The specific embodiment
The preparation of embodiment 1 medicine of the present invention
Get Fructus Buchananiae latifoliae 120g, Rhizoma Chuanxiong 500g decocts with water, and is prepared into granule.
The preparation of embodiment 2 medicines of the present invention
Get Fructus Buchananiae latifoliae medical material 120g, water boiling and extraction 2 times, each 1.5h, 8 times of amounts merge medicinal liquid, filter, and filtrate is concentrated into relative density 1-1.05 (60 ℃), adds ethanol to concentration of alcohol 75%, leaves standstill 1h, the filtering precipitation, 60 ℃ of drying under reduced pressure of filtrate are to doing, promptly.
Get Rhizoma Chuanxiong medical material 500g, adopt percolation to extract medical material solvent ratios amount: 8 times, time: 24h, number of times: 2 times, solvent: 70% ethanol, merge percolate, 60 ℃ of decompression recycling ethanols are got concentrated solution (1g raw medicinal herbs/ml), cross the D101 macroporous adsorbent resin of handling well (internal diameter 1.8cm, high 9cm, volume 23ml), flow velocity is 2BV/h, absorption 1h washes flow velocity 2BV/h earlier with 5 times of water gagings, and then, control flow velocity 2BV/h with 6 times of amounts of 30% ethanol eluting.Collect eluent, reclaim ethanol, 60 ℃ of drying under reduced pressure are to doing, promptly.
Get above-mentioned two kinds of extracts, PEG400 and propylene glycol are an amount of, after being uniformly dispersed, fill the preparation soft capsule, 35 ℃ of dryings, promptly.
Below prove beneficial effect of the present invention by pharmacodynamics test.
The test for intestinal absorption of turning up of test example 1 isolated rat
With commercially available helicide sheet, Fructus Buchananiae latifoliae extract, Fructus Buchananiae latifoliae and medicine of the present invention (Fructus Buchananiae latifoliae 120g; Rhizoma Chuanxiong 500g) is object of study, adopts the isolated rat test for intestinal absorption of turning up, investigate its absorbing state, to estimate Fructus Buchananiae latifoliae and Rhizoma Chuanxiong component compatibility (by embodiment 2 preparations) its bioavailability situation of change afterwards.
1.1 experiment material
Day island proper Tianjin high performance liquid chromatograph (LC-10ATvp infusion pump, SPD-10Avp UV-detector, CT-10ASvp column oven); High performance liquid chromatography work station (Zhejiang intelligence reaches N2000); BP211D type electronic analytical balance (buy in German Sartorius company); 79HW-1 type constant temperature blender with magnetic force (Jintan City, Jiangsu Jin Cheng state wins experimental apparatus factory); Oxygen cylinder; Effusion meter; Teat glass.
Fructus Buchananiae latifoliae (purchasing) in lotus pond medical material market, Chengdu; Helicide reference substance (lot number: 100385-200401 purchases in Nat'l Pharmaceutical ﹠ Biological Products Control Institute); Acetonitrile (chromatographically pure is bought the company in Fisher); Water (redistilled water, self-control); Micropore filter (0.45 μ m buys the company in Millipore); Other reagent is analytical pure; SD rat (♂, 180~220g).
1.2 content assaying method
1.2.1 chromatographic condition
Chromatographic column: F door Luna-C
18(4.6 * 250mm, 5 μ m); Mobile phase: acetonitrile-water-glacial acetic acid (8: 91: 1); Flow velocity: 1.0mlmin
-1Column temperature: 25 ℃; Detect wavelength: 270nm; Sample size: 10 μ l; Sensitivity: AUFS=0.01; Theoretical cam curve is calculated according to helicide should not be lower than 8000.
1.2.2 standard curve is drawn
Getting accurate compound concentration is 120.8 μ g.ml
-1Standard solution, measure with above-mentioned chromatographic condition sample introduction, (μ g) is X with sample size, peak area (mv*min) is Y, does linear regression, the results are shown in Table 1, regression equation is: Y=3 * 10
6X+15283, helicide is in 0.0031~1.2080 μ g scope internal linear relation good (r=0.9998).
Table 1. linearity and range
The test for intestinal absorption 1.3 isolated rat turns up
Before the experiment with rat overnight fasting (can freely drink water), after disconnected cervical vertebra is put to death, open the abdominal cavity along ventrimeson after, the taking-up jejunum places the tyrode's solution of ice bath, aerating oxygen, removal mesentery.After cleaning with ice-cold tyrode's solution, intestinal segment is softly overturn with little Glass rod, make intestinal mucosa towards outside, serosa side is inside, ligation intestinal anus side will be fixed in sample tap on the intestinal.Fill with blank bench teat liquid as acceptable solution from sample tap to enteral with syringe, then it is vertically put into test liquid, experimental provision is seen Fig. 1, and aerating oxygen in test tube, whole device are put into 37 ℃ of waters bath with thermostatic control and experimentized.Respectively at 10,15,30,45,60min, the 0.5ml that takes a sample in the intestinal capsule, the filtering with microporous membrane of 0.45 μ m, the filtrate ice bath is preserved, and measures according to the said method sample introduction, replenishes 37 ℃ of blank bench teat liquid of 0.5ml simultaneously in the intestinal capsule.
1.4 evaluation index
With accumulation infiltration capacity, multiple is that index is estimated the absorbing state of each medicine.
1.5 date processing
Accumulative total transit dose (Q), multiple (R) adopt following formula to calculate:
(Q: the accumulation infiltration capacity of time t; Helicide concentration in the Cn:t time reception tank; V: liquor capacity in the reception tank; Vi: sample volume; Helicide concentration in the reception tank before the Ci:t time; Q sheet: the accumulation infiltration capacity of helicide sheet; C sheet: the initial concentration of helicide sheet; R: the multiple of comparing with helicide sheet accumulation infiltration capacity)
1.6 result and analysis
By the result as can be known, the accumulation infiltration capacity of Fructus Buchananiae latifoliae is 2.25 times of helicide sheet, and the accumulation infiltration capacity of component compatibility is 6.44 times of helicide sheet, illustrates that the absorbing state of Fructus Buchananiae latifoliae extract is better than helicide, and behind the Rhizoma Chuanxiong (processed) component compatibility, its bioavailability significantly improves.
The test for intestinal absorption of turning up of table 2. isolated rat
Test example 2 medicines are to the influence of mice autonomic activities
With commercially available helicide sheet, Fructus Buchananiae latifoliae extract, Rhizoma Chuanxiong (processed) extract, Fructus Buchananiae latifoliae and medicine of the present invention (Fructus Buchananiae latifoliae 120g: Rhizoma Chuanxiong 500g) be object of study, adopt the influence of medicine to the mice autonomic activities, investigate its drug effect, with its pharmacodynamic change situation behind evaluation Fructus Buchananiae latifoliae and the Rhizoma Chuanxiong component compatibility.
2.1 experiment material
KM mice (♂, 18~22g); ZZ-6 type mice autonomic activities tester; Electronic analytical balance.
2.2 animal grouping
With picric acid animal is numbered, adopts random digit that animal is carried out the stratified random grouping.
Grouping of table 3. animal and administration
2.3 animals administer
This experimental animal feeding began the prospective adaptation environment 3 days in pharmacological evaluation animal observation ward of Chengdu University of Traditional Chinese Medicine in experiment, normally gave diet and water.And before beginning, formal experiment writes down the autonomic activities number of times of respectively organizing in the mice 5min with ZZ-6 type mice autonomic activities tester, respectively each group mice is carried out the ip administration according to " animal grouped table ", behind 6d administration 45min, write down the autonomic activities number of times of respectively organizing in the mice 5min with ZZ-6 type mice autonomic activities tester.
2.4 evaluation index
Observation index is the autonomic activities rate of change, that is:
2.5 date processing
Experimental data is represented with mean ± S.D., adopts SPSS software, statistical test one factor analysis of variance, test level α=0.05 or 0.01.
2.6 result and analysis
By the result as can be known, helicide sheet, each dosage group of Fructus Buchananiae latifoliae, each dosage group of compatibility all have remarkable inhibitory action (p<0.05) to the mice autonomic activities; And each dosage group of Rhizoma Chuanxiong (processed) does not all have inhibitory action.Fructus Buchananiae latifoliae height, middle dosage group significantly are better than helicide sheet (p<0.05) to mice autonomic activities inhibitory action; Compatibility height, middle dosage group significantly are better than helicide sheet (p<0.05) to mice autonomic activities inhibitory action; Under the same dose, the compatibility group significantly is better than Fructus Buchananiae latifoliae group (p<0.05) to mice autonomic activities inhibitory action, illustrates that the drug effect of Fructus Buchananiae latifoliae extract is better than the helicide sheet, and with the Rhizoma Chuanxiong (processed) component compatibility after, its drug effect significantly improves.
Table 4. medicine to the influence of mice autonomic activities (mean ± S.D., n=10)
Test example 3 medicines are to the influence of mouse sleep time
With commercially available helicide sheet, Fructus Buchananiae latifoliae extract (pressing the method preparation of embodiment 2), Rhizoma Chuanxiong (processed) extract, Fructus Buchananiae latifoliae and medicine of the present invention (Fructus Buchananiae latifoliae 120g: Rhizoma Chuanxiong 500g, by embodiment 2 preparations) be object of study, adopt the influence of medicine to the mice autonomic activities, investigate its drug effect, with its pharmacodynamic change situation behind evaluation Fructus Buchananiae latifoliae and the Rhizoma Chuanxiong component compatibility.
3.1 experiment material
KM mice (♂, 18~22g); Pentobarbital sodium; Electronic analytical balance.
3.2 animal grouping
With content under " test example 2 " item.
3.3 animals administer
This experimental animal feeding began the prospective adaptation environment 3 days in pharmacological evaluation animal observation ward of Chengdu University of Traditional Chinese Medicine in experiment, normally gave diet and water.Respectively each group mice is carried out the ip administration according to " animal grouped table ", (50mg/kg 0.1ml/10g), writes down each treated animal length of one's sleep in 6d administration 45min pneumoretroperitoneum injection pentobarbital sodium.
3.4 evaluation index
To be sleeping index more than the mice righting reflex loss 1min, be sleep time from righting reflex loss to recovery time.
3.5 date processing
With content under " test example 2 " item.
3.6 result and analysis
By the result as can be known, helicide sheet, each dosage group of Fructus Buchananiae latifoliae, each dosage group of compatibility all have significant prolongation effect (p<0.05) to mouse sleep time; And all not prolongation effects of each dosage group of Rhizoma Chuanxiong (processed).Fructus Buchananiae latifoliae height, middle dosage group significantly are better than helicide sheet (p<0.05) to mouse sleep time prolongation effect; Compatibility height, middle dosage group significantly are better than helicide sheet (p<0.05) to mouse sleep time prolongation effect; Under the same dose, the compatibility group significantly is better than Fructus Buchananiae latifoliae group (p<0.05) to mouse sleep time prolongation effect, illustrates that the drug effect of Fructus Buchananiae latifoliae extract is better than the helicide sheet, and with the Rhizoma Chuanxiong (processed) component compatibility after, its drug effect significantly improves.
Table 5. medicine to the influence of mouse sleep time (mean ± S.D., n=10)
To sum up, turn up test for intestinal absorption, medicine to the influence of mice autonomic activities, the medicine experiment confirms such as influence to mouse sleep time by isolated rat, according to big Rhizoma Chuanxiong side, adopt Fructus Buchananiae latifoliae and Rhizoma Chuanxiong to carry out bioavailability and the drug effect (p<0.05) that component compatibility can significantly strengthen helicide.
Test example 4 Drug therapy nitroglycerin of the present invention cause the test of rat migraine
1 experimental technique
This experimental animal feeding began the prospective adaptation environment 3 days in pharmacological evaluation animal observation ward of Chengdu University of Traditional Chinese Medicine in experiment, normally gave diet and water.Press table 6 and prevent ig administration 3d before modeling, 4d is except that blank group subcutaneous injection equal-volume normal saline, and when daily weight subcutaneous injection Nitro-Bid 10mg/kg modeling 1 time, it is subcutaneous that subcutaneous injection is positioned right shoulder by rat for all the other each groups.After the modeling about 3min, animal occurs that ears are rubescent, forelimb is frequently scratched one's head, climb phenomenons such as cage increased frequency, dysphoria, and this phenomenon continues about 3h, occurs curling oneself up then, and movable minimizing state shows the success of migraine animal model replication.Behind the modeling 4h, 10% chloral hydrate (0.35ml/100g) ip anesthesia, take out cerebral tissue, must on the ice platform, carry out during operation, take out whole brain tissue's (rejecting cerebellum) in the 1min, with ice-cold normal saline flushing, remove bloodstain, filter paper is wiped away dried, cuts the brain stem and the hypothalamus part that contain rapheal nuclei, locus coeruleus, drop in the liquid nitrogen immediately and solidify, standby; Prepare brain tissue homogenate during mensuration, measure the content of cerebral tissue 5-hydroxy tryptamine (5-HT), calcitonin-gene-related peptide (CGRP).
Grouping of table 6. animal and dosage
2 evaluation indexes
Content with cerebral tissue 5-hydroxy tryptamine (5-HT), calcitonin-gene-related peptide (CGRP) is that index is estimated the drug effect of each medicine.
3 date processing
Adopt SPSS software, statistical test is with t check and single factor F are checked test level α=0.05 in groups.
4 interpretations of result
By 5-HT and CGRP index respectively organize data analysis as can be known, between 1. blank group and the model group significant difference (P<0.05) is arranged, illustrate that the animal model modeling successfully.2. Fructus Buchananiae latifoliae, Fructus Buchananiae latifoliae+Rhizoma Chuanxiong all and between the model group have significant difference (P<0.05), illustrate that Fructus Buchananiae latifoliae, Fructus Buchananiae latifoliae+Rhizoma Chuanxiong all have good therapeutical effect to the migraine due to the nitroglycerin.In addition, Fructus Buchananiae latifoliae+Rhizoma Chuanxiong effect is better than single with Fructus Buchananiae latifoliae (P<0.05), and disclosing Rhizoma Chuanxiong can increase drug effect.
Table 7. nitroglycerin cause rat migraine animal model experiment result (mean ± S.D., n=10)
Annotate:
★: compare P<0.05 with model
△: compare P<0.05 with Fructus Buchananiae latifoliae
In sum, medicine of the present invention uses raw material Fructus Buchananiae latifoliae, Rhizoma Chuanxiong compatibility, has synergistic function, can be used for the treatment headache, and as angioneurotic headache and migraine, insomnia, drug effect is clear and definite, provides a kind of new selection for clinical.
The best proportioning screening test of test example 5 medicine materials of the present invention
1 experimental technique
With content under " test example 4 " item, wherein, the animal grouping is as shown in table 8 with dosage.
Grouping of table 8. animal and dosage
2 evaluation indexes
With content under " test example 4 " item.
3 date processing
With content under " test example 4 " item.
4 interpretations of result
By 5-HT and CGRP index respectively organize data analysis as can be known, between 1. blank group and the model group significant difference (P<0.05) is arranged, illustrate that the animal model modeling successfully.2. proportioning 1,2,3 all and between the model group has significant difference (P<0.05).3. efficacy strength, proportioning 3>proportioning 1>proportioning 2 discloses proportioning 3 best results.
Table 9. nitroglycerin cause rat migraine animal model experiment result (mean ± S.D., n=10)
Annotate:
★: compare P<0.05 with model group
To sum up, medicine of the present invention uses raw material Fructus Buchananiae latifoliae, Rhizoma Chuanxiong with 120: 500 ratio compatibilities, have best potentiation.