The specific embodiment;
Preparation example 1: the preparation of Carthamus yellow, total salvianolic acid
Get flos carthami 5kg, 30 times of water gaging percolation filter, with absorption with macroporous adsorbent resin (volume ratio of extracting solution and resin 3: 1),, continue then with 5 column volumes of deionized water eluting with 2 column volumes of deionized water eluting, collect eluent, the absorption of reuse polyamide, elder generation is washed till colourless with deionized water, use 95% ethanol elution of meta-alkalescence then, collect eluent, concentrate on the rotary evaporator and remove ethanol, lyophilization obtains Carthamus yellow, and wherein hydroxyl carthamin yellow A-containing 75.8%.
Get red rooted salvia 5kg, pulverize, 30 times of amount sour water percolation extract, filter, adsorb (volume ratio of extracting solution and resin 3: 1) with macroporous adsorbent resin, with deionized water eluting 3-5 column volume, 5 column volumes of the alcoholic solution eluting of reuse 30%, collect eluent, the adjusting pH value is 2-5, concentrates ethanol, lyophilizing, get total salvianolic acid, wherein contain salvianolic acid B 80.4%.
Preparation example 2: the preparation of Carthamus yellow, total salvianolic acid
Get flos carthami 5kg, 30 times of water gaging percolation filter, with absorption with macroporous adsorbent resin (volume ratio of extracting solution and resin 3: 1), with 2 column volumes of deionized water eluting, continue then to collect eluent with 5 column volumes of deionized water eluting, concentrating the back is immobile phase with the polydextran gel, separate with the deionized water eluting, collect the S-A Hydroxysafflor yellow A composition, concentrate on the rotary evaporator, lyophilization obtains Carthamus yellow, and wherein hydroxyl carthamin yellow A-containing 77.5%.
Get red rooted salvia and pulverize, 15 times of amount 60% acetone carry out percolation, collect percolate, concentrate, do not distinguish the flavor of to there being acetone, after water dilutes, carry out absorption with macroporous adsorbent resin (medical material amount and amount of resin 1: 1), with deionized water eluting 3-5 column volume, 5 column volumes of the alcoholic solution eluting of reuse 30%, collect eluent, concentrate ethanol, lyophilizing gets total salvianolic acid, wherein contains salvianolic acid B magnesium salt 72.8%.
Preparation example 3: the freeze-dried powder that preparation is made up of Carthamus yellow and total salvianolic acid
Under cleaning condition, get Carthamus yellow 190g, total salvianolic acid 10g, be dissolved in 1000ml water for injection, add 100g mannitol, stirring and dissolving, ultrafiltration obtains apyrogenic clear liquor, press the lyophilizing of freeze-dried powder technology, make the freeze-dried powder that contains Carthamus yellow 190mg, total salvianolic acid 10mg.
Preparation example 4: the freeze-dried powder that preparation is made up of Carthamus yellow and total salvianolic acid
Under cleaning condition, get Carthamus yellow 10g, total salvianolic acid 190g, be dissolved in 1000ml water for injection, stirring and dissolving, ultrafiltration obtains apyrogenic clear liquor, press the lyophilizing of freeze-dried powder technology, make the freeze-dried powder that contains Carthamus yellow 10mg, total salvianolic acid 190mg.
Preparation example 5: the freeze-dried powder that preparation is made up of Carthamus yellow and total salvianolic acid
Under cleaning condition, get Carthamus yellow 20g, total salvianolic acid 80g, be dissolved in 1000ml water for injection, add the 100g low molecular dextran, stirring and dissolving, ultrafiltration obtains apyrogenic clear liquor, press the lyophilizing of freeze-dried powder technology, make the freeze-dried powder that contains Carthamus yellow 20mg, total salvianolic acid 80mg.
Preparation example 6: the freeze-dried powder that preparation is made up of Carthamus yellow and total salvianolic acid
Under cleaning condition, get Carthamus yellow 80g, total salvianolic acid 20g, be dissolved in 1000ml water for injection, add the 100g low molecular dextran, stirring and dissolving, ultrafiltration obtains apyrogenic clear liquor, press the lyophilizing of freeze-dried powder technology, make the freeze-dried powder that contains Carthamus yellow 80mg, total salvianolic acid 20mg.
Preparation example 7: the freeze-dried powder that preparation is made up of Carthamus yellow and total salvianolic acid
Under cleaning condition, get Carthamus yellow 25g, total salvianolic acid 75g, be dissolved in 1000ml water for injection, stirring and dissolving, ultrafiltration obtains apyrogenic clear liquor, press the lyophilizing of freeze-dried powder technology, make the freeze-dried powder that contains Carthamus yellow 25mg, total salvianolic acid 75mg.
Test example 1: the influence that various dose Carthamus yellow, total salvianolic acid, compositions are damaged the rat local cerebral ischemia
(1) material:
Carthamus yellow, total salvianolic acid, compositions: by preparation example 1 preparation.
Red tetrazolium: U.S. Sigma company product, face with preceding and be made into 4% solution with normal saline.
Laboratory animal: regular grade SD rat, male, body weight 280g-350g, natural drug Engineering Technical Research Centre zoopery center, Shandong Province provides.The quality certification number: No. 200106005, Shandong kinoplaszm word.
(2) method and result:
Animal is divided into sham operated rats (normal saline) at random, model control group (normal saline), nimodipine group (Nim, 1.0mg/kg), Carthamus yellow small dose group (1.5mg/kg), dosage group (3.5mg/kg) in the Carthamus yellow, the heavy dose of group of Carthamus yellow (45mg/kg), total salvianolic acid small dose group (2mg/kg), dosage group (3.5mg/kg) in the total salvianolic acid, the heavy dose of group of total salvianolic acid (60mg/kg), compositions low dose [Carthamus yellow (1.5mg/kg)+total salvianolic acid (2mg/kg)], every group of 1O of compositions heavy dose [Carthamus yellow (45mg/kg)+total salvianolic acid (60mg/kg)] is only.After the fasting 12 hours, and chloral hydrate (350mg/kg, i.p.) anesthesia separates right carotid, and folder closes in the neck, common carotid artery, external carotid artery proximal part and distal end ligation, cut off the centre.The external carotid artery free-end is pulled to internal carotid artery in alignment, bolt line (selecting diameter 0.24mm nylon wire for use, length 5.0cm) is inserted into intracranial by external carotid artery, stop when meeting slight resistance, insertion depth is about 2cm.Ligation external carotid artery opening, and open the common carotid artery bulldog clamp, the disinfection and stitching wound causes right side middle cerebral artery ischemia model; Sham operated rats is only carried out the separation (above experiment is all carried out at 23 ℃~25 ℃) of right carotid, internal carotid artery, external carotid artery.Each treated animal intravenous injection relative medicine of postoperative.Press document [Liu Xiaoguang, Xu Lina, a kind of rat brain medium-sized artery model that can estimate thrombolytic and anti-thrombolytic after 24 hours, Acta Pharmaceutica Sinica, 1995,30:662] described method and standard is observed and the behavior disorder of record rat: (A) carry the Mus tail and observe forelimb flexing situation, stretch to ground as two forelimb symmetries, count 0 fen, the wrist flexing occurs as operation offside forelimb and count 1 fen, the elbow flexing is counted 2 fens, the shoulder inward turning is counted 3 fens, existing wrist flexing and/or elbow flexing have shoulder inward turning person again, count 4 fens.(B) animal is placed on the plane earth, push away both shoulders respectively, check resistance to side shifting.As bilateral resistance equity and strong, count 0 fen, as resistance descender when the operation offside promotes, according to decline degree difference be divided into gently, in, weigh three degree, count 1,2 and 3 fen respectively.(C) the two forelimbs of animal are put on the wire netting, observed the muscular tension of two forelimbs.Two muscle of anterior limb tension force equities and strong person count 0 fen.Count 1,2 and 3 fen according to operation offside muscular tension decline degree difference equally.(D) animal has ceaselessly to a side person of turn-taking, and counts 1 fen.According to the standard scoring, full marks are 11 minutes, and mark is high more, and expression animal behavior obstacle is serious more.
Put to death rat behind the behavior scoring, get brain, remove olfactory bulb, cerebellum and low brain stem, crownly be cut into 5, the brain sheet takes on a red color after normal structure is dyed with red tetrazolium (TTC) dyeing, and blocking tissue is white in color, taking a picture in dyeing back, asks the infarct size ratio with Chinese Aero-Space university pathological image analysis software.Data are used
Expression is carried out statistical procedures with F check between group.
The result is as shown in table 1, and ischemia is after 24 hours, and the model group rat shows tangible behavior disorder, and tangible kitchen range shape ischemic region also appears in rat cerebral tissue, reaches about 25% of full brain.
1.5mg/kg Carthamus yellow and 2mg/kg total salvianolic acid group are failed to improve the rat behavior obstacle, are reduced ischemic areas; 3.5mg/kg Carthamus yellow and 3.5mg/kg total salvianolic acid group have all been improved the rat behavior obstacle to a certain extent, have been reduced ischemic areas.
Compositions small dose group [Carthamus yellow (1.5mg/kg)+total salvianolic acid (2mg/kg)] is improved the rat behavior obstacle very significantly, is reduced ischemic areas, compare p<0.01 with model group, and significant difference (P<0.05) is more also arranged with 3.5mg/kg Carthamus yellow, 3.5mg/kg total salvianolic acid.Carthamus yellow and total salvianolic acid have synergism in the prompting compositions.
Compositions heavy dose [Carthamus yellow (45mg/kg)+total salvianolic acid (60mg/kg)] is improved the rat behavior obstacle extremely significantly, is reduced ischemic areas, compares P<0.001 with model group.
The influence that table 1 Carthamus yellow, total salvianolic acid, compositions are damaged the rat local cerebral ischemia
Group |
Behavior disorder |
Ischemic areas (%) |
The heavy dose of group of the heavy dose of group of dosage group total salvianolic acid composition small dose group composition in the heavy dose of group of the dosage group carthamin yellow total salvianolic acid small dose group total salvianolic acid in the sham-operation group model control group Nim group carthamin yellow small dose group carthamin yellow |
0 11.50±1.31 7.15±2.66
**10.22±3.16 8.80±1.07
*6.86±3.17
**10.03±3.58 8.79±2.42
*6.58±1.96
**6.67±2.35
**#△ 5.03±1.55
*** |
0 25.41±4.22 17.33±5.68
**22.38±8.95 19.23±2.45
*12.91±4.32
**23.14±6.27 19.56±3.75
*12.37±4.48
**13.34±7.56
**#△ 10.22±2.56
*** |
Compare with model control group
*P<0.05,
*P<0.01,
* *P<0.001.
Compare #P<0.05 with dosage group in the Carthamus yellow
Compare with dosage group in the total salvianolic acid
△P<0.05
Test example 2: different proportioning pharmaceutical compositions are to the influence of rat local cerebral ischemia damage
Carthamus yellow, total salvianolic acid, compositions: by preparation example 2 preparations.
Red tetrazolium: U.S. Sigma company product, face with preceding and be made into 4% solution with normal saline.
Laboratory animal: regular grade SD rat, male, body weight 280g-350g, natural drug Engineering Technical Research Centre zoopery center, Shandong Province provides.The quality certification number: No. 200106005, Shandong kinoplaszm word.
(2) method and result:
Animal is divided into sham operated rats (normal saline) at random, model control group (normal saline), compositions A[Carthamus yellow (10mg/kg)+total salvianolic acid (0mg/kg)], compositions B[Carthamus yellow (9.5mg/kg)+total salvianolic acid (0.5mg/kg)], compositions C[Carthamus yellow (8mg/kg)+total salvianolic acid (2mg/kg)], compositions D[Carthamus yellow (5mg/kg)+total salvianolic acid (5mg/kg)], compositions E[Carthamus yellow (2mg/kg)+total salvianolic acid (8mg/kg)], composition F [Carthamus yellow (0.5mg/kg)+total salvianolic acid (9.5mg/kg)], compositions G[Carthamus yellow (0mg/kg)+total salvianolic acid (10mg/kg)] 10 every group.Make rat local cerebral ischemia model and carry out neural behavior scoring, the dyeing of brain sheet by method shown in the experimental example 1.
Data are used
Expression is carried out statistical procedures with F check between group.
The result is as shown in table 2, and ischemia is after 24 hours, and the model group rat shows tangible behavior disorder, and tangible kitchen range shape ischemic region also appears in rat cerebral tissue, reaches about 25% of full brain.The pharmaceutical composition (10mg/kg) of different proportionings all has significant protective effect (P<0.05 or P<0.01) to rat cerebral ischemia.
The different proportioning pharmaceutical compositions of table 2 are to the influence of rat local cerebral ischemia damage
Group |
Behavior disorder |
Ischemic areas (%) |
Sham-operation group model control group composition A composition B composition C composition D composition E composition F composition G |
0 10.6±1.1 9.1±1.2
* 8.4±1.1
** 7.0±0.8
**#
△ 7.2±0.7
**#
△ 7.1±0.9
**#
△ 8.5±0.8
* 8.9±0.7
* |
0 25.6±4.5 22.9±2.1
* 22.5±2.1
* 18.2±1.8
**#
△ 20.1±1.4
**#
△ 21.2±1.3
**#
△ 22.3±1.6
* 22.6±1.1
* |
Compare with model control group
*P<0.05,
*P<0.01.
Compositions A group is #P<0.05 relatively
Compositions G organizes relatively
△P<0.05
Test example 3: Carthamus yellow, total salvianolic acid, compositions are to the influence of rat heart muscle ischemic injuries
(1) material:
Carthamus yellow, total salvianolic acid, compositions: by preparation example 2 preparations.
Painstaking effort pellet (compound Salviae Miltiorrhizae) injection: Guizhou Shenqi Pharmaceutical Co., Ltd.
Chlorination nitro blue tetrazolium (N-BT) is provided by Military Medical Science Institute medical supply station.
Laboratory animal: regular grade Wistar rat, male, body weight 280g-350g, natural drug Engineering Technical Research Centre zoopery center, Shandong Province provides.The quality certification number: No. 200106005, Shandong kinoplaszm word.
(2) method and result:
Animal is divided into sham operated rats (normal saline) at random, model control group (normal saline), FUFANG DANSHEN ZHUSHEYE group (DS, 10mg/kg), Carthamus yellow small dose group (1.5mg/kg), dosage group (3.5mg/kg) in the Carthamus yellow, the heavy dose of group of Carthamus yellow (45mg/kg), total salvianolic acid small dose group (2mg/kg), dosage group (3.5mg/kg) in the total salvianolic acid, the heavy dose of group of total salvianolic acid (60mg/kg), compositions low dose [Carthamus yellow (1.5mg/kg)+total salvianolic acid (2mg/kg)], 10 every group of compositions heavy doses [Carthamus yellow (45mg/kg)+total salvianolic acid (60mg/kg)].After the fasting 12 hours, limbs II lead electrocardiogram is surveyed in ip urethane (1.2g/kg) anesthesia.Cut off left front fur, iodine tincture and alcohol disinfecting, along left border of sternum 1cm place, cut off thoracic wall muscle and two ribs, open the thoracic cavity rapidly, expose heart, the ligation left coronary artery is put back to heart immediately between arterial cone and left auricle, squeezes the thoracic cavity air, use the mosquito forceps closed-chest, cause Model Rats with Acute Myocardial Ischemia.Each treated animal intravenous injection relative medicine of postoperative.1.5h, 3h electrocardiogram before the record administration and after the administration take out heart behind the 6h, after cleaning with cold saline, and-20 ℃ of refrigerator freeze overnight.Next day, refrigerated heart is cut into 5 by ligation place to apex uniform thickness, immerse in the freshly prepared 0.5%NBT phosphate buffer (pH 7.4).37 ℃ of water-bath jolting 10~15min.Blot the dyeing liquor of slice surface with filter paper, separate the coloured portions and the part of being unstained, weigh the compute infarct size.Infarct size (%)=infarction part weight/(non-infarction part weight+infarction part weight) * 100%.
The result is as shown in table 3, and myocardial ischemia is after 6 hours, and tangible kitchen range shape ischemic region appears in the model group rat heart muscle, reaches about 25%.1.5mg/kg Carthamus yellow and 2mg/kg total salvianolic acid group are failed to improve the rat electrocardio, are reduced ischemic areas; 3.5mg/kg Carthamus yellow and 3.5mg/kg total salvianolic acid group all reduce rising, the minimizing ischemic areas of the limb lead electrocardiogram J point that is caused by myocardial ischemia to a certain extent; Compositions small dose group [Carthamus yellow (1.5mg/kg)+total salvianolic acid (2mg/kg)] reduces rising, the minimizing ischemic areas of limb lead electrocardiogram J point very significantly, compare P<0.01 with model group, and significant difference (P<0.05) is more also arranged with 3.5mg/kg Carthamus yellow, 3.5mg/kg total salvianolic acid; Compositions heavy dose [Carthamus yellow (45mg/kg) total salvianolic acid (60mg/kg)] reduces rising, the minimizing ischemic areas of limb lead electrocardiogram J point extremely significantly, compares P<0.001 with model group.
Table 3 Carthamus yellow, total salvianolic acid, compositions are to the influence of rat heart muscle ischemic injuries
Grouping |
Infarct size (%) |
The J point raises (10 * mv) |
Normally |
1.5h |
3h |
The heavy dose of group of the heavy dose of group of dosage group total salvianolic acid composition small dose group composition in the heavy dose of group of the dosage group carthamin yellow total salvianolic acid small dose group total salvianolic acid in the model control group danshen injections group carthamin yellow small dose group carthamin yellow |
25.6±4.3 17.8±6.2
*22.5±8.5 19.4±3.5
*12.8±4.5
**23.3±6.4 19.7±3.5
*12.6±4.5
**13.6±7.8
**#△ 10.5±2.7
*** |
0.8±0.6 0.8±0.6 0.7±0.6 0.7±0.5 0.7±0.7 0.8±0.7 0.7±0.6 0.8±0.6 0.8±0.5 0.7±0.6 |
6.7±2.3 6.0±1.5
*6.5±1.3 5.8±1.4
*5.0±1.6
**6.4±1.5 5.7±1.8
*4.9±2.1
**4.7±1.6
**#△ 4.3±1.7
*** |
6.1±1.4 5.1±1.3
*5.8±1.9 5.2±1.8
*4.3±2.0
**5.7±2.1 5.1±1.7
*4.1±1.8
**4.0±1.6
**#△ 3.7±1.9
*** |
Compare with model control group
*P<0.05,
*P<0.01,
* *P<0.001.
Compare #P<0.05 with dosage group in the Carthamus yellow
Compare △ P<0.05 with dosage group in the total salvianolic acid