CN1145230A - High content ginko leaves flavone lactone extract and its prepn. method - Google Patents
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Abstract
A gingko leaf-flavone lactone extract for protecting cardiovascular organism and improving its function contains total flavone equal to or greater than 44% (flavone glucoside equal to or higher than 24%), lactone equal to or greater than 6% and controlling ginkgolic acid equal to or less than 10 ppm.
Description
The invention belongs to high content ginko leaves flavone lactone extract and preparation method thereof.
Semen Ginkgo (Gin kgo biloba L.) is a Ginkgoaceae Ginkgo Semen Ginkgo kind of plant.Folium Ginkgo is the conventional Chinese medicine material, have the gas of astringing the lung, relieving asthma cough, the turbid function of leukorrhagia stopping, can be used for diseases such as cough and asthma due to lung deficiency, coronary heart disease, angina pectoris.Active component in the Folium Ginkgo mainly is flavonoid and lactone, and they have the obvious treatment effect to improving cardiovascular, cerebrovascular and peripheral vessels.
The medical science man has done a large amount of research and developments to Folium Ginkgo both at home and abroad, find mainly to contain flavone compound in the Folium Ginkgo, flavone, flavonol and glycoside thereof are wherein arranged, catechin and bisflavones, next is the lactone that contains sesquiterpene and diterpene, also has a spot of organic acid, alkaloid and other chemical compound in addition.At present existing many Folium Ginkgo extractum preparations, but because preparation technology's difficulty, thereby this class preparation only limits to contain flavonoid glycoside 〉=24%, and a certain amount of lactone to the requirement of content and quality, ignored other effective flavone active component, do not collect free flavone; In addition, existing preparation does not carry out limit examine to the ginkgoic acid that is easy to generate zest and allergen matter yet.So existing Folium Ginkgo extractum preparation has been subjected to certain restriction on drug effect.
The objective of the invention is to develop the multiple effective flavone active ingredient in the Folium Ginkgo, develop a kind of high content ginko leaves flavone lactone extract.
The invention discloses a kind of high content ginko leaves flavone lactone extract, have another name called " ginkgo flavone and lactone " (EGB50), main component content is as follows: total flavones 〉=44%, (wherein flavonoid glycoside 〉=24%), lactone 〉=6%, control ginkgoic acid≤10ppm.
High content ginko leaves flavone lactone extract pharmacological research proof of the present invention has than the significant protection body, improves function the heart, cerebrovascular.
One, high content ginko leaves flavone lactone extract is to the therapeutical effect research of Cor Canitis muscle infarction
1, for test agent:
High content ginko leaves flavone lactone extract (EGB50) is a light brown powder shape solid, and it is standby to be made into the suspension of 6mg/ml and 20mg/ml concentration with 1% tragcanth during use.
2, experimental technique:
It is that 30mg/kg and 100mg/kg are oral that dosage is selected in this experiment for use.Positive controls selects for use nifedipine 5mg/kg oral.
3, experimental procedure:
With Canis familiaris L. with 3% pentobarbital sodium 30mg/kg intraperitoneal anesthesia, per os inserts No. 12 stomach tubes and makes administrable then, per os inserts anesthesia endotracheal tube (Curity simultaneously, internal diameter 9.55mm, Thailand produces), connect artificial respirator, with 30 times/minute frequencies, practiced artificial respiration than 1.5: 1 during breathing, regulating tidal volume is 15-20cmH to breathing malleation
2O.Animal lain on the left side be fixed on the operating-table, separate femoral vein in right side groin place, insert conduit and make transfusion usefulness, open breast in left side thoracic wall the 4th, the 5th intercostal, and cut off the 5th rib, and open the thoracic cavity with dilator, cut off pericardium apart from vagus nerve 2cm place along neural trend, then pericardium is sutured in thoracic wall, fully exposes heart; At the free coronary artery of left anterior descending coronary artery the 3rd branch root, its underpass, to sew up in heart surface with cardiovascular not damaged line sewing needle by four jiaos of made 30 multiple spot epicardial leads of document, electrode spacing 8mm, trace normal limb lead I, II, III and augmented limb lead aVR, aVL, aVF6 synchronously and lead, trace 30 electrocardiograms in surface then, electrocardiogram calibration 8mm/mv when tracing limb lead, the epicardial electrogram calibration is 1mm/mv, chart speed 25mm/s.Preceding 2 minutes of ligation is from femoral vein iv lignocaine 5mg/kg, then with the Dextrose and Sodium Chloride Inj. continuous drip that adds lignocaine 0.2mg/ml.1 minute, 10 member record epicardial electrogram after the ligation, be administration with 10 minutes before.In ligation after 10 minutes, gavage test specimen from stomach tube by the 5ml/kg volume, negative control group gavages normal saline with volume, respectively at administration 0.5,1,1.5,2,2.5 3 hour record epicardial electrogram are measured the high summation of each point ECG ST section (the cardiac diagnosis lead number (N-ST) of value of ∑-ST) and ST section rising＞2mv.Put to death animal at the 3rd hour, take out heart rapidly, weigh up weight whole-heartedly, cut atrium and right ventricle, weigh up left ventricular mass, put into the refrigerator quick-freezing 1 hour, under the coronary ligation line, parallel coronary sulcus, heavy back such as ventricle are cut into 5, put into 0.5% nitro blue tetrazolium (N-BT) solution, in 37 ℃ of water bath with thermostatic control jolting dyeing 10 minutes, normal myocardium dyed and is skipper, the infarct cardiac muscle is not colored as light yellow, the careful infraction part of downcutting weighs up infarct weight, calculates infarct weight and accounts for the percentage ratio that reaches left ventricular mass whole-heartedly.
4, result:
1. to the influence of degree of myocardial ischemia:
After the ligation 10 minutes, (∑-ST) obviously raise and reaches 180.4 ± 56.7mv the ECG ST section summation.After the administration 30 minutes, even (∑-ST) highly significant descends Folium Ginkgo extractum 30mg/kg group degree of myocardial ischemia, 201 ± 59mv reduces to 147 ± 67mv before the administration, 60 ± 29mv descends, with before the administration and the equal highly significant of normal saline group comparing difference (P＜0.01), the administration of Folium Ginkgo extractum 100mg/kg group after 30 minutes also highly significant reduce degree of myocardial ischemia, (∑-ST) reduce to 97 ± 44mv from 199 ± 85mv, 102 ± 40mv descends, with before the administration and the relatively more equal P of matched group＜0.01, degree of myocardial ischemia continues to descend after two groups, continue to administration action time after 3 hours, Folium Ginkgo extractum high dose group reduces the effect of degree of myocardial ischemia greater than low dose group, but two group difference nonsignificances (P＞0.05).Positive control nifedipine group is in administration after 30 minutes, ∑-ST descends, compare significant difference (P＜0.05) with physiology saline group, but with comparing no significant difference (P＞0.05) before the administration, to 1.5 hours, produce significant difference (P＜0.05) with comparing before the administration, with comparing then difference highly significant (P＜0.01).Negative control group, give normal saline after ∑-ST continues rising, to 1 hour near peak value, but with comparing nonsignificance (P＞0.05) before the administration.Experimental result sees Table one.
Experimental result shows that the oral degree of myocardial ischemia that the Canis familiaris L. coronary ligation is caused of " EGB50 " 30 and 100mg/kg has significant therapeutic effect.
2. to the influence of myocardial ischemia scope (N-ST):
After the ligation 10 minutes, the N-ST meansigma methods was 25.6 ± 2.6 points.
Oral 30 minutes of " EGB50 " 30 and 100mg/kg, N-ST reduces to 21.3 ± 3.0 declines 3.0 ± 1.5 and reduces to 20.0 ± 4.5 from 26.2 ± 2.5 from 24.3 ± 3.4 respectively and descends 6.2 ± 3.7, with comparing before the administration, difference highly significant (P＜0.01), compare with matched group, significant difference (P＜0.05), N-ST continues slowly to descend after 1 hour, with reaching matched group before the administration relatively, the equal highly significant of difference (P＜0.01).Myocardial ischemia scope decline acting duration surpasses 3 hours, and the action intensity high dose group is greater than low dose group, but there was no significant difference (P＞0.05).Behind oral 1 hour of the nifedipine, the myocardial ischemia scope with administration before and matched group compare, highly significant descend (P＜0.01) appears.Normal saline group myocardial ischemia scope does not have significant change (P＞0.05) in time.Experimental result sees Table two.
Experimental result shows that " EGB50 " 30 and 100mg/kg are oral to reduce the myocardial ischemia scope very significantly.
3. high content ginko leaves flavone lactone extract is learned the quantitative tissue of myocardial infarct size and is detected influence:
Show that with N-BT dyeing " EGB50 " is consistent with the epicardial electrogram measurement result to the influence of myocardial infarct size." EGB50 " 30 and 100mg/kg group all can reduce myocardial infarct size, the infarct/whole-heartedly and the ratio of infarct/left chamber are with matched group relatively, descend all extremely significantly (P＜0.01), the effect of 100mg/kg group is organized greater than 30mg/kg, and two groups relatively there were significant differences (P＜0.05).The nifedipine group also reduces infarction size (P＜0.01) very significantly.Experimental result sees Table three.
Two, high content ginko leaves flavone lactone extract (EGB50) is to the influence of coronary ligation Canis familiaris L. isolated culture myocardial oxygen consumption.
1, for test agent: EGB50 and nifedipine, the same.
2, experimental technique:
With laboratory animal, after administration, put to death in 3 hours, take out heart rapidly, in low temperature Tai Shi (Tyrodes) liquid, wash most congestion, core muscle infarction district center position and normal myocardium of left ventricle tissue each one, on the ice bath dish, cut into slices, on electronic balance, accurately take by weighing 60mg, put into the 10ml glass syringe respectively, add freshly prepared myocardium Incubating Solution (tyrode's solution) 5.0ml, drain air, seal outlet with rubber cap, balance is 30 minutes in 37 ℃ of waters bath with thermostatic control, and often rotates.The Incubating Solution partial pressure of oxygen is measured with Corning161 type blood gas analyzer, and calibrating gas suppressed zero and standard value are used in the instrument preheating before measuring after 1 hour.During experiment, measure with fresh Incubating Solution earlier and hatch preceding partial pressure of oxygen 2 times, measure fresh Incubating Solution partial pressure of oxygen 2 times again after sample determination is intact, totally 4 measured values are average as hatching preceding partial pressure of oxygen with front and back, each tissue incubation's liquid was surveyed secondary respectively after sample had been hatched, average as hatching the back oxygen partial pressure value, the two difference is this cardiac muscular tissue's oxygen consumption, represents with mmHgO.
3, experimental result:
Experimental result sees Table four.
Normal saline matched group normal myocardium oxygen consumption highly significant is greater than infarcted myocardium (P＜0.01), and " EGB50 " and nifedipine group organize oxygen consumption not have significant difference (P＞0.05) for two kinds." EGB50 " 30mg/kg after po3 hour the normal myocardium oxygen consumption be 30.3 ± 2.2mmHgO, 100mg/kg is 26.8 ± 3.4mmHgO, 36.7 ± 2.9mmHgO compares with physiology saline group, equal highly significant (P＜0.01) descends, " EGB50 " 100mg/kg organizes normal oxygen consumption and the 30mg/kg group compares, and there were significant differences (P＜0.05).Nitre benzene heavy stone used as an anchor also significantly reduces normal myocardium oxygen consumption (P＜0.05).All there is not significant difference between each group of infarcted myocardium oxygen consumption.
Experimental result shows that " EGB50 " can significantly reduce the normal myocardium oxygen consumption, and action intensity and dosage positive correlation.
Statistical result shows that " EGB50 " can significantly reduce the normal myocardium oxygen consumption, and action intensity and dosage positive correlation.
The statistical test burst data t method of inspection.
3, conclusion:
Experimental result proves, high content ginko leaves flavone lactone extract of the present invention (EGB50) can significantly reduce the degree of myocardial ischemia of coronary ligation Canis familiaris L., dwindle the myocardial ischemia scope, action intensity and dosage positive correlation, it is consistent with the epicardial electrogram result that quantitative tissue is learned testing result, increase with medication dose, infarct area significantly reduces, and proves that the myocardial infarction that the Canis familiaris L. coronary artery ligation is caused has the obvious treatment effect.Experimental result also shows; should " EGB50 " make normal myocardium tissue oxygen consumption produce the decline of dose dependent; the effect of 30mg/kg group is significantly organized greater than nifedipine 5mg/kg; the effect of prompting " EGB50 " treatment myocardial infarction still can be by reducing oxygen consumption and then producing myocardium protecting action except that its direct coronary artery dilating circulation.Experimental result is also found, " EGB50 " 30mg/kg PO, and the improvement of epicardial electrogram degree of ischemia is organized greater than nifedipine 5mg/kg.
(n=6/group of the oral impact to CAL dog surface ecg ST lift-off value summation of table one " EGB50 "; X ± SD, mV) (mg/kg) 0.5 1 1.5 2 2.5 3 physiological saline 5ml/kg 148 ± 21 158 ± 29a, 186 ± 49a, 185 ± 48a, 190 ± 45a, 187 ± 45a, 186 ± 46a nifedipine, 5 169 ± 30 131 ± 42ae, 143 445ae, 133 ± 32be, 122 ± 29be, 122 ± 37be, 122 ± 35beEGB50,30 201 ± 59 147 ± 67cf, 138 ± 62cf, 131 ± 61cf, 126 ± 59cf, 116 ± 50cf, 108 ± 48cf before the administration of (h) sample behind the dosed administration
100 199±85 97±44cf 115±79cf 90±50cf 76±44cf 78±38cf 79±32cf
AP＞0.05, bP＜0.05, cP＜0.01 is with comparing before the administration
DP＞0.05, cP＜0.05, fP＜0.01 are with physiology saline group relatively
Table two " EGB50 " oral to coronary artery ligation Canis familiaris L. surface ecg ST section raise count summation influence (n=6 only/group, X ± SD, mV)
Behind the dosed administration before the administration of (h) sample
(mg/kg) 0.5 1 1.5 2 2.5 3 physiological saline 5ml/kg 24.7 ± 1.0 24.3 ± 1.0a, 25.2 ± 1.2a, 25.0 ± 1.1a, 24.3 ± 1.6a, 23.7 ± 2.3a, 24.0 ± 2.0a nifedipine, 5 27.2 ± 2.4 24.0 ± 3.2ad, 23.0 ± 2.8cf, 22.5 ± 2.3cf, 22.2 ± 1.9cf, 22.2 ± 1.8ce, 21.3 ± 2.0ceEGB50,30 24.3 ± 3.4 21.3 ± 3.0ce, 19.7 ± 3.1cf, 19.5 ± 3.6cf, 20.7 ± 3.0cf, 19.0 ± 3.3cf, 19.0 ± 2.8ce
100 26.2±2.5 20.0±4.5bf 19.2±5.1cf 18.7±4.9bf 17.5±4.8cf 17.7±5.0ce 17.5±3.6cf
AP＞0.05, bP＜0.05, cP＜0.01 is with comparing before the administration
DP＞0.05, eP＜0.05, fP＜0.01 are with physiology saline group relatively
Table three " EGB50 " is to N-BT dyeing mensuration myocardial infarct size
Influence (n=6 only/group, the infarct/left chamber of the group dosage infarct of X ± SD)/whole-heartedly
(mg/kg) (%) (%) normal saline 5ml/kg 12.7 ± 2. 19.5 ± 3.4 nifedipine 5 4.7 ± 1.0c 7.1 ± 1.3cEGB50 30 5.7 ± 0.9c 8.6 ± 1.3c
100 3.5 ± 1.8ce, 5.4 ± 2.9ceaP＞0.05, bP＜0.05, dP＞0.05 is compared with physiology saline group in cP＜0.01, eP＜0.05, fP＜0.01 is organized relatively with " EGB50 " 30mg/kg
Table four " EGB50 " is to coronary ligation Canis familiaris L. isolated culture myocardial oxygen consumption
Influence (n=6 only/group, X ± SD)
Dosage myocardial oxygen consumption (mmHgO) group
(mg/kg) normal myocardium infarcted myocardium normal saline 5ml/kg 36.7 ± 2.9 26.7 ± 4.7 nifedipine 5 33.3 ± 2.3b 30.3 ± 5.6aEGB50 30 30.3 ± 2.2c 30.2 ± 6.6a
100 26.8 ± 3.4ce, 27.2 ± 4.0adaP＞0.05, bP＜0.05, dP＞0.05 is compared with physiology saline group in cP＜0.01, eP＜0.05, fP＜0.01 is organized relatively with " EGB50 " 30mg/kg
Three, high content ginko leaves flavone lactone extract is to the protective effect (medicine is the same) of animal acute cerebral ischemia
1, to the influence of acute cerebral ischemia in rats:
Oral " EGB50 " 50 of rat, 200, behind continuous 8 days of the 500mg/kg, underwent operative is burnt vertebral artery, and close bilateral common carotid arteries with the bulldog clamp folder and cause cerebral ischemia, after 5 minutes, loosening bulldog clamp irritates blood flow again, reach the EEG after irritating again before and after the record ischemia, finding as a result: the matched group ischemia is irritated back EEG again and is recovered slowly, only return to 67% of normal level to 60 ', " EGB50 " 200mg/kg, the effect of 500mg/kg group is obvious, and irritating 10 ' EBG more just has obvious recovery, shows that " EGB50 " can obviously improve the EEG ischemic that causes behind the rat blocking-up bilateral common carotid arteries and change.(seeing Table five)
2, to the influence of Canis familiaris L. acute cerebral ischemia:
Animal is obeyed " EGB50 " 50mg/kg, 250mg/kg in advance, 10 days, get himself blood before the experiment and prepare blood clot, inject its common carotid artery and cause the cerebral embolism ischemia, measure cerebral blood flow (CBF) then, cerebral vascular resistance (CVR) and serum lactate dehydrogenase (SLD) (LDH) content are made positive control with ligustrazine.The result shows that the CBF that Canis familiaris L. oral " EGB50 " can significantly cause anti-cerebral ischemia reduces, and CVR increases, can obviously reduce the LDH release increase that cerebral ischemia causes, its effect and ligustrazine quite or better, and its effect is relevant with dosage, high dose " EGB50 " acts on and obviously is better than low dosage.Illustrate that " EGB50 " can improve cerebral circulation, has the better protect effect to acute cerebral ischemia.(seeing Table six, seven, eight) four, high content ginko leaves flavone lactone extract are to the influence of rabbit blood rheological parameters
" EGB50 " external, obviously reduces the viscoelasticity of rabbit blood with the concentration of 30 μ g/ml, 60 μ g/ml, slows down it and advance the speed, and prolongs the recalcification time of the viscoelastic amount of touching.In observing its experiment to the blood coagulation process influence, can see that also " EGB50 " of above-mentioned concentration can obviously prolong blood coagulation time, the freezing rate that slows down delays the process of whole blood coagulation." EGB50 " also has the obvious suppression effect to high and low concentration ADP and collagen-induced platelet aggregation, and can promote the depolymerization after low concentration ADP causes platelet aggregation.These results suggest " EGB50 " have prevention thrombosis and promote thromboclastic effect.(seeing Table nine, ten, 11,12)
Table five EGB50 is to the influence of rat cerebral ischemia EEG
Group dosage number of animals EEG current potential % (X ± SD)
Ischemia is irritated again
G/kg 5 10 30 60 contrasts 10 17.2 ± 12.3 46.6 ± 25.1 50.7 ± 21.8 60.7 ± 24.2 ligustrazine groups 0.15, (iv) 10 16.9 ± 6.60 66.3 ± 35.6 93.2 ± 16.7**, 93.1 ± 6.7EGB50 0.05, (po) 10 14.2 ± 10.5 61.3 ± 10.2 77.7 ± 20.4 852 ± 14,9EG,B50 0.20, (po) 10 27.1 ± 25.1 93.0 ± 36**, 100.0 ± 60**, 100.0 ± 6.0**EGB50 0.50, (po) 10 16.1 ± 13.8 83.0 ± 31 90.0 ± 16.7** 93.9 ± 16.3
* P＜0.05 of comparing with matched group, * * P＜0.01
Table six EGB50 is to the Pedis Canitis blood flow, (CBF) influence before the group N dosed administration approach ischemia CBF behind the ischemia, (ml/min.100g), (only), (mg/kg) 1 ' 5 ' 30 ' 60 ' 90 ' 120 ' contrast, 6 N.S i.v. 251.31 88.33 90.74 134.64 127.45 127.88 129.20
± 94.80 ± 33.18## ± 44.28## ± 73.49# ± 73.53# ± 73.15# ± 72.93# ligustrazine 6 20 i.v. 213.82 204.08 204.20 202.53 177.00 161.20 154.47
±68.39 ±62.90** ±65.02** ±59.44 ±47.88 ±44.16 ±38.37EGB50 6 50 p.o 232.95 179.03 201.02 208.35 202.40 192.49 176.92
±88.50 ±74.81* ±87.85* ±84.12 ±82.29 ±89.69 ±83.07EGB50 6 250 p.o. 169.85 136.63 156.37 166.05 152.75 148.80 140.17
Before ± 59.00 ± 38.25* ± 54.64* ± 80.01 ± 75.54 ± 71.69 ± 66.32 and the ischemia than #P＜0.05, ##P＜0.01 and contrast ratio * P＜0.05, * * P＜0.01
Table seven " EGB50 " Pedis Canitis vascular resistance (CVR) influence before the group N dosed administration approach ischemia CVR (KPa ml/min) .100g behind the ischemia, X ± SD (only) (mg/kg) 1 ' 5 ' 30 ' 60 ' 90 ' 120 ' contrast 6 N.S i.v. 0.06 0.14 0.14 0.12 0.12 0.12 0.12
± 0.03 ± 0.05## ± 0.04## ± 0.04# ± 0.04# ± 0.05 4# ± 0.04# ligustrazine 6 20 i.v. 0.08 0.06 0.06 0.08 0.08 0.09 0.09
±0.04 ±0.03** 0.03** ±0.02 ±0.02 ±0.02 ±0.02EGB50 6 50 p.o 0.07 0.08 0.08 0.08 0.08 0.08 0.10
±0.03 ±0.03* ±0.04* ±0.04* ±0.04 ±0.04 ±0.06EGB50 6 250 p.o. 0.08 0.09 0.09 0.09 0.10 0.11 0.11
Before ± 0.02 ± 0.01* ± 0.02* ± 0.03 ± 0.04 ± 0.05 ± 0.05 and the ischemia than #P＜0.05, ##P＜0.01 with according to than * P＜0.05, * * P＜0.01
Table eight " EGB50 " to dog serum lactic acid dehydrogenase (LDH) influence before the group N dosed administration approach ischemia LDH behind the ischemia (unit/1 00ml, X ± SD) (only) (mg/kg) 1 ' 5 ' 30 ' 60 ' 90 ' 120 ' contrast 6 N.S i.v. 187.6 520.6 530.5 588.8 615.0 710.6 850.6
± 3.8 ± 400.2 ± 432.8 ± 432.2* ± 456.8* ± 566.2* ± 618.0* ligustrazine 6 20 i.v. 180.7 230.7 221.0 216.1 207.4 231.3 258.8
±63.2 ±130.4 ±115.5 ±110.4 ±98.8 ±1?40.8 ±180.0EGB50 6 50 p.o 160.7 289.6 260.6 202.8 190.4 186.9 210.5
±94.2 ±180.8 ±158.2 ±100.6 ±90.8 ±88.8 ±110.4EGB50 6 250 p.o. 203.1 220.3 246.6 228.8 230.0 230.6 236.3
± 110.4 ± 128.6 ± 160.6 ± 130.6 ± 138.5 ± 120.0 ± 136.8 with ischemia before than * P＜0.05
Table nine " EGB50 " is to the viscoelastic influence of rabbit blood
Number of animals blood drug level Tg TG Ua (G) Ua (g) Gmax Gnax medicine n ug/ml mim dydc/cm
2Dydc/cm
2
* 10
3* 10
3Matched group 8 290 334 21.54 5.09 5.19 7.72
± 132 ± 132 ± 15.13 ± 1.62 ± 1.39 ± 1.90 ligustrazine 8 100 281 313 26.11 6.53 6.36 8.57
±137 ±132 ±14.73 ±2.87 ±2.62 ±3.12EGB50 8 30 344 366 19.24 4.36 4.34 6.47
±171 ±1.77 ±14.11 ±211 ±181 ±1.36EGB50 8 300 431 494 14.18 3.67 3.71 4.69
±2.48 ±2.57 ±11.39 ±1.46 ±1.80 ±1.32*EGB50 8 600 424 353 11.33 334 291 402
± 258 ± 282 ± 9.34 ± 1.02* ± 136* ± 1.76** compares * P＜0.05, * * P＜0.01 with matched group
Table ten " EGB50 " is to the influence of rabbit blood process of setting
A contraction rate medicine ug/ml min units/min min units/min contrasted 8 0.58 ± 0.07 26.78 ± 15.25 3.35 ± 0.94 6.27 ± 2.31 ligustrazines 8 10.0 0.72 ± 0.12 35.67 ± 15.67 3.02 ± 1.00 8.20 ± 3.73EGB50 8 5.0 0.80 ± 0.26 21.37 ± 10.6 34.12 ± 1.20 6.35 ± 2.65EGB50,8 30.0 1.13 ± 0.49* 18.58 ± 9.11 5. ± 1.23*, 4.12 ± 1.71EGB50,8 60.0 1.45 ± 0.74*, 15.08 ± 6.63 5.92 ± 1.33** 4.00 ± 1.00 with control group comparison * P＜0.05, * * P＜0.01 when the number of animals blood concentration was coagulated because of time solidification speed peak
Table ten EGB50 is to low concentration ADP (5 * 10
-7G/ml) induce
The inhibitory action of platelet aggregation
Plasma drug level is assembled percentage rate medicine g/m1 1min 2mmin 3mmin contrast 41.3 ± 6.5 51.6 ± 4.9 50.5 ± 8.4EGB50 1 * 10
-439.2 ± 5.64 6.5 ± 8.9 40.6 ± 8.4EGB50 2 * 10
-433.6 ± 4.0* 37.3 ± 4.7* 28.7 ± 10.4*EGB50 3 * 10
-425.5 data all use X ± SD to represent that (2) * compares P＜0.01 (3) number of animals n=8 with matched group in ± 7.0* 30.1 ± 7.4*1 15.4 ± 11.1* (1) table
Accumulative aggregating agent prepared therefrom half-inhibition concentration (ug/ml) the half depolymerization concentration (ug/ml) that influences of two pairs of different aggregating agent prepared therefrom induced platelets of table ten
EGB50 aspirin EGB505 * 10
-6G/mlADP 410.5 10.45 * 10
-7Numerical value is the meansigma methods of 8 animals in g/mlADP 445.0 409.0 collagens 281.7 tables
Table ten three ginkgo flavone and lactone and (EGB50) product comparison both at home and abroad
Project EGB50 Tebenin Tebonin protects peaceful
-forte
The powerful tebonin of tebonin
Total flavones 44%+---quality flavonoid glycoside 24%++++ standard lactone 6%+-+-
Ginkgoic acid＜10ppm+-+-drug effect cardiovascular system (Canis familiaris L.)+---the research cerebrovascular system+++-
(rat, Canis familiaris L.) toxicity acute toxicity test++++ the research long term toxicity test
Canis familiaris L.+++-
Rat+++-the safety research result of high content ginko leaves flavone lactone extract of the present invention is as follows: 1, the oral LD of acute toxicity
50About 15g/kg, lumbar injection LD
50Be 767mg/kg.2, the long term toxicity rat reaches 1100mg/kg for " EGB50 " maximum dose level, and continuous irrigation stomach 90 days is not seen obvious influence to rat appetite, activity situation and body weight, and obvious influence is not seen in inspections such as blood picture, urine biochemistry, histopathology yet.
The Beagle Canis familiaris L. reaches 600mg/kg for " EGB50 " high dose, and continuous 90 days, each treated animal was movable normal, and it is good to ingest, and hematology, serum biochemistry, the every index of routine urinalysis and the result of histopathologic examination are all with the matched group no significant difference.Drug withdrawal middle of the month of convalescent period, every index of each treated animal is all no abnormal.3, general pharmacology " EGB50 " does not have obvious influence to the spontaneous activity of mice.
" EGB50 " is to blood pressure heart rate, electrocardiogram and the respiratory frequency of Canis familiaris L.
, the degree of depth etc. all do not have obviously influence.
The above results shows that high content ginko leaves flavone lactone extract of the present invention has than the significant protection body, improves function the heart, cerebrovascular, can effectively improve degree of myocardial ischemia, reduce blood muscular tissue oxygen consumption, improve cerebrovascular EEG ischemic symptom, the antagonism cerebral blood flow reduces, obviously reduce blood visco elasticity, prolong blood coagulation time etc., thereby the prevention and the therapeutical effect of the heart, cerebrovascular disease all is better than existing product; All show safety, avirulence through toxicological security research, acute toxicity and long term toxicity.
Another object of the present invention provides the preparation method of high content ginko leaves flavone lactone extract of the present invention.
Adopted poisonous and hazardous flammable solvent in the existing Folium Ginkgo extractum preparation method, and technology is loaded down with trivial details, operation inconvenience, for example Chinese patent medicine magazine VOL14, NO6, the technology that " extracting the method for flavone compound from Folium Ginkgo " of report in 1992 introduced.And can not make high content ginko leaves flavone lactone extract with these methods.
The invention discloses a kind of preparation method of high content ginko leaves flavone lactone extract.
The preparation method of high content ginko leaves flavone lactone extract of the present invention is that Folium Ginkgo coarse powder water and hydrophilic solvent are extracted, and makes high content ginko leaves flavone lactone extract after concentrated, precipitation, filtration, column chromatography for separation, drying.
The concrete enforcement of the preparation method of high content ginko leaves flavone lactone extract of the present invention is that exsiccant ginkgo leaf powder is broken into coarse powder, cross 60 mesh sieves, water of doubly measuring with crude drug in whole weight 5-30 or hydrophilic solvent heating and refluxing extraction or merceration percolation extract, heat is carried 2-3 time, each 1-3 hour, or cold extraction 1 time, 48-88 hour, to be evaporated to proportion under 60 ℃ of conditions be 1.15-1.25 in being lower than with extracting solution then, the 1-5 times of water gaging dissolving that adds primary dose, left standstill 36-72 hour, filter, filtrate is not by being column chromatography 1-2 time of carrier, water and water one pure mixed solvent eluting again with the separating medium of ion-exchange group, collect eluent, after being lower than under 60 ℃ the condition concentrating under reduced pressure or spray drying, make high content ginko leaves flavone lactone extract.
Hydrophilic solvent described in the preparation method of high content ginko leaves flavone lactone extract of the present invention is aliphatic lower alcohol or lower ketones etc.
It is wherein said that what be used for column chromatography is not macroporous adsorbent resin, silica gel, aluminium oxide, polyamide, kieselguhr, active carbon or cellulose etc. with the separating medium of ion-exchange group.And these media can single use also can mix use.
Wherein said is aliphatic lower alcohol as the alcohol in the water-pure mixed solvent of column chromatography eluent, and water is the arbitrary proportion of 99-1: 1-99 with the ratio of alcohol.
High content ginko leaves flavone lactone preparation method of the present invention, easy and simple to handle, do not have harsh equipment requirements, be suitable for the scale suitability for industrialized production.
The high content ginko leaves flavone lactone extract that makes with the inventive method can be used as various oral formulations such as oral liquid, tablet, granule, hard capsule or soft capsule etc.; Active ingredient in the crude drug of injection such as liquid drugs injection or powder pin etc. and external preparation.
Example 1, preparation high content ginko leaves flavone lactone extract.
The dry coarse powder 1kg of Folium Ginkgo adds 70% ethanol 5kg, and reflux 2 hours is filtered, and repeats heat and carries totally 3 times.Merging filtrate, 50 ℃ are evaporated to proportion 1.25, add water 3kg, dissolving, left standstill 36 hours, filter, filtrate adds to macroporous adsorbent resin, and (HP 20,0.5kg) and polyamide (60 orders, 0.5kg) mixed column, respectively with water 1kg, 10% ethanol 1kg, 50% ethanol 2kg, 95% ethanol 2kg eluting, 10%, 50%, 95% ethanol elution is merged 50 ℃ of concentrating under reduced pressure, lyophilization, get Folium Ginkgo extract powder 0.017kg, contain total flavones 44.7% (wherein total flavone glucoside 27.8%), total lactone 6.8%, ginkgoic acid＜10ppm.Example 2, the dry coarse powder 10kg of Folium Ginkgo add 50% ethanol 60kg, and reflux 1 hour is filtered, and residue adds 50% ethanol 50kg, and reflux 1 hour is filtered.Merging filtrate, 60 ℃ are evaporated to proportion 1.2, add water 40kg dissolving, left standstill 72 hours, filter, filtrate add to macroporous adsorbent resin (C1300,10kg) post is respectively with water 10kg, 30% ethanol 10kg, 60% ethanol 10kg eluting, 60 ℃ of 30% ethanol elution are evaporated to nothing alcohol flavor, concentrated solution add to polyamide (60 orders, 10kg) post is respectively with water 10kg, 10% ethanol 10kg, 80% ethanol 10kg eluting, with water and 80% ethanol elution, and aforementioned 60% ethanol elution merges 60 ℃ of concentrating under reduced pressure, spray drying, get Folium Ginkgo extract powder 0.14kg, contain total flavones 45.6% (wherein total flavone glucoside 31.4%), total lactone 7.2%, ginkgoic acid＜10ppm.Example 3, the dry coarse powder 50kg of Folium Ginkgo adds 60% ethanol and mixed thoroughly moistening 4 hours, puts in the percolation bucket, with 60% ethanol 1000kg percolation 78 hours, merge percolate, 60% is evaporated to proportion 1.15, adds water 100kg dissolving, left standstill 48 hours, filter, filtrate adds to macroporous adsorbent resin, and (DA 201,50kg) post, respectively with water 50kg, 10% ethanol 50kg, 20% ethanol 50kg, 50% ethanol 100kg is eluting successively, merge 10% and 20% ethanol elution, 60 ℃ are evaporated to nothing alcohol flavor, and concentrated solution adds to polyamide column (60 orders, 30kg) post, water 50kg, 10% ethanol 50kg, 20% ethanol 50kg, 70% ethanol 100kg difference is eluting successively, with water and 70% ethanol elution and 60 ℃ of concentrating under reduced pressure of aforementioned 50% ethanol elution merging, spray drying gets Folium Ginkgo extract powder 0.6kg, contains total flavones 46.8% (wherein total flavone glucoside 30.1%), total lactone 6.8%, ginkgoic acid＜10ppm.The dry coarse powder 100kg of example 4, Folium Ginkgo adds 50% ethanol 1000kg, and reflux 2 hours is filtered, and residue is with 50% ethanol 1000kg, reflux 2 hours, filtration.Merging filtrate, 60 ℃ are evaporated to proportion 1.2, add water 300kg dissolving, left standstill 48 hours, filter, filtrate adds to macroporous adsorbent resin (DA201,30kg) and polyamide (60 orders, 20kg) mixed column is respectively with water 100kg, 30% ethanol 100kg, 60% ethanol 100kg, 90% ethanol 100kg is eluting successively, and 30%, 60%, 90% ethanol elution is merged, 60 ℃ of concentrating under reduced pressure, dry, pulverizing get Folium Ginkgo extract powder 1.5kg, contain total flavones 47.2% (wherein total flavone glucoside 24.8%), total lactone 6.3%, ginkgoic acid＜10ppm.
Claims (5)
1, a kind of high content ginko leaves flavone lactone extract, the Main Ingredients and Appearance that it is characterized in that this Folium Ginkgo extractum is for containing total flavones 〉=44%, (wherein flavonoid glycoside 〉=24%), lactone 〉=6%, ginkgoic acid＜10ppm.
2, a kind of preparation method of high content ginko leaves flavone lactone extract as claimed in claim 1, it is characterized in that this preparation method is that exsiccant ginkgo leaf powder is broken into coarse powder, cross 60 mesh sieves, water of doubly measuring with crude drug in whole weight 5-30 or hydrophilic solvent heating and refluxing extraction or the merceration filter method of filtering is extracted, heat is carried 2-3 time, each 1-3 hour, or cold extraction 1 time, 48-88 hour, to be evaporated to proportion under 60 ℃ of conditions be 1.15-1.25 in being lower than with extracting solution then, add 1-5 times of water gaging dissolving of primary dose, left standstill 36-72 hour, filter, filtrate is not by being column chromatography 1-2 time of carrier with the separating medium of ion-exchange group, water and water one pure mixed solvent eluting are again collected eluent, in being lower than concentrating under reduced pressure under 60 ℃ the condition, make high content ginko leaves flavone lactone extract after the drying.
3, the preparation method of high content ginko leaves flavone lactone extract according to claim 2 is characterized in that wherein said hydrophilic solvent is aliphatic lower alcohol or lower ketones.
4, the preparation method of high content ginko leaves flavone lactone extract according to claim 2 is characterized in that wherein said what be used for column chromatography is not macroporous adsorbent resin, silica gel, aluminium oxide, polyamide, kieselguhr, active carbon or cellulose etc. with the separating medium of ion-exchange group.And these media can single use also can mix use.
5, the preparation method of high content ginko leaves flavone lactone extract according to claim 2, it is characterized in that the wherein said alcohol that is used for the water-pure mixed solvent of column chromatography eluent is aliphatic lower alcohol, water is the arbitrary proportion of water: alcohol=99-1: 1-99 with the mixed proportion of alcohol.
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| US6030621A (en) * | 1998-03-19 | 2000-02-29 | De Long; Xie | Ginkgo biloba composition, method to prepare the same and uses thereof |
| CN1118510C (en) * | 2000-04-26 | 2003-08-20 | 中国石化上海石油化工股份有限公司 | Process for preparing mother particles of polypropylene resin containing gingko leaf extract |
| WO2007048333A1 (en) | 2005-10-26 | 2007-05-03 | Shanghai Biochip Co., Ltd. | The use of ginkgo biloba extract in preparation of a composition for lowering cholesterol |
| CN101194918B (en) * | 2007-05-09 | 2011-04-27 | 广西师范大学 | Technique for removing ginkgolic acid in folium ginkgo extract |
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| CN105412167A (en) * | 2015-11-27 | 2016-03-23 | 陕西嘉禾生物科技股份有限公司 | Method for extracting and separating ginkgo flavone and ginkgolide from ginkgo leaves |
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| CN102133243B (en) * | 2011-03-09 | 2013-06-05 | 上海诺德生物实业有限公司 | Folium ginkgo extract containing 6-hydroxykynurenic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1102331A (en) * | 1993-11-04 | 1995-05-10 | 梁欣 | Method for extracting ginkgo extract from ginkgo leaf |
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- 1995-09-15 CN CN95111763A patent/CN1073848C/en not_active Expired - Lifetime
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| US6030621A (en) * | 1998-03-19 | 2000-02-29 | De Long; Xie | Ginkgo biloba composition, method to prepare the same and uses thereof |
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| US6632460B2 (en) | 1998-03-19 | 2003-10-14 | Shanghai Xingling Sci. & Tech. Pharmaceutical Co., Ltd. | Ginkgo biloba composition, method to prepare the same and uses thereof |
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| WO2007048333A1 (en) | 2005-10-26 | 2007-05-03 | Shanghai Biochip Co., Ltd. | The use of ginkgo biloba extract in preparation of a composition for lowering cholesterol |
| CN101194918B (en) * | 2007-05-09 | 2011-04-27 | 广西师范大学 | Technique for removing ginkgolic acid in folium ginkgo extract |
| CN102295651A (en) * | 2011-07-01 | 2011-12-28 | 北京双鹤高科天然药物有限责任公司 | Extraction and separation method of general flavone and total lactones in ginkgo leaf |
| CN105412167A (en) * | 2015-11-27 | 2016-03-23 | 陕西嘉禾生物科技股份有限公司 | Method for extracting and separating ginkgo flavone and ginkgolide from ginkgo leaves |
| CN105412167B (en) * | 2015-11-27 | 2020-01-21 | 陕西嘉禾生物科技股份有限公司 | Method for extracting and separating ginkgetin and bilobalide from folium Ginkgo |
| CN110974856A (en) * | 2019-12-02 | 2020-04-10 | 晨光生物科技集团股份有限公司 | Method for controlling free flavone in ginkgo leaf extract |
| CN110974856B (en) * | 2019-12-02 | 2021-12-10 | 晨光生物科技集团股份有限公司 | Method for controlling free flavone in ginkgo leaf extract |
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| CN1073848C (en) | 2001-10-31 |
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