwei drug.

A kind of preparation method for the treatment of the Chinese medicine composition of ischemia apoplexy is a raw material with the Radix Astragali 10～20g, Radix Angelicae Sinensis 10～18g, Radix Scrophulariae 8～10g, Flos Lonicerae 8～15g, Herba Dendrobii 8～15g, Radix Glycyrrhizae 6～10g, and its preparation process is as follows.

(1) get Radix Angelicae Sinensis 10～18g and carry out carbon dioxide supercritical fluid extraction, collect extract, standby;

(2) medicinal residues behind the Radix Angelicae Sinensis carbon dioxide abstraction and the Radix Astragali 10～20g, Radix Scrophulariae 8～10g, Flos Lonicerae 8～15g, Herba Dendrobii 8～15g, Radix Glycyrrhizae 6～10g are merged, carry out ethanol extraction, obtain ethanol extract;

(3) with the medicinal liquid behind the above-mentioned ethanol extract recovery ethanol, concentrate, centrifugal, water washing and precipitating, washing liquid and filtrate merge, and discard precipitation, and supernatant circulates behind upper prop behind the absorption with macroporous adsorbent resin and goes up sample, uses ethanol elution, obtains ethanol elution;

(4) with the ethanol elution decompression recycling ethanol, concentrate, crushed after being dried becomes the dry extract powder;

(5) at last (1), (4) two components that step obtains are merged, promptly obtain Chinese medicine composition of the present invention.

More particularly, the method for preparing Chinese medicine composition of the present invention is, is raw material with Radix Astragali 8g, Radix Scrophulariae 8g, Flos Lonicerae 8g, Herba Dendrobii 8g, Radix Glycyrrhizae 8g, and its step is as follows:

(1) get Radix Angelicae Sinensis 8g and carry out carbon dioxide supercritical fluid extraction collection extract, standby;

(2) medicinal residues behind the carbon dioxide supercritical fluid extraction of Radix Angelicae Sinensis and Radix Astragali 8g, Radix Scrophulariae 8g, Flos Lonicerae 8g, Herba Dendrobii 8g, Radix Glycyrrhizae 8g are merged, with 70% alcohol reflux 2 times, 50% alcohol reflux 1 time, the 1st 8 times of amounts, 2nd, 36 times of amounts, each 1h, three times alcohol extract merges, decompression recycling ethanol and thin film concentration get alcohol extract to there not being the alcohol flavor;

(3) with above-mentioned alcohol extract, concentrate, centrifugal, discard precipitation, supernatant circulates behind upper prop or upper prop behind the SP-825 type absorption with macroporous adsorbent resin 12h and goes up sample 4 times, with 60% ethanol eluting successively;

(4) with above-mentioned ethanol elution, decompression recycling ethanol concentrates, drying, pulverize the dry extract powder;

(5) (1), (4) two components that step obtains are merged, promptly obtain Chinese medicine composition of the present invention.

The carbon dioxide supercritical fluid extraction thing of above-mentioned steps (1) Radix Angelicae Sinensis is a volatile ingredient; Main component is total flavones, total saponins, astragaloside, chlorogenic acid, ferulic acid etc. in the dry extract of step (4).

Chinese medicine composition of the present invention can be made oral solid formulations such as tablet, granule, micropill and capsule with conventional formulation method.

3. beneficial effect

(1) said composition is at the treatment ischemia apoplexy, choose corresponding pharmacodynamics screening model and compound recipe, simplify prescription, and the main position of further having determined compositions is that the Radix Angelicae Sinensis volatile ingredient separates mixed extracts such as containing total saponins, total flavones, total phenolic acid with full side's mixed extract through macroporous adsorbent resin, remove volatile ingredient and contained polysaccharide composition and other water-solubility impurities of prescription drug in the Flos Lonicerae, greatly reduced taking dose.

(2) adopt CO ₂-SFE obtains volatility active component in the Radix Angelicae Sinensis, because the extraction separation temperature is low, has kept unsettled active component preferably, has improved curative effect.

(3) effective part group of employing macroporous adsorbent resin separation and concentration compound medicine, method is easy, and the feasibility of suitability for industrialized production is better.

(4) full side's mixed extract separates positions, mixed extract middle part such as containing total saponins, total flavones, total phenolic acid through macroporous adsorbent resin and forms basic clear and definite and proportion of composing relative fixed.The combined extracts dissolubility is good, hygroscopicity is little, is convenient to preparation.

Four, the specific embodiment

Embodiment 1: the preparation method of the Chinese medicine composition of treatment ischemia apoplexy

Prescription: Radix Astragali 8g, Radix Angelicae Sinensis 8g, Radix Scrophulariae 8g, Flos Lonicerae 8g, Herba Dendrobii 8g, Radix Glycyrrhizae 8g

The preparation method step is as follows:

(1) get the Radix Angelicae Sinensis drying, powder 20 orders drop into extraction kettle, 2 extraction-containers and jar and heat respectively or cool off, and when the extraction kettle temperature reaches 40 ℃, the temperature of extraction-container I reaches 50 ℃, when the temperature of extraction-container II reaches 35C, opens CO ₂Gas cylinder when extraction kettle pressure reaches 20MPa, when the pressure of extraction-container II is 6MPa, begins cycling extraction, CO ₂Flow is about 18kg/h, from the extraction-container discharging, collects extract behind the extraction 2h;

(2) medicinal residues behind the carbon dioxide abstraction of Radix Angelicae Sinensis and Radix Astragali 8g, Radix Scrophulariae 8g, Flos Lonicerae 8g, Herba Dendrobii 8g, Radix Glycyrrhizae 8g merge, with 70% alcohol reflux 2 times, 50% alcohol reflux 1 time, the 1st 8 times of amounts, 2nd, 36 times of amounts, each 1h, three times alcohol extract merges, and the decompression thin film concentration is to there not being the alcohol flavor;

(3) alcohol extract that obtains with above-mentioned steps 2 removes alcohol, concentrates, and is centrifugal, discards precipitation, and supernatant circulates behind upper prop or upper prop behind the SP-825 type absorption with macroporous adsorbent resin 12h and goes up sample 4 times, and 60% ethanol is eluting successively;

(4) with above-mentioned ethanol thing eluent, decompression recycling ethanol concentrates (0.08MPa, 60 ℃), vacuum drying (0.08MPa, 70 ℃), pulverize the extractum powder;

(5) two combinations that (1), (4) step are obtained merge, and promptly obtain Chinese medicine composition of the present invention.

Embodiment 2, TONGSAIMAI compositions coagulate the therapeutical effect of cerebral infarction due to the medium-sized artery rat to electricity

1. experiment purpose: observe the TONGSAIMAI compositions is coagulated cerebral infarction due to the medium-sized artery rat to electricity therapeutical effect.

2. experiment material

2.1 animal: the SD rat, male, 250-300g is available from Nanjing Medical University's animal center.

2.2 medicine: medicine all seals, and puts shady and cool dry place and preserves, be mixed with liquid after, put 4 ℃ of preservations.

TONGSAIMAI compositions: lot number is provided by Jiangsu Southern Chinese Medicine Large Pharmaceutical Co., Ltd.: 20030926, every gram extractum contains crude drug 10.43 grams, and the day for human beings restrains with crude drug amount 51.84;

TONGSAIMAI PIAN extractum: lot number is provided by Jiangsu Southern Chinese Medicine Large Pharmaceutical Co., Ltd.: 20030120, every gram extractum contains crude drug 6.67 grams, and the day for human beings restrains with crude drug amount 42.02;

Positive drug group (nimodipine tablet): produce by Shanxi Yabao Pharmaceutical Co., Ltd., lot number 030905, the day for human beings is with crude drug amount 90-360 milligram;

2.3 medicine preparation, dosage and grouping

The TONGSAIMAI PIAN group (1.69g extractum amount/kgd), TONGSAIMAI compositions high dose group (2.66g extractum amount/kgd), dosage group in the TONGSAIMAI compositions (1.33g extractum amount/kgd), (0.44g extractum amount/kgd), positive drug group (nimodipine tablet) (32mg/kgd) the above-mentioned medicine of respectively organizing all is configured to desired concn with distilled water to TONGSAIMAI compositions low dose group.The administration volume is: 1ml/100g.

3. method and result

3.1 get 160 of body weight 250～300g SD male rats, method [1] by Tamura etc. is carried out rat brain medium-sized artery electric coagulation, each Mus is with 10% chloral hydrate anesthesia (4ml/kg, lumbar injection), with the mid point of lateral position along right external auditory canal and right eye outer canthus line, cut the about 2cm of skin perpendicular to line, successively separating muscle is removed cerebral dura mater, exposes medium-sized artery, confirm that the back electricity coagulates tractus olfactorius to one section middle cerebral artery between the inferior cerebral vein,, the layer-by-layer suture wound is injected 5000 units/ml penicillin sodium (3ml/kg that protects from infection, lumbar injection), postoperative steams again and raises.Observe postoperative 4h, 8h, 24h influence to its behavior scoring.Standards of grading: 1. carry overhead about 1 chi of Mus tail, observe the forelimb situation.Stretch to ground symmetrically as two forelimbs, count 0 fen.Wrist flexing, elbow flexing, shoulder inward turning or three occur if any left fore and all have, be chosen as 1,2,3,4 fen respectively.2. with on the sliding floor of animal horizontalization, push away a left side or the right side respectively) take on to side shifting, check the resistance that opposing promotes.Normal rat bilateral resistance symmetry.When right shoulder is mobile to the left, find the resistance descender,, be chosen as 1,2,3 fen according to the difference of decline degree (light, in, heavy); 3. animal two forelimbs are put on the wire netting, observed the muscular tension of two forelimbs.Normal rat two muscle of anterior limb tension force symmetries.The obvious descender of left fore muscular tension is taken place, and according to the weight of decline degree, is chosen as 1～3 fen.(4) animal not have the person of turn-taking be 0 minute, the turn-take meter 1 minute of behavior of an oriented side, oriented not stall of a side circle person counted 2 fens.According to above standard scoring, full marks 12 minutes, mark is high more, illustrates that the behavior disorder of animal is serious more, gets 10 fens above persons and further tests.Rat is divided into 7 groups at random, is respectively sham operated rats, TONGSAIMAI PIAN group, model group, positive drug group, TONGSAIMAI compositions senior middle school low dose group, administration every day secondary, continuous seven days.

3.2 getting blood, carotid artery carries out hemorheology test and PAgT.

Get rat carotid artery blood, place calparine pipe and the plastic test tube that fills 3.8% sodium citrate, carry out hemorheology and learn test and PAgT.

Get whole blood with calparine pipe, get about 1.5ml the zero graduation that adds packed cell volume Guan Zhongzhi top with capillary burette, leave standstill 1 hour after, observe the length (leukocyte is not counted in) of red blood cell decreased; Packed cell volume is with reference to Wen's method, will survey centrifugal 30 minutes of the packed cell volume pipe 3000rpm of erythrocyte sedimentation rate, reads the height of erythrocyte post.

Other gets whole blood 0.8mL, detects whole blood viscosity on the blood viscosity tester, and 2000RPM got supernatant gained blood plasma in centrifugal 10 minutes and detects the blood plasma viscosity then.And on the blood clotting analyzer, detect prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT) with blood plasma.

With ADP is derivant, by the turbidimetry for Determination platelet aggregation rate.9: 1 mixings of whole blood and anticoagulant (3.8% sodium citrate), centrifugal 8 minutes of 1000rpm, obtain upper strata platelet rich plasma (PRP), surplus blood centrifugal 10 minutes again with 3500rpm, platelet poor plasma (PPP), get 250 μ l PRP and place in the opacity tube, 37 ℃ of incubations 5 minutes, in PRP, add the ADP (making its final concentration is 10mmol/L) of 10 μ l subsequently, on platelet aggregation instrument, detect maximum agglutination rate.The results are shown in Table.

3.3 get brain with being about to the rat broken end, behind removal olfactory bulb, cerebellum and the low brain stem, freezing ten minutes, be cut into five.The 1st cutter is before brain in the middle of the utmost point and the optic chiasma line; The 2nd cutter is at the optic chiasma position; The 3rd cutter is at the infundibular stalk position; The 4th cutter is between the infundibular stalk and the posterior lobe tail utmost point.Place 5ml to contain 0.05%2,4 the brain sheet rapidly then, in PBS (10%) solution of 5-triphenyltetrazolium chloride (TTC), 37 ℃ of lucifuge water-bath temperature are incubated 10min, dyed after, normal cerebral tissue is and attacks rare redness, and blocking tissue is white in color, and boundary is clearly demarcated.Infraction assessment of indices: take out and blot, be tiled in order on the black paper, take pictures, print, calculate the infraction index as follows with filter paper.Infraction index=focus of infarct paper heavy (g)/infraction side brain paper heavy (g),

Use Microsoft Excel software to carry out date processing to all data, and statistic analysis result.

3.4 histopathologic slide checks: repeat 3.1 tests, behind the sacrifice of animal, get its cerebral tissue and place 10% formalin internal fixation, routine is drawn materials, paraffin embedding, slice thick 4～5 μ m, HE dyeing, the pathology professional reads sheet, according to the pathological changes light and heavy degree, sxemiquantitative is "+" successively, " ++ ", " +++", " ++ ++, no pathological changes normal structure is labeled as "-", and keeping the score respectively again is 1 minute; 2 minutes, and 3 minutes, 4 minutes; 0 minute, all marks except that blood vessel hypertrophy, macrophage hypertrophy that add up drew total points.Blood vessel hyperplasia, macrophage hypertrophy are the performance of injury repairing, thus from total points, deduct, according to the revised total points of every example, calculate (X ± SD) the dividing equally of every group every animal, the high more prompting of score value damage is serious more, otherwise then illustrates damage after drug treating and alleviate, and treatment is effective.Get cerebral tissue and do remainder behind the pathological section, weigh, make 10% homogenate with the pH7.0PBS buffer, 4 ℃ of centrifugal 30min of 3000r/min get supernatant and detect indexs such as NO, iNOS, total NOS, SOD, MDA.

Table 1: the TONGSAIMAI compositions is coagulated the influence (X ± SD) of cerebral infarction hemorheology of rat due to the medium-sized artery to electricity

Group	Example number (only)	Whole blood viscosity (mpas)
						200s -1	30s -1	5s -1	1s -1
		Dosage group Tongsaimai composition high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group Tongsaimai composition low dose group Tongsaimai composition	12 10 11 11 11 11 11	5.49±2.36 4.03±0.34 * 4.13±0.35 * 4.22± 0.47 4.33± 0.54 4.37±0.45 4.34± 0.53	7.46±2.97 5.38± 0.44 ** 5.56±0.49 * 5.71±0.50 5.77±0.63 5.88±0.55 5.90±0.54					13.33±4.78 9.24± 1.03 ** 9.76± 1.18 ** 10.02± 0.90 * 9.98±1.29 * 10.26±1.13 10.15±0.87	31.44±10.73 20.93± 3.48 ** 22.40± 3.94 ** 23.40±3.34 * 22.54±4.26 * 23.34±3.33 * 22.90± 2.34 **

Compare with model group, ^*P＜0.05, ^*P＜0.01.Compare with TONGSAIMAI PIAN, ^#P＜0.05, ^##P＜0.01.Down together.

Table 2 TONGSAIMAI compositions is coagulated the influence (X ± SD) of cerebral infarction hemorheology of rat due to the medium-sized artery to electricity

Group	The example number	Plasma viscosity (mpas)	Erythrocyte sedimentation rate (mm)	Hematocrit (%)
					Dosage group high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group low dose group	12 10 11 11 11 11 11	1.43±0.19 1.39±0.10 1.47±0.09 1.42±0.11 1.40±0.06 1.47±0.11 1.46±0.11	0.83±0.49 1.05±0.72 1.41±1.87 0.82±0.25 0.95±0.72 1.77±1.78 1.55±1.54	41.42±5.21 37.3±3.23 * 38.73±2.76 38.64±2.54 38.27±3.50 38.91±4.37 39±3.32

Compare with model group, ^*P＜0.05, ^*P＜0.01.

Table 3 TONGSAIMAI compositions is coagulated the influence (X ± SD) of cerebral infarction hemorheology of rat due to the medium-sized artery to electricity

Group	The example number	PT(s)	TT(s)	APTT(s)
					Dosage group high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group low dose group	12 10 11 11 11 11 11	19.94±1.60 22.48±6.85 21.23±4.73 19.43±2.82 17.82±1.97 ** 18.53±2.07 23.11±12.46	30.90±4.64 35.92±8.87 36.75± 12.56 31.52±4.71 30.12±3.76 31.92±6.53 32.91± 10.22	19.72±2.85 21.76±13.48 22.98±15.95 20.06±2.40 18.87±2.63 19.26±2.90 23.90±12.34

Compare with model group, ^*P＜0.05, ^*P＜0.01.

Table 4 TONGSAIMAI compositions is coagulated the influence (X ± SD) of cerebral infarction PAgT due to the medium-sized artery to electricity

Group	The example number	Dosage	Platelet maximum agglutination rate (%)
				Dosage group high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group low dose group	12 10 11 11 11 11 11	- - 32mg/kg·d 1.69g/kg·d 0.44g/kg·d 1.33g/kg·d 2.66g/kg·d	47.80±2.94 43.22±5.13 * 43.38±3.72 * 46.22±6.13 40.66±3.93 ** 38.15±3.55 ** 37.56±5.39 **

Compare with model group, ^*P＜0.05, ^*P＜0.01.

The TONGSAIMAI compositions has reduction effect preferably to whole blood viscosity, and platelet aggregation is also had certain inhibitory action.Illustrate that it can make the blood stasis state obviously recover, and has the effect of invigorating blood circulation.Influence to plasma viscosity, erythrocyte sedimentation rate, hematocrit and thrombin time (TT), APTT (APTT), prothrombin time (PT) is little,

Table 5 TONGSAIMAI compositions is coagulated due to the medium-sized artery cerebral infarction to electricity and is blocked exponential influence (X ± SD)

Group	The example number	Dosage	Infraction index (%)
				Dosage group high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group low dose group	11 11 10 10 8 11 9	- - 32mg/kg·d 1.69g/kg·d 0.44g/kg·d 1.33g/kg·d 2.66g/kg·d	5.95±0.89 0 4.09±0.85 ** 4.96±1.25 5.17±0.99 4.37±1.13 ** 4.56±1.83 *

Compare with model group, ^*P＜0.05, ^*P＜0.01.

Show that by table 5 result TONGSAIMAI compositions high dose is compared with model group with middle dosage group, the infraction index has remarkable decline, compares there was no significant difference with the TONGSAIMAI PIAN group.Illustrate that the TONGSAIMAI compositions has certain therapeutical effect to the rat ischemia apoplexy.

Table 6 TONGSAIMAI compositions is coagulated the (X ± SD) of the influence of mediator in the cerebral infarction rat cerebral even slurry due to the medium-sized artery to electricity

Group	The example number	NO(μ mol/gprot)	MDA(nmol/mgprot)	SOD(U/mgprot)
					Dosage group high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group low dose group	9 11 10 8 8 12 9	3.05±1.90 1.15±1.14 * 1.31±0.92 * 2.15±1.86 0.59±0.33 * 1.66±1.66 1.18±1.00 *	3.09±1.28 1.63±1.11 2.52±1.44 1.76±0.46 * 1.85±2.12 2.79±2.12 3.20±0.88	21.16±2.52 24.81±3.60 * 22.68±3.49 * 21.92±1.56 23.25±1.55 24.88±4.53 * 26.89±1.54 **

Compare with model group, ^*P＜0.05, ^*P＜0.01.

Nitric oxide (NO) is as a kind of new neurotransmitter, produce by nitricoxide synthase (NOS) degraded arginine, it has the extremely short half-life, by the penetrating cell membrane of dispersion, in born of the same parents, permeate into outside the born of the same parents rapidly, extensively act on pericaryon, tip, glial cell and endotheliocyte etc., but the nitric oxide of excessive and imbalance synthesizes and then causes cell injury even death, when central nervous system's ischemia, can cause nerve injury, thereby bring out the synthetic excessive nitric oxide of nitricoxide synthase (NOS), the central nervous system is had direct neurotoxicity, be the major reason of the tardy property of cerebral ischemia neural cell injury.From last table result as can be seen, the TONGSAIMAI compositions can significantly reduce the NO content that raises because of ischemia in the rat cerebral tissue, thereby alleviates the neurocyte toxic action of NO, keeps the unconventional maincenter courier effect of NO performance in the brain network information is transmitted.In the aerobe object, all there is a large amount of superoxide ions to generate at any time in addition.Superoxide ion has higher chemism, and biological cell is had certain toxicity.It can with hydroperoxidation, generate the stronger hydroxyl radical free radical (OH) of toxicity.Superoxide ion and hydroxyl radical free radical attack biomembrane can change the structure and the permeability of film; The attack chromosome can make chromosome degraded, fracture.They also can attack many cellular contents, make it lose biological activity and function.The polyunsaturated fatty acid (Polyunsaturated fatty acid PUFA) that oxygen-derived free radicals can be attacked in the biomembrane causes lipid peroxidation, and therefore form lipid peroxide such as aldehyde radical (MDA), ketone group, hydroxyl and new oxygen-derived free radicals etc.So, if superoxide ion content is high unusually in the organism or in a certain tissue, will cause disease, in addition dead.SOD is the enzyme of catalysis superoxide anion (O2-) dismutation reaction.Its existence is relevant with biological intravital Detoxication, the height indirect reaction of SOD vigor body remove the ability of free radical, and the height indirect reaction of MDA the body cell order of severity that attacked by free radical.From last table result as can be seen, the TONGSAIMAI compositions can obviously increase superoxide dismutase (SOD) vigor, reduces malonaldehyde (MDA) content, and the rat ischemia apoplexy is had the obvious treatment effect.

Table 7 TONGSAIMAI compositions is coagulated the (X ± SD) of the influence of mediator in the cerebral infarction rat cerebral even slurry due to the medium-sized artery to electricity

Group	The example number	Total NOS (U/mgprot)	INOS(U/mgprot)	cNOS(U/mgprot)
					Dosage group high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group low dose group	11 11 9 11 9 10 11	1.50±0.21 0.94± 0.23 ** 1.06±0.57 0.92±0.58 ** 1.05±0.57 1.17±0.83 1.33±0.66 *	1.29±0.34 0.63±0.13 ** 0.58±0.19 ** 0.48±0.16 ** 0.66±0.21 ** 0.67±0.12 ** 0.53±0.21 **	0.21±0.32 0.31±0.20 0.48±0.45 0.44±0.52 0.39±0.45 0.48±0.84 0.72±0.65 **

Compare with model group, ^*P＜0.05, ^*P＜0.01.

NOS has two kinds of forms: structural type NOS (constructive NOS, cNOS) and induced NOS (inducible NOS, iNOS).CNOS is the physiology existence form of NOS, and the synthetic NO time is short, amount is few; INOS level under physiological condition is very low, and the local inflammation cell is stimulated by various factors under the cerebral ischemic condition, and iNOS great expression, activation can discharge NO for a long time in a large number, so become the major reason of the tardy property of neurocyte damage after the cerebral ischemia.The expression of inducible nitric oxide synthase (iNOS) is arranged at the neutrophilic granulocyte of the blood vessel of ischemic area and infiltration, glial cell and macrophage also produce iNOS when cerebral ischemia in addition, and the NO and the by-product hyponitric acid thereof that are produced by iNOS can cause neuronal death by destroying neuronal structure albumen.The neuron NO of neuronal NOS (nNOS) generation can impel the neural poison of glutamic acid mediation in addition, thereby causes nerve cell death.By contrast, the NO of endotheliocyte NOS (eNOS) generation has protective effect to cerebral ischemia.Experiment finds that the TONGSAIMAI compositions can raise Mus brain eNOS activity, and downward modulation iNOS and total NOS improve function of nervous system, points out this medicine to alleviate the cerebral ischemia infringement by protection cerebrovascular endothelium.

Table 8 TONGSAIMAI compositions is to the influence of cerebral infarction rat brain

Group	Brain number (n)	The degree score value of brain damaged
			Dosage group high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group low dose group	8 8 8 8 8 8 8	3.38±1.77 0.63±0.52 2.38±1.06 2.50±1.38 2.63±1.19 2.50±1.07 1.63±1.06

Histopathology is observed, and model group rat cerebral tissue has downright bad in various degree, and downright bad place glial cell has hypertrophy in various degree, and sees that macrophage hypertrophy, glial nodule form glial cell and interstitial edema.Positive drug and TONGSAIMAI compositions are alleviating brain tissue impairment in varying degrees, and its effect is followed successively by heavy dose of group, positive drug group, middle dosage group, former dosage form group, small dose group by descending order.Sham operated rats biopsy tissues pathological change is not obvious, and model group brain tissue impairment degree is the most serious, illustrates that the TONGSAIMAI compositions has protective effect to cerebral tissue.

4. conclusion

Show from above result, the TONGSAIMAI compositions has the obvious treatment effect to the rat ischemia apoplexy, can obviously reduce infarct size, increase superoxide dismutase (SOD) vigor, reduce malonaldehyde (MDA) and nitric oxide (NO) content, rising endothelial nitric oxide synthase (NOS) content reduces inducible nitric oxide synthase (NOS) content.

Histopathology is observed explanation TONGSAIMAI compositions has protective effect to cerebral tissue.

Embodiment 3, TONGSAIMAI compositions are to the protective effect of middle cerebral artery thromboembolism rat model

1. experiment material

1.1 medicine and reagent

With embodiment 2.

1.2 laboratory animal

Cleaning level male SD rat, body weight 240g～310g is provided by west, Shanghai pul-Bi Kai laboratory animal company limited.

2. statistical method

Experimental data is expressed as X ± SD, and measurement data is relatively used grade preface value method (Ridit method) with Student T check comparative statistics difference between the behavior rank groups.

3. test method

3.1 experimental program: adopt internal carotid artery bolt collimation method to prepare this model.Adopt the method for Longa to be improved rat oral gavage administration, continuous 5 days, 1h is with 10% chloral hydrate anesthesia (350mg/kg after the administration in the 5th day, ip), face upward the position fixing with operating-table on, the cervical region median incision, after cutting skin, passivity is separated, and finds out common carotid artery (CCA), continues to separate downwards and ligation external carotid artery (ECA) and each branch's (occipital artery of external carotid artery, superior thyroid artery, lingual artery and facial artery), peel off vagus nerve gently, isolate internal carotid artery (ICA) and arteria pterygopalatina, the ECA proximal part is equipped with line, closes ICA and CCA with the bulldog clamp folder, cut an osculum at ECA apart from the ICA2mm place, one nylon wire (diameter 0.24mm, head end is handled through naked light, it is slick spherical that it is melt into) is inserted ECA and enters CCA, the light bundle is equipped with line, prevent hemorrhagely, cut off ECA, unclamp the bulldog clamp of ICA, traction ECA, make itself and ICA approximately in line, the pumpback nylon wire makes it along going into ICA gently, (attention avoids nylon wire to enter arteria pterygopalatina, until slight resistance is arranged to continue propelling downwards.This moment, visible ICA stretched, the nylon wire insertion depth is about 18mm, shows that nylon wire has passed middle cerebral artery (MCA) The initial segment, arrives anterior cerebral artery (ACA) near-end, all blood of having blocked MCA comprise the blood supply from ICA and ACA and posterior cerebral artery for the source.Unclamp the CCA bulldog clamp, tighten line fully, stay the long the end of a thread of 1cm outward, skin suture.Steam again and raise.

Nylon wire is gently drawn in perfusion again behind the ischemia 3h when pouring into again, and nylon wire is extracted outside the cranium, and blood flow is logical again, prunes nylon wire, and skin suture steams again nature and feeds.Above process is all carried out under room temperature constant (24～25 ℃) situation, is beneficial to estimate the cerebral ischemia situation.Observation is to the influence of focal cerebral ischemia in rats behavior scoring and cerebral infarct size.Above process is all carried out under room temperature constant (24～25 ℃) situation, is beneficial to estimate the cerebral ischemia situation.Observation is to the influence of focal cerebral ischemia in rats behavior scoring and cerebral infarct size.

3.2 rat experiment cerebral ischemia behavior scoring method

With reference to the method for Bederson etc. the behavioral deficiency of postoperative animal is carried out rank scores, grade scale is as follows:

The animal symptom	Grade
		Carry tail when unsettled, two forelimbs of animal all stretch to the floor direction and do not have other row defectives and carry tail when unsettled, the operation offside forelimb of animal shows as wrist elbow flexing, the shoulder inward turning, the elbow abduction, being close to thoracic wall places animal on the smooth flat, pushing hands art side is takeed on when resistance reduces the animal walking freely to side shifting the time, to performing the operation to the side ring commentaries on classics or turn-taking	0 grade 1 grade 2 grades 3 grades

Rating fraction is high more, shows that the behavioral deficiency of animal is serious more.

3.3TTC cerebral infarct size is measured in dyeing

Cerebral infarct size is measured in TTC dyeing ^[3]: 24 hours broken ends are got brain behind the MCAO, and brain is put (2～3 ℃) 10min in the ice-cold normal saline, remove olfactory bulb, cerebellum and low brain stem after, make not stagnant blood dirt of surface with the normal saline flushing brain, blot on every side moisture after, along the crown four blade of cutting, be cut into five.First cutter is before brain in the middle of the utmost point and the optic chiasma line; Second cutter is at the optic chiasma position; The 3rd cutter is at the infundibular stalk position; Four blade is between the infundibular stalk and the posterior lobe tail utmost point.Rapidly the brain sheet is put then in the phosphate buffer solution that 5ml contains 1%TTC, the lucifuge temperature was incubated 30 minutes, wherein stirred once every 7～8 minutes, dyed after, normal cerebral tissue is rose, and blocking tissue is white in color, and boundary is clearly demarcated.After temperature is incubated and finished the brain sheet is taken a picture, cut the calculating of weighing of red white colour district.Then according to the weight area method, calculate five brain sheets area of totally 10 planar gross areas and infarct area respectively, obtain the percentage ratio that the infarct area accounts for the hemisphere gross area, i.e. the infraction rate.

3.4 the mensuration of brain water content: behind the sacrifice of animal, take out brain and claim to place 115 ℃ of electrically heated drying cabinets to dry to constant weight cerebral tissue after the weight in wet base, claim dry weight, calculate brain water content by following formula:

Water content (%)=[(weight in wet base-dry weight)/weight in wet base] * 100%

Cerebral index=(cutaneous horn weight/body weight) * 100

3.5 blood plasma 6-K-PGF _{1 α}, TXB ₂, the ET assay: the carotid artery blood-letting, indometacin-EDTA.Na anticoagulant adds an amount of aprotinin simultaneously, 3000 commentaries on classics/min centrifugal separation plasmas are put-20 ℃ of preservations.Put with PLA General Hospital Science and Technology Development Center during test and the test kit that provided is provided (date of manufacture is on April 23rd, 2004, testDate is on May 9th, 04), put rabbit test center by Nanjing No.1 Hospital and detect, instrument is that Finland produces Wizard 1470 γ calculating instruments.

3.6 animal grouping and administration

Rat is divided into 7 groups at random, i.e. sham operated rats, nimodipine group, high, medium and low dosage of model group TONGSAIMAI and TONGSAIMAI PIAN group.Every group 10, gastric infusion, continuous 5 days, underwent surgery in the 5th day, postoperative 30min is administered once again.

3.7 tissue pathological slice

Repeat 3.1 tests, behind the sacrifice of animal, get its cerebral tissue and place 10% formalin internal fixation, routine is drawn materials, paraffin embedding, slice thick 4～5 μ m, HE dyeing, the pathology professional reads sheet, and according to the pathological changes light and heavy degree, sxemiquantitative is "+" successively, " ++ ", " +++", " ++ ++ ", no pathological changes normal structure is labeled as "-", keeping the score respectively again is 1 minute, 2 minutes, and 3 minutes, 4 minutes, 0 minute, all marks that add up drew total points, calculate (X ± SD) the dividing equally of every group every animal, the high more prompting of score value damage is serious more, otherwise then illustrates damage after drug treating and alleviate, and treatment is effective.

4. result

4.1 behavioristics's scoring: when 4h and 24h, delayed ischemic neurological deficits has in various degree all appearred in all experimental animals.During 4h, behavioristics's score of each administration treated animal and model group comparing difference do not have significance (P＜0.05=: behind 24h, behavioristics's scoring of each dosage treated animal of TONGSAIMAI compositions is all on a declining curve, and significantly be lower than model group, learn by statistics and handle, difference has significance, and (P＜0.05, P＜0.01=sees Table 9.Prompting TONGSAIMAI compositions can be improved behavioristics's obstacle of animal pattern.

The influence of table 9 TONGSAIMAI compositions to the behavioristics of Focal Cerebral Ischemia Reperfusion rat is marked

Group	The example number	Dosage (g/1000g)	Behavioristics's scoring
					4h	24h
			Tongsaimai combination object height in the low Tongsaimai composition of model sham-operation Nimodipine TONGSAIMAI PIAN Tongsaimai composition	10 10 10 10 10 10 10			- - 2×10 -3 2 2 1 0.5	2.6±0.52 0 2.6±0.52 2.6±0.52 2.6±0.52 2.6±0.52 2.5±0.53	2.5±0.53 0 1.8±0.79 * 1.9±0.88 1.8±0.79 * 1.7±0.82 * 1.4±0.52 **

Annotate: compare with model group: ^*: P＜0.05, ^*: P＜0.01.

4.2 the influence to cerebral infarct size and infraction rate: cerebral infarct size and the infraction rate of each administration group rat all are starkly lower than model group, learn by statistics and handle, and difference has significance, and (P＜0.05～P＜0.01=sees Table 10.Prompting TONGSAIMAI compositions can obviously be dwindled the cerebral infarct size of rat model, reduces the infraction rate.

4.3 the influence to brain water content and cerebral index: rat model cerebral index and water content are all apparently higher than sham operated rats, TONGSAIMAI compositions high dose can reduce the cerebral index of rat model, high, middle dosage all can reduce the brain water content of animal pattern, compare with model group, difference all has significance (P＜0.05 P＜0.01), sees Table 3.Prompting TONGSAIMAI compositions can reduce the water content of ischemic tissue of brain, alleviates cerebral edema.

The influence of table 10. pair all kitchen range cerebral ischemia re-pouring rat cerebral infarction areas and infraction rate (X ± SD)

Group	The example number	Dosage (g/kg)	Infarct size (cm 2)	Infraction rate (%)	Suppression ratio (%)
						Tongsaimai combination object height in the low Tongsaimai composition of model sham-operation Nimodipine TONGSAIMAI PIAN Tongsaimai composition	10 10 10 10 10 10 10	- - 2×10 -3 2 2 1 0.5	3.08±1.41 0 1.35±1.52 * 1.36±1.61 * 1.43±1.54 * 1.31±135 * 0.47±0.53 **	26.38±11.36 0 11.87±13.08 * 12.03±14.33 * 12.10±12.51 * 11.81±12.20 * 4.44±4.54 **	55.00 54.38 54.15 55.24 83.16

Annotate: compare with model group: ^*: P＜0.05, ^*: P＜0.01.

The influence of table 11. pair Focal Cerebral Ischemia Reperfusion rat brain index and brain water content (X ± SD)

Group	The example number	Dosage (g/kg)	Cerebral index	Water content (%)
					Tongsaimai combination object height in the low Tongsaimai composition of model sham-operation Nimodipine TONGSAIMAI PIAN Tongsaimai composition	10 10 10 10 10 10 10	- - 2×10 -3 2 0.5 1 2	0.530±0.011 0.470±0.037 ** 0.496±0.029 * 0.501±0.051 0.508±0.044 0.522±0.029 0.486±0.045 *	80.46±1.19 78.38±0.90 * 79.04±1.45 * 79.21±1.40 79.28±1.63 79.16±1.52 * 79.08±1.21 *

Annotate: compare with model group: ^*: P＜0.05, ^*: P＜0.01.

4.4 to blood plasma 6-K-PGF _{1 α}, TXB ₂, ET content influence: TXB in the Focal Cerebral Ischemia Reperfusion rat model blood plasma ₂, ET content obviously raises, and with the sham operated rats comparing difference significance (P＜0.05=arranged; Except that nimodipine, each administration group blood plasma TXB ₂, ET content all has a declining tendency, wherein middle and high dose group can reduce TXB in the blood plasma ₂Content, middle dosage and former dosage form group can reduce the content of ET in the blood plasma, (P＜0.05=sees Table 12 with the model group comparing difference significance all.

Table 12 couple Focal Cerebral Ischemia Reperfusion rat plasma 6-K-PGF _{1 α}, TXB ₂, ET content influence (X ± S)

Group	The example number	Dosage (g/kg)	6-K-PGF 1α、	TXB 2、	ET
						Tongsaimai height in the low Tongsaimai of model sham-operation Nimodipine TONGSAIMAI PIAN Tongsaimai	10 10 10 10 10 10 10	- - 2×10 -3 2 0.5 1 2	800.38±462.47 611.59±416.13 668.84±393.36 1008.88±369.61 780.03±426.85 900.48±411.69 660.06±333.02	821.58±453.83 317.29± 260.24 ** 749.62±698.24 716.74±450.90 484.79±531.58 362.64±220.31 * 370.52±315.76 *	46.25±11.43 33.19±12.73 * 44.02±16.22 34.09±13.09 * 35.80±18.48 28.99±15.95 * 35.64±17.76

PGI ₁, TXA ₂Adjusting to platelet and vascular function has great importance, TXA ₂Mainly synthetic in platelet microsome, have very strong vasoconstriction and promote the platelet aggregation effect.PGI ₁Synthetic by vascular endothelial cell, but vasodilator, anticoagulant.Under the normal condition, both keep dynamic equilibrium that normal function, the adjusting blood flow of keeping blood vessel had great importance, and both dysequilibriums are to cause platelet aggregation, thrombosis and angiospastic major reason under the pathological state.TXA ₂About 30 seconds of biological half-life is metabolized to the TXB of non-activity rapidly ₂PGI ₁About 3 minutes of biological half-life is metabolized to 6-K-PGF _{1 α}Because PGI ₁And TXA ₂The character instability is difficult to direct mensuration, all measures 6-K-PGF inside and outside the native land _{1 α}And TXB ₂As judging PGI ₁And TXA ₂The index of concentration.ET is a kind of adjusting peptide and the somatomedin that is distributed widely in cardiovascular system, is the at present known the strongest vasoconstrictive and the most persistent humoral factor that causes, by the vascular endothelial cell secretion, participates in the adjusting of blood flow.Clinical and experimentation shows that the ET secretion increases during cerebral ischemia.This experiment shows each administration group blood plasma TXB ₂, ET content all has a declining tendency, wherein middle and high dose group can reduce TXB in the blood plasma ₂Content, middle dosage and former dosage form group can reduce the content of ET in the blood plasma, with the model group comparing difference significance are arranged all.

Table 13 TONGSAIMAI compositions is to the influence of cerebral infarction rat brain

Group	Brain number (n)	The degree score value of brain damaged
			Dosage group high dose group in the model group sham-operation group positive drug group TONGSAIMAI PIAN group low dose group	6 6 5 5 6 5 6	5.8±0.4 0.4±0.54 2.6±1.82 3.8±2.17 3.17±2.04 2.2±1.64 2.33±2.9

Histopathology is observed, line bolt method cerebral infarction rat model, and it is downright bad in various degree to show as cerebral tissue, glial cell and interstitial edema.Positive drug and TONGSAIMAI compositions are having the brain tissue impairment of alleviating effect in varying degrees to the apoplexy model, and its effect is followed successively by the heavy dose of group of TONGSAIMAI compositions, middle dosage group, positive drug group, small dose group by descending order.Sham operated rats tissue pathologies change is not obvious, and former dosage form group brain tissue impairment degree is lighter than model group, but attaches most importance to than other groups.

5. conclusion

Show that from above result the TONGSAIMAI compositions can be improved behavioristics's obstacle of animal pattern, can obviously dwindle the cerebral infarct size of rat model, reduce the infraction rate: reduce the water content of ischemic tissue of brain, alleviate cerebral edema.Each administration group blood plasma TXB ₂, ET content all has a declining tendency, wherein middle and high dose group can reduce TXB in the blood plasma ₂Content, middle dosage and former dosage form group can reduce the content of ET in the blood plasma, with the model group comparing difference significance are arranged all.Histopathology is observed, line bolt method cerebral infarction rat model, and it is downright bad in various degree to show as cerebral tissue, glial cell and interstitial edema.Positive drug and TONGSAIMAI compositions are having the brain tissue impairment of alleviating effect in varying degrees to the apoplexy model, and its effect is followed successively by the heavy dose of group of TONGSAIMAI compositions, middle dosage group, positive drug group, small dose group by descending order.Sham operated rats tissue pathologies change is not obvious, and former dosage form group brain tissue impairment degree is lighter than model group, but attaches most importance to than other groups.

Embodiment 4, TONGSAIMAI compositions extractum are to the influence of rat thrombus in vivo

[experiment purpose]

Behind the ligation postcava, cause vascular damaged, bloodstream blocking forms the postcava thrombosis, observes the influence of TONGSAIMAI compositions extractum to thrombosis length, weight.

[experiment material]

1. animal: the healthy SD rat, about 300g, male and female half and half are available from Nanjing Medical University's Experimental Animal Center.Production licence number: (SCXK) 2002-0031.

2. medicine: TONGSAIMAI compositions extractum, Nanjing University of Traditional Chinese Medicine's new drug and preparation research chamber, marine drug research center provide, and clinical people's consumption 51.84g/ day, every gram extractum is equivalent to crude drug amount 7.92g.Medical university's Pharmaceutical limited company provides in the south.

The former dosage form of TONGSAIMAI, clinical people's consumption 6.3g extractum/day, every gram extractum is equivalent to crude drug amount 6.67g.Medical university's Pharmaceutical limited company provides in the south.

Pentobarbital sodium: Shanghai chemical reagents corporation of Chinese Medicine group advances to divide lot number: F20020405.

3. equipment: the some covers of operating theater instruments.

[experimental technique]

Get 72 of healthy SD rats, be divided into 6 groups at random, 12 every group.Model group (equivalent distilled water), positive drug group (nimodipine g/kgd), large, medium and small three the dosage groups of TONGSAIMAI compositions extractum (2.679g/kgd, 1.3395g/kgd, 0.4465g/kgd).

Administering mode is a gastric infusion, and once a day, administration is 10 days altogether.Last administration fasting the previous day.After the last administration 1 hour, rat was with pentobarbital sodium 40mg/kg intraperitoneal injection of anesthesia, and fixedly postabdomen sterilization of dorsal position is opened the abdominal cavity from ventrimeson, exposes and also separates postcava, in the place's ligation of left renal vein lower horizontal, and sews up the abdominal cavity.After the ligation 2 hours, reopen the abdominal cavity, 2cm clamps postcava with mosquito forceps in the place below ligation, takes out this section blood vessel, and official jargon is cut in stringer open rapidly, and removal of thromboses on filter paper dips in and does unnecessary bloodstain, weighing weight in wet base.Put into 37 ℃ of oven dryings then 24 hours.The weighing dry weight.

The The data EXCEL software that obtains carries out F check and T check.

[experimental result]

TONGSAIMAI compositions extractum has certain inhibitory action to the postcava thrombosis that ligation damage tube wall causes, and sees Table 14.

Table 14. TONGSAIMAI compositions extractum is to the influence of rat postcava thrombosis (X ± SD)

Group	Dosage (g/kgd)	The example number	Thrombosis length (mm)	Wet weight of thrombus (mg)	Thrombosis dry weight (mg)
						Dosage heavy dose in the former dosage form low dose of model group positive drug	- 0.032 27.77 13.89 4.63	11 12 11 12 11 12	10.89±3.97 6.12±4.61 * 8.60±2.84 8.56±3.50 7.27±2.62 * 7.12±3.74 *	34.45±13.73 14.58±13.03 ** 24.18±8.33 * 19.33±11.22 ** 19.27±7.50 ** 17.08±7.13 **#	9.55±2.54 4.67±4.66 ** 7.36±2.25 * 6.58±3.94 * 6.73±2.69 * 6.00±3.36 **

Compare with model group, ^*P＜0.05, ^*P＜0.01.Compare with former dosage form, ^#P＜0.05.

[experiment conclusion]

TONGSAIMAI compositions extractum has certain inhibitory action to the postcava thrombosis that ligation damage tube wall causes.

The influence that the mice thrombus in vivo that embodiment 5, TONGSAIMAI compositions are brought out collagen protein-epinephrine forms

[experiment purpose]

Collagen protein and epinephrine all are the platelet aggregation derivants, and mouse mainline etc. bring out the interior lung of body and fasten plug, and thrombosis causes hemiplegia and death, and observing TONGSAIMAI compositions extractum influences it.

[experiment material]

1. animal: the ICR mice, 18-22g, male and female half and half are available from Nanjing Medical University's Experimental Animal Center.Production licence number: (SCXK) 2002-0031.

2. medicine and reagent: the TONGSAIMAI compositions, Nanjing University of Traditional Chinese Medicine's new drug and preparation research chamber, marine drug research center provide, clinical people's consumption 51.84g crude drug/day, every gram extractum is equivalent to crude drug amount 10.43g.The former dosage form of TONGSAIMAI, clinical people's consumption 6.3g extractum/day, every gram extractum is equivalent to crude drug amount 6.67g.Medical university's Pharmaceutical limited company provides in the south.Lot number:

Aspirin, Hunan pharmaceutical factory, lot number: 990402.

Epinephrine inj, 1mg/ml, Tianjin gold credit aminoacid company limited is produced lot number: 0306031.

The II collagen type, Sigma company produces, and Beijing nation decides the packing of Tyke biotech company.

3. equipment: little rat holder, 1ml syringe.

[experimental technique]

Get 144 of mices, be divided into 6 groups at random, every group 24, male and female half and half: model group (distilled water 10ml/kgd), the former dosage form group of TONGSAIMAI (the 16.39g crude drug/kgd), positive drug group (aspirin 50mg/kgd), large, medium and small three the dosage groups of TONGSAIMAI compositions (40.08,20.04,6.68g crude drug/kgd).Continuous irrigation stomach 7 days is irritated the long-pending 10ml/kg of being of body of stomach.1h after the last administration, tail vein injection epinephrine-collagen protein mixes liquid 0.1ml/10g body weight.Observe mortality rate in the mice 5 minutes.The result adopts X 2 test, the comparable group differences.

Attached: the preparation of collagen protein-epinephrine mixing derivant: as to take by weighing collagen protein 30mg, be soaked in the 3-4ml normal saline more than 2 hours homogenate, add normal saline 40ml, centrifugal 10 minutes of 3000rpm gets supernatant, other adds epinephrine 1ml, adds normal saline to 50ml.

[experimental result]

The inhibitory action of the postcava thrombosis that TONGSAIMAI compositions extractum causes ligation damage tube wall sees Table 15;

Table 15 TONGSAIMAI is to the influence of collagen protein-epinephrine induced mice thrombus in vivo (X ± SD)

Group	Dosage (g/kgd)	The example number	Dead mouse number in 5 '	Mouse death rate (%) in 5 '
					Dosage low dose in the former dosage form heavy dose of model group positive drug	- 0.05 16.39 40.08 20.04 6.68	24 21 22 23 23 21	16 2 7 11 7 11	66.67 9.52 ** 39.13 * 47.83 30.43 ** 47.62

Compare with model group, ^*P＜0.05, ^*P＜0.01.

[experiment conclusion]

The TONGSAIMAI compositions has inhibitory action to platelet aggregation in the caused mice body of collagen protein-epinephrine, thereby suppresses the formation of its thrombus in vivo.

Claims (5)

1, a kind of preparation method for the treatment of the Chinese medicine composition of ischemia apoplexy is a raw material with the Radix Astragali 10～20g, Radix Angelicae Sinensis 10～18g, Radix Scrophulariae 8～10g, Flos Lonicerae 8～15g, Herba Dendrobii 8～15g, Radix Glycyrrhizae 6～10g, and its preparation process is as follows:

(1) gets Radix Angelicae Sinensis 10～18g and carry out co_2 supercritical fluid extraction, collect extract;

(2) medicinal residues behind the Radix Angelicae Sinensis carbon dioxide abstraction and the Radix Astragali 10～20g, Radix Scrophulariae 8～10g, Flos Lonicerae 8～15g, Herba Dendrobii 8～15g, Radix Glycyrrhizae 6～10g merge, and carry out ethanol extraction, obtain ethanol extract;

(3) with the medicinal liquid behind the above-mentioned ethanol extract decompression recycling ethanol, concentrate centrifugal, water washing and precipitating, washing liquid and filtrate merging, discard precipitation, supernatant obtains ethanol elution with ethanol elution behind absorption with macroporous adsorbent resin;

(4) with the ethanol elution decompression recycling ethanol, concentrate, crushed after being dried gets the dry extract powder;

(5) component that above-mentioned steps (1), (4) is obtained merges, and promptly obtains Chinese medicine composition of the present invention.

2. the preparation method of Chinese medicine composition according to claim 1 is characterized in that in the step (2) earlier with 70% alcohol reflux 2 times, afterwards with 50% alcohol reflux 1 time, and the 1st 8 times of amounts, the 2nd, 36 times of amounts are extracted 1h at every turn.

3. the preparation method of Chinese medicine composition according to claim 1 is characterized in that on clear liquid circulate in the step (3) sample 4 times, with 60% ethanol eluting successively behind upper prop or upper prop behind the SP-825 type absorption with macroporous adsorbent resin 12h.

4. the preparation method of Chinese medicine composition according to claim 1 is characterized in that the drying means in the step (4) is spray drying or vacuum drying.

5. the preparation method of the Chinese medicine composition of claim 1 can be made into tablet, granule, micropill, capsule with the method.