CN107243037A - Application of the field pennycress general flavone in treatment focal cerebral ischemia medicine is prepared - Google Patents
Application of the field pennycress general flavone in treatment focal cerebral ischemia medicine is prepared Download PDFInfo
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Abstract
The present invention relates to application of the field pennycress general flavone in treatment focal cerebral ischemia medicine is prepared, it can effectively solve to extract field pennycress general flavone from field pennycress, realize application problem of the field pennycress general flavone as sole active agent in treatment focal cerebral ischemia medicine is prepared, described field pennycress general flavone is, field pennycress is ground into coarse powder, aether backflow extracts degreasing, the field pennycress dregs of a decoction volatilize ether, plus the immersion of the ethanol of mass concentration 80%, refluxing extraction, filtering, decompression filtrate recycling ethanol is to without alcohol taste, plus distilled water is dispersed to the dispersion liquid equivalent to the 0.3g/mL containing crude drug amount, the type macroporous absorbent resins of AB 8 on dispersion liquid, distilled water is used successively, the ethanol of mass concentration 10%, the ethanol elution of mass concentration 80%, collect 80% ethanol eluate, ethanol is recovered under reduced pressure to without alcohol taste, freeze-drying, crush, obtain field pennycress general flavone, field pennycress general flavone of the present invention is effective for treatment focal cerebral ischemia, the new application and medical value of field pennycress are opened up, it is the big innovation on medicine.
Description
Technical field
The present invention relates to medicine, particularly a kind of field pennycress general flavone answering in treatment focal cerebral ischemia medicine is prepared
With.
Background technology
Focal cerebral ischemia refers to transient ischemic attack (TIA), and arteria carotis or Vertebro-basilar System occur of short duration
Property blood supply it is not enough, cause focal cerebral ischemia to cause burst, transience, invertibity neurological dysfunction.Breaking-out continues
Several minutes, generally recovered completely in 30 minutes, it is small more than 2 to leave the performance of slight neurologic impairment often, or CT and MRI is aobvious
Show brain tissue ischemia sign.TIA is apt to occur in 34~65 years old, and over-65s account for 25.3%, and male is more than women.Morbidity is unexpected, many
Fallen ill when Body Position Change, hyperactivity hyperkinesia, neck are rotated or bent and stretched suddenly.Morbidity absence of aura, there is transient nerve
System positions sign, and general unconscious obstacle lasts 5~20 minutes, can recurrent exerbation, it is but general completely extensive in 24 hours
It is multiple, no sequel.
Field pennycress (Classification system：Thlaspi arvense Linn.), Thlaspi, penny cress are called, is Papaverales herba thlaspis genus
Under plant, be herbaceous plant, pungent, hardship has strong foot odour taste, has clearing heat and detoxicating, removing blood stasis and expelling pus during for Chinese medicine infusion
The effects such as, for appendicitis, dysentery, enteritis, hepatitis, eye conjunctivitis, postpartum blood stasis stomachache, carbuncle swells furunculosis.But so far there are no
Application of the general flavone extracted from field pennycress in anti-ischemic therapy treats focal cerebral ischemia medicine.
The content of the invention
For above-mentioned situation, to overcome the defect of prior art, it is always yellow that the purpose of the present invention is just to provide a kind of field pennycress
Application of the ketone in treatment focal cerebral ischemia medicine is prepared, can effectively solve to extract field pennycress general flavone from field pennycress, real
Application problem of the existing field pennycress general flavone as sole active agent in treatment focal cerebral ischemia medicine is prepared.
The technical scheme that the present invention is solved is that a kind of field pennycress general flavone is in treatment focal cerebral ischemia medicine is prepared
Using described field pennycress general flavone is that field pennycress is ground into coarse powder, and aether backflow extracts degreasing, discards ether；Field pennycress
The dregs of a decoction volatilize ether, plus the immersion of the ethanol of mass concentration 80%, refluxing extraction, filtering, decompression filtrate recycling ethanol to without alcohol taste, plus
Distilled water is dispersed to AB-8 type macroporous absorbent resins on the dispersion liquid equivalent to the 0.3g/mL containing crude drug amount, dispersion liquid, first with distillation
Water elution, discards aqueous；After with the ethanol elution of mass concentration 10%, eluent is discarded；The ethanol elution of mass concentration 80% is used again,
80% ethanol eluate is collected, ethanol is recovered under reduced pressure to without alcohol taste, is freeze-dried, crushes, obtain field pennycress general flavone.
The present invention can effectively solve to extract field pennycress general flavone from field pennycress, and lack effective for treating focal brain
Blood, has opened up the new application and medical value of field pennycress, is the big innovation on medicine, there is significant economic and social benefit.
Embodiment
Embodiment below in conjunction with concrete condition for the present invention elaborates.
The present invention is in specific implementation, a kind of field pennycress general flavone answering in treatment focal cerebral ischemia medicine is prepared
With described field pennycress general flavone is that field pennycress was ground into the coarse powder of 20-30 mesh sieves, first adds the ether of 10 times of weight to return
Stream extracts 1h, and degreasing discards ether；The field pennycress dregs of a decoction volatilize ether, plus 10 times of weight of field pennycress, the ethanol of mass concentration 80%
0.5h, refluxing extraction 1h are soaked, filtering obtains first time filtrate；The dregs of a decoction add 10 times of weight of field pennycress, the second of mass concentration 80% again
Alcohol, refluxing extraction 1h, filtering obtains second of filtrate, merges filtrate twice, and ethanol is recovered under reduced pressure to without alcohol taste, plus distillation moisture
AB-8 type macroporous absorbent resins on the dispersion liquid equivalent to the 0.3g/mL containing crude drug amount, dispersion liquid are dissipated to, are first steamed with 2 times of column volumes
Distilled water is eluted, and discards aqueous；After with 4 times of column volumes, the ethanol elution of mass concentration 10%, eluent is discarded；Again with 6 times of cylinders
The long-pending, ethanol elution of mass concentration 80%, collects 80% ethanol eluate, and ethanol is recovered under reduced pressure to without alcohol taste, freeze-drying, powder
It is broken, obtain field pennycress general flavone, field pennycress general flavone mass content more than 51%.
The effect of field pennycress general flavone prepared by the present invention has anti-focal cerebral ischemia through experiment, effective for preparing
The medicine of focal cerebral ischemia is treated, is to treat the innovation on focal cerebral ischemia medicine, and through Patrinia scaniosaefolia general flavone to rat office
The influence experiment of stove cerebral ischemia re-pouring model achieves extraordinary advantageous effects, and relevant experimental data is as follows：
1 material
1.1 animal
Wistar rats, SPF grades, male is provided by Lukang Medical Co., Ltd., Shandong.Quality certification number：
37005400000023, animal experimental center credit number：SYXK (Henan) 2015-0005, ethics lot number：DWLL15010017,
Job qualification certificate number：201302001.
1.2 medicines and reagent
Patrinia scaniosaefolia general flavone of the present invention；
Nimodipine tablet, Shanxi Yabao Pharmaceutical Group Corp., lot number：140861；
NAOLUOTONG JIAONANG, Jilin Jinbao Pharmaceutical Co., Ltd., lot number：150401；
Physiological saline, Henan Shuan Hehuali pharmaceutcal corporation, Ltds, lot number C115052005-2；
Penicillin, Huabei Pharmaceutic Co., Ltd, lot number：F4013504；
Formalin, analyzes pure, Yantai City Chemical Co., Ltd., lot number in pairs：20150701；
Chloraldurate, Tianjin recovery fine chemistry industry research institute, lot number 20141025；
Medical ethanol (alcohol), big disinfectant preparation Co., Ltd of Xinxiang City three, lot number：20150520；
Red tetrazolium (TTC), Chinese East China Normal University chemical plant (Shanghai), lot number：04102711；
Disodium hydrogen phosphate, Tianjin Zhi Yuan chemical reagent Co., Ltd, lot number：20141110；
Sodium dihydrogen phosphate, the factory of Tianjin chemical reagent three, lot number：20110108；
ICAM-1 (ICAM-1) ELISA detection kit, R＆D companies, lot number：20150901A；
Interleukin-1 ' beta ' (IL-1 β) ELISA detection kit, R＆D companies, lot number：20150901A；
Interleukin-6 (IL-6) ELISA detection kit, R＆D companies, lot number：20150901A；
Tumor necrosis factor α (TNF-α) ELISA detection kit, R＆D companies, lot number：20150901A；
Bcl-2 correlation X protein (Bax) ELISA detection kits, R＆D companies, lot number：20150801A；
The B cell lymphoma factor 2 (Bcl-2) ELISA detection kit, R＆D companies, lot number：20150801A；
Cysteine proteinase-3 (Caspase-3) ELISA detection kit, R＆D companies, lot number：20150901A；
Bioengineering Research Institute, lot number are built up in Coomassie brilliant blue kit, Nanjing：20150821；
Superoxide dismutase (SOD) detection kit, Bioengineering Research Institute, lot number are built up in Nanjing：20150821；
MDA (MDA) detection kit, Bioengineering Research Institute, lot number are built up in Nanjing：20150901；
Bioengineering Research Institute, lot number are built up in ATP enzyme kit, Nanjing：20150826；
1.3 instrument
Line bolt, the dense Bioisystech Co., Ltd of West Beijing, production number：2838-A4；
Electronic balance：Shanghai Precision Scientific Apparatus Co., Ltd, model：FA2204B；
Electronic scale：Shanghai Precision Scientific Apparatus Co., Ltd, model：YP1201N；
Table-type low-speed autobalance centrifuge：Changsha Xiang Zhi centrifuges Instrument Ltd., model：TDL-40B；
Water bath with thermostatic control, Shanghai Yiheng Scientific Instruments Co., Ltd, model：HWS12；
ELIASA, BIO-RAD companies of the U.S., model：BIORAD-680；
Adjustable pipette, Shanghai Lei Bo Analytical Instrument Co., Ltd.
2 methods
2.1 modelings and administration
Healthy rat 98 is taken, male is normal to raise 3 days, weigh and be randomly divided into 7 groups, every group 14, respectively do evil through another person
Shu Zu ﹑ model groups, Nimodipine group, brain network lead to group, large, medium and small dosage Patrinia scaniosaefolia general flavone group.Nimodipine group (positive control
Medicine, dosage is 20mg/kg, equivalent to 10 times of clinical dosage), brain network leads to group, and (positive control drug, brain network leads to, to medicament
Measure as 500mg/kg, 10 times of quantity)；Large, medium and small dosage Patrinia scaniosaefolia general flavone group (200mg/kg, 100mg/kg, 50mg/
kg).Sham-operation group and model group difference gavage same volume physiological saline (each group gavage volume is 2ml/100g).Daily administration
1 time, successive administration 7d.
It can't help in 8 fasting of the 6th day evening after water, the 7th day 8 a.m. batch weighing, administration 1h, use 10% chloraldurate
(0.3ml/100g) intraperitoneal injection of anesthesia, hits exactly clip to the left in neck with eye scissors, successively separates left common carotid (CCA)
And it is standby to wear both threads, it is standby that separation external carotid artery (ECA) wears single line, ligatures external carotid artery, ligatures arteria carotis communis proximal part,
One brief summary of distal end tip-tap can cut off wide small of about 0.2mm through being defined in arteria carotis communis by line bolt at bifurcated about 5mm
Mouthful, line bolt is inserted, enters internal carotid through arteria carotis communis furcation, upwards deeply to bifurcated above 18-20mm, until there is resistance
Power, that is, block arteria cerebri media porch, ligatures and gently extracts bolt line after arteria carotis communis proximal part, 2h out to slightly resistance, realizes
Reperfu- sion, sets up middle cerebral artery occlusion-Reperfu- sion (MCAO) animal model.Sham-operation group only exposure left side blood vessel, does not do slotting
23~25 DEG C of room temperature is kept in line processing, surgical procedure.
2.2 Testing index and detection method
2.2.1 nervous symptoms score
After all rat Reperfu- sion 22h, Neurological deficits (Longa point systems), standards of grading are carried out：Impassivity work(
Symptom can be lacked, activity is normal：0 point；It is unable to full extension offside fore paw：1 point；Occur turn-taking to hemiplegia side when creeping：2 points；
During walking, body rolls to hemiplegia：3 points；Spontaneous it can not walk, the loss of consciousness：4 points；It is dead：5 points.Score as 0 point and 5 points
Person rejects；
2.2.2 brain tissue materials and processing
After rat broken end, brain tissue is stripped rapidly, is put into -20 DEG C of low temperature refrigerators and cools down 15min, take out brain tissue, go
Fall the remainder after olfactory bulb, cerebellum, low brain stem, being cut into 3 parts along coronal-plane, (Part I is preced with for best optic chiasma before brain
In face of shape at 1mm, Part II is that 1mm locates before and after optic chiasma coronal-plane, and about 2mm thin slice, Part III is optic chiasma hat
Brain tissue behind shape face at 1mm to tail end).
2.2.3 prepared by brain tissue homogenate
Part I brain tissue is taken, sagittal cuts left side brain tissue (plug wire bolt side), after physiological saline cleaning, assay balance
Its weight of precise, with physiological saline (ml)：Brain tissue (g)=9: the ice-cold physiological saline that 1 ratio is added, filling
10% brain homogenate is made of glass homogenizer in the large beaker of ice cube, 4 DEG C, 3000r/min centrifugation 10min take supernatant point
Dress, -20 DEG C of refrigerator Cord bloods determine IL-6, IL-1 β, TNF-α, Bcl-2, Bax, Casp-3, ICAM-1, ATP in brain homogenate
Enzyme, SOD, MDA level.
2.2.4 brain infarction area is determined
Part II brain section is taken to be put into rapidly so that in the 1%TTC solution of pH=7.2 phosphate buffers configuration, 37 DEG C are kept away
Light is incubated 15min, stirs therebetween once, in favor of the two-sided abundant dyeing of brain section.Brain piece is taken out after dyeing, 10% good fortune is positioned over
It is kept in dark place and takes pictures in your Malin's solution, the brain tissue of non-infarct is rose after dyeing, and the brain tissue of infarct is white.
Analyzed through image analysis system and calculate Infarct area, the percentage for asking it to account for the gross area.
2.2.5HE dyeing and immunohistochemical staining
Part III brain tissue, routine paraffin wax embedding carries out HE dyeing, and immunohistochemistry staining method carries out NGF and NF κ
Bp65 is dyed.
2.2.6 in brain tissue IL-6, IL-1 β, TNF-α, Bcl-2, Bax, Casp-3, ICAM-1 assay method
Cleaning Principle, operating procedure：With Serum Neuron Specific Enolase (NSE) in experiment one.
2.2.7 method is determined in Coomassie brilliant blue protein quantification side
Sample pre-treatments：10% brain tissue homogenate, 1 is pressed with physiological saline:4 are diluted to 2% tissue homogenate, to be measured.
| Blank tube | Standard pipe | Determine pipe | |
| Distilled water (mL) | 0.05 | ||
| 0.563/L protein standards liquid (mL) | 0.05 | ||
| Sample (mL) | 0.05 | ||
| Coomassie brilliant blue nitrite ion (mL) | 3.0 | 3.0 | 3.0 |
Add after the corresponding reagent of each pipe and mix, stand 10min, distilled water zeroing, 1cm optical paths at 595nm, survey each
Pipe OD values.
Calculation formula
2.2.8 the assay method of ATPase in Brain Tissue
Atpase assay Operations Sequence Chart in brain homogenate
The preparation of A, B, C intermixture before test
| Guan Hao | A pipe amount of liquid | B pipe amount of liquid | C pipe amount of liquid |
| Reagent one (μ l) | 130×(n+2) | 130×(n+2) | 90×(n+2) |
| Reagent two (μ l) | 40×(n+2) | 40×(n+2) | 40×(n+2) |
| Reagent three (μ l) | |||
| Reagent four (μ l) | 40×(n+2) | 40×(n+2) | 40×(n+2) |
| Reagent five (μ l) | 40×(n+2) | ||
| Reagent six (μ l) | 40×(n+2) | 40×(n+2) | 40×(n+2) |
| Mix reagent total amount (μ l) | 250×(n+2) | 250×(n+2) | 250×(n+2) |
| Often pipe answers liquid absorption (μ l) | 250 | 250 | 250 |
Enzymatic reaction
Mix, 3000-4000r/min centrifugation 10min take the μ l of supernatant 200 to determine phosphorus.
Mix, 37 DEG C of water-baths 30 minutes are cooled to room temperature, 660nm at, 1cm optical paths, distilled water returns to zero colorimetric.
Note：A pipes are control tube；B pipes are Na+K+- ATP enzyme pipe；C pipes are Mg++- ATP enzyme pipe；Standard pipe concentration is 0.5 μ
mol/ml
Calculation formula：
ATP vigor (μm olPi/mgprot/hour)=(determining pipe OD values-control tube OD values)/standard pipe OD in tissue
The homogenate proteins concentration (mgprot/ml) of sample extension rate * 6/ in value * standard pipes concentration × reaction system
2.2.9 in brain tissue SOD assay method
Measuring principle：Ultra-oxygen anion free radical can be produced after xanthine and xanthine oxidase interreaction, it is oxidized
Azanol formation nitrite, aubergine can be showed in the presence of developer, its absorbance is surveyed with ultraviolet specrophotometer.
SOD has single-minded inhibitory action to ultra-oxygen anion free radical, and the nitrite of formation is reduced, and with colorimetric method, determines the suction of pipe
Shading value is less than the absorbance of control tube, sets up formula by standard pipe measured value, calculates sample SOD vigor.
The measure of total SOD (T-SOD) vigor：
Mix, room temperature is placed 10 minutes, at wavelength 50nm, 1cm optical path cuvettes, distilled water zeroing, colorimetric.
SOD vigor=(control tube absorbance-measure pipe absorbance)/control tube absorbance/50%* is anti-in tissue homogenate
Answer protein concentration (mgprot/ml) in liquid cumulative volume/sampling amount (ml)/tissue
Mgprot is milligram albumen number.
2.2.10 in brain tissue MDA assay method
Measuring principle：Lipid peroxide degraded contains Bing bis- Chuo (MDA) in producing, MDA contracts with thiobarbituricacidα- (TBA)
Red product can be formed after conjunction, the product has maximum absorption band at 532nm.
Operation order
Tightened and taken out after test tube mouthful, 95 DEG C of water-bath 40min with antistaling film after mixing, flow water cooling, 3500-4000r/
Min centrifuges 10min.Taken with liquid-transfering gun at supernatant, 1cm optical paths 532nm, each pipe OD values are adjusted in distilled water zeroing.
MDA contents (nmol/mgprot)=(determining pipe absorbance-measure blank tube absorbance) * standard items are dense in tissue
Degree/(standard pipe absorbance-standard blank tube absorbance)/protein content (mgprot/ml)
3 statistical procedures methods
Data analysis carries out the statistical procedures of data information with the statistical packages of SPSS 19.0, and measurement data is with averagely
Number ± standard deviationRepresent, compare between each group and use one-way analysis of variance, the neat person's least significant difference of variance test
(LSD) method, heterogeneity of variance person is examined with Games-Howell methods, and ranked data are examined with Radit.
4 results
(result is shown in for the influence of 4.1 pairs of Focal Cerebral Ischemia-Reperfusion in Rats model death rates and clinic nerve function assay
Table 6)
Influence of the table 6 to the Focal Cerebral Ischemia-Reperfusion in Rats model death rate and clinic nerve function assay
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from Table 6, the death rate highest of model group, Nimodipine group, brain network lead to group, large, medium and small dosage Patrinia scaniosaefolia
The death rate of general flavone group rat decreases, and illustrates that administration each group can reduce Focal Cerebral Ischemia Reperfusion in various degree
The death rate of rat model, reduces brain tissue impairment, protects brain tissue.Compared with sham-operation group, model group rats nerve work(
Can missing scoring significantly rise (P<0.01), prompting modeling success；With model group ratio, brain network leads to group, Nimodipine group, heavy dose
The scoring of Patrinia scaniosaefolia general flavone group rat afunction significantly reduces (P<0.01) in, low dose of Patrinia scaniosaefolia general flavone group rat god
(P is substantially reduced through afunction scoring<0.05), illustrate that there is administration each group different degrees of improvement focal cerebral ischemia to fill again
Note the effect of rat brain neural status.
4.2 pairs of Focal Cerebral Ischemia-Reperfusion in Rats model brain homogenate IL-6, IL-1 β, influence (results of TNF-α content
It is shown in Table 7)
Table 7 is to Focal Cerebral Ischemia-Reperfusion in Rats model brain homogenate IL-6, IL-1 β, the influence of TNF-α content
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from Table 7, with sham-operation group ratio, focal cerebral ischemia-reperfusion injury rat model brain tissue IL-6, IL-1 β,
TNF-α level significantly rises (P<0.01) modeling success, is illustrated；With model group ratio, Nimodipine group, brain network lead to group, big agent
Amount Patrinia scaniosaefolia general flavone group rat cerebral tissue IL-6 levels significantly reduce (P<0.01) in, low dose of Patrinia scaniosaefolia general flavone group rat brain
Tissue IL-6 levels substantially reduce (P<0.05)；With model group ratio, Nimodipine group, brain network lead to group, large, medium and small dosage Patrinia scaniosaefolia
General flavone group rat cerebral tissue IL-1 β levels significantly reduce (P<0.01)；With model group ratio, Nimodipine group, brain network lead to group, big
Dosage Patrinia scaniosaefolia general flavone group rat cerebral tissue TNF-α level significantly reduces (P<0.01), middle dosage Patrinia scaniosaefolia general flavone group rat brain
Tissue T NF- alpha levels substantially reduce (P<0.05), low dose of Patrinia scaniosaefolia general flavone group rat cerebral tissue TNF-α level has reduction trend
(P ＞ 0.05).Illustrate that administration each group has reduction focal cerebral ischemia-reperfusion injury rat model brain tissue IL-6, IL-1 β, TNF-α
Level, reduce inflammation effect of the reaction to brain tissue impairment.
Influence (the result of 4.3 pairs of Focal Cerebral Ischemia-Reperfusion in Rats model brain homogenate Bax, Bcl-2, Casp-3 contents
It is shown in Table 8)
Influence of the table 8 to Focal Cerebral Ischemia-Reperfusion in Rats model brain homogenate Bax, Bcl-2, Casp-3 content
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from Table 8, with sham-operation group ratio, model group rats brain tissue Bax, Caspase-3 level is significantly raised
(P<0.01), the significantly raised (P of Bcl-2 levels<0.05) modeling success, is illustrated；With model group ratio, Nimodipine group, brain network lead to
Group, large, medium and small dosage Patrinia scaniosaefolia general flavone group rat cerebral tissue Bax levels significantly reduce (P<0.01)；With model group ratio, Buddhist nun is not
Horizon group, brain network lead to group, large, medium and small dosage Patrinia scaniosaefolia general flavone group rat cerebral tissue Bcl-2 levels and significantly raise (P<0.01)；
With model group ratio, Nimodipine group, brain network lead to group, big, middle dosage Patrinia scaniosaefolia general flavone group rat cerebral tissue Caspase-3 levels and shown
Write reduction (P<0.01), low dose of Patrinia scaniosaefolia general flavone group rat cerebral tissue Caspase-3 levels substantially reduce (P<0.05), explanation
Administration each group can increase in various degree suppresses apoptosis gene expression in Level In Rats With Focal Cerebral Ischemia model brain tissue, reduction promotees
Apoptosis gene is expressed, and suppresses Apoptosis, so as to protect brain tissue, alleviates symptoms of cerebral ischemia.
4.4 pairs of Focal Cerebral Ischemia-Reperfusion in Rats model brain infarction area percentages, shadows of brain homogenate ICAM-1 levels
Ring (the results are shown in Table 9)
Table 9 is to Focal Cerebral Ischemia-Reperfusion in Rats model brain infarction area percentage, the shadow of brain homogenate ICAM-1 levels
Ring
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from Table 9, with sham-operation group ratio, model group rats brain tissue ICAM-1 levels, brain infarction area percentage
Than significantly rise (P<0.01) modeling success, is illustrated；With model group ratio, it is total that Nimodipine group, brain network lead to group, heavy dose of Patrinia scaniosaefolia
Flavones group rat cerebral tissue ICAM-1 levels significantly reduce (P<0.01) in, low dose of Patrinia scaniosaefolia general flavone group rat cerebral tissue
ICAM-1 levels substantially reduce (P<0.05)；With model group ratio, Nimodipine group, brain network lead to group, heavy dose of Patrinia scaniosaefolia general flavone
Group rat cerebral infarction area percentage significantly reduces (P<0.01), middle dosage Patrinia scaniosaefolia general flavone group rat cerebral infarction area percentage
Substantially reduce (P<0.05), low dose of Patrinia scaniosaefolia general flavone group rat cerebral infarction area percentage has reduction trend (P ＞ 0.05), says
Bright administration each group has different degrees of reduction Focal Cerebral Ischemia Reperfusion rat model brain tissue ICAM-1 levels, reduces brain
The effect of infarct size.
4.5 pairs of Focal Cerebral Ischemia-Reperfusion in Rats model brain homogenate Na+-K+-ATP enzyme activities and Mg++-ATP enzyme activities
Influence (the results are shown in Table 10)
Table 10 is to Focal Cerebral Ischemia-Reperfusion in Rats model brain homogenate Na+-K+-ATP enzyme activities and Mg++-ATP enzyme activity
The influence of power
Note：With model group ratio, * * P<0.01, * P<0.05
As can be seen from Table 10, with sham-operation group ratio, model group rats brain tissue Na+-K+-ATP enzymes and Mg++-ATP enzymes
Vigor significantly reduces (P<0.01) modeling success, is illustrated；With model group ratio, Nimodipine group, brain network lead to group, heavy dose of Patrinia scaniosaefolia
General flavone group rat cerebral tissue Na+-K+-ATP enzyme activities significantly raise (P<0.01) in, low dose of Patrinia scaniosaefolia general flavone group it is big
Significantly raised (the P of murine brain Na+-K+-ATP enzyme activities<0.05)；Nimodipine group, brain network lead to group, large, medium and small dosage Patrinia scaniosaefolia
General flavone group rat cerebral tissue Mg++-ATP enzyme activities significantly raise (P<0.01), illustrate that administration each group can rise in various degree
High Focal Cerebral Ischemia Reperfusion rat model brain tissue ATP enzyme vigor.
The influence (the results are shown in Table 11) of 4.6 pairs of Focal Cerebral Ischemia-Reperfusion in Rats model brain homogenate MDA and SOD levels
Influence of the table 11 to Focal Cerebral Ischemia-Reperfusion in Rats model brain homogenate MDA and SOD level
Note：With model group ratio, * * P<0.01, * P<0.05
It can be seen from upper table 11 compared with sham-operation group, MDA contents are notable in model group rats brain tissue homogenate
Rise, SOD contents significantly reduce (P<0.01) modeling success, is illustrated；Compared with model group, brain network leads to group, Nimodipine group, big
MDA levels significantly reduce (P in dosage Patrinia scaniosaefolia general flavone group brain tissue<0.01) in, in low dose of Patrinia scaniosaefolia general flavone group brain tissue
MDA levels substantially reduce (P<0.05)；Brain network leads to SOD in group, Nimodipine group, big, middle dosage Patrinia scaniosaefolia general flavone group brain tissue
Level significantly raises (P<0.01), the significantly raised (P of SOD levels in low dose of Patrinia scaniosaefolia general flavone group brain tissue<0.05), prompting loses
Sauce general flavone can mitigate Focal Cerebral Ischemia rat model brain tissue peroxidization, protect brain tissue.
The shadow of 4.7 pairs of Focal Cerebral Ischemia-Reperfusion in Rats model brain tissue NGF, NF-KBp65 immuno positive expressions
Ring (the results are shown in Table 12)
Table 12 is to Focal Cerebral Ischemia-Reperfusion in Rats model brain tissue NGF, NF-KBp65 immuno positive expression
Influence
Note：With model group ratio, * * P<0.01, * P<0.05
With sham-operation group ratio, model group rats brain tissue NGF, NF-KBp65 immuno positive it can be seen from upper table 12
Expression significantly raises (P<0.01) modeling success, is illustrated；With model group ratio, brain network leads to group, Nimodipine group, large, medium and small
Dosage Patrinia scaniosaefolia general flavone group rat cerebral tissue NGF immuno positives expression significantly raises (P<0.01)；With model group ratio, brain
It is notable that network leads to group, Nimodipine group, heavy dose of Patrinia scaniosaefolia general flavone group rat cerebral tissue NF-KBp65 immuno positive expressions
Reduce (P<0.01), middle dosage Patrinia scaniosaefolia general flavone group rat cerebral tissue NF-KBp65 immuno positives expression substantially reduces (P
<0.05), low dose of Patrinia scaniosaefolia general flavone group rat cerebral tissue NF-KBp65 immuno positives expression has reduction trend (P ＞
0.05), illustrate that each administration group can promote cranial nerve cell NGF to express, suppress NF-KBp65 expression, prevention rat cranial nerve is thin
Born of the same parents are denatured or dead, so as to maintain cranial nerve normal function, mitigate the damage caused by cerebral ischemia re-pouring.
4.8 influence (the results are shown in Table 14) to the pathological change of Focal Cerebral Ischemia Reperfusion rat model brain tissue cortical area
To Focal Cerebral Ischemia Reperfusion rat model brain tissue cortical area Histopathological Studies (see annex 1)：Sham-operation
Group cerebral cortex is without oedema, and nerve cell is normal；Model group Neurons of Cerebral Cortex oedema, large stretch of neuronal necrosis, infarct
Area accounts for more than the 2/3 of cortex；The logical group cerebral cortex partial nerve edema of brain network, a small number of neuronal necrosis, infarct size
Within account for cortex 1/3；Nimodipine group cerebral cortex partial nerve edema, a small number of neuronal necrosis, infarct size is accounted for
Within the 1/3 of cortex；Heavy dose of Patrinia scaniosaefolia general flavone group Neurons of Cerebral Cortex oedema, a small number of neuronal necrosis, infarct size
Within account for cortex 1/3；Middle dosage Patrinia scaniosaefolia general flavone group Neurons of Cerebral Cortex oedema, a small number of neuronal necrosis, infarct face
Product is accounted within the 1/3~2/3 of cortex；Low dose of Patrinia scaniosaefolia general flavone group Neurons of Cerebral Cortex oedema, large stretch of neuronal necrosis,
Infarct size is accounted within the 1/3~2/3 of cortex.
Influence of the table 13 to Focal Cerebral Ischemia Reperfusion rat model brain tissue cortical area pathological change
"-" cerebral cortex is without oedema, and nerve cell is normal；"+" Neurons of Cerebral Cortex oedema, a small number of neurons are bad
Extremely, infarct size is accounted within the 1/3 of cortex；" ++ " Neurons of Cerebral Cortex oedema, neuronal necrosis in blocks, infarct size is accounted for
Within the 1/3~2/3 of cortex；" +++ " Neurons of Cerebral Cortex oedema, large stretch of neuronal necrosis, infarct size accounts for cortex
More than 2/3.
Examined through Ridit, with sham-operation group ratio, model group has significant statistical significance (P<0.01) model group, is illustrated
There is significant pathological change, modeling success in rat cerebral tissue cortical area；With model group ratio, brain network, which leads to group, Nimodipine group, to be had
Significant statistical significance (P<0.01)；Large, medium and small dosage Patrinia scaniosaefolia general flavone group has obvious statistical significance (P<0.05),
Illustrate the pathology damage for the improvement office's property made cerebral ischemia re-pouring rat model brain tissue cortical area that each administration group can be different degrees of
Degree, protects brain tissue.
The influence (the results are shown in Table 15) of 4.9 pairs of Focal Cerebral Ischemia-Reperfusion in Rats model brain tissue hippocampus pathological changes
It is as follows to Focal Cerebral Ischemia Reperfusion rat model brain tissue hippocampus Histopathological Studies：Sham-operation group brain
Hippocampal tissue is without oedema, and nerve cell is normal；Model group cerebral hippocampus tissue edema, large stretch of neuronal necrosis, infarct size is accounted for
More than the 2/3 of hippocampal tissue；The logical group cerebral hippocampus tissue edema of brain network, a small number of neuronal necrosis, infarct size accounts for hippocampal tissue
1/3 within；Nimodipine group cerebral hippocampus tissue edema, a small number of neuronal necrosis, infarct size account for the 1/3 of hippocampal tissue with
It is interior；Heavy dose of Patrinia scaniosaefolia general flavone group cerebral hippocampus tissue edema, a small number of neuronal necrosis, infarct size accounts for the 1/3 of hippocampal tissue
Within；Middle dosage Patrinia scaniosaefolia general flavone group cerebral hippocampus tissue edema, a small number of neuronal necrosis, infarct size accounts for the 1/ of hippocampal tissue
Within 3~2/3；Low dose of Patrinia scaniosaefolia general flavone group cerebral hippocampus tissue edema, neuronal necrosis in blocks, infarct size accounts for hippocampus group
Within 1/3~2/3 knitted.
Influence of the table 14 to Focal Cerebral Ischemia Reperfusion rat model brain tissue hippocampus pathological change
"-" cerebral hippocampus tissue is without oedema, and nerve cell is normal；"+" cerebral hippocampus tissue edema, a small number of neurons are bad
Extremely, infarct size is accounted within the 1/3 of hippocampal tissue；" ++ " cerebral hippocampus tissue edema, neuronal necrosis in blocks, infarct size is accounted for
Within the 1/3~2/3 of hippocampal tissue；" +++ " cerebral hippocampus tissue edema, large stretch of neuronal necrosis, infarct size accounts for hippocampus group
More than 2/3 knitted.
Examined through Ridit, with sham-operation group ratio, model group has significant statistical significance (P<0.01) model group, is illustrated
There is significant pathological change, modeling success in rat cerebral tissue's hippocampus；With model group ratio, brain network leads to group, Nimodipine group agent
Measurer is statistically significant (P<0.01)；Large, medium and small dosage Patrinia scaniosaefolia general flavone group has obvious statistical significance (P<
0.05) disease for the improvement office property the made cerebral ischemia re-pouring rat model brain tissue hippocampus that each administration group can be different degrees of, is illustrated
Degree of injury is managed, brain tissue is protected.
5 conclusions
By observing clinic nerve function assay of the Patrinia scaniosaefolia general flavone to Focal Ischemia-Reperfusion in Rats model, death
IL-6, IL-1 β in rate, brain infarction area percentage, brain tissue, TNF-α, Bcl-2, Bax, Casp-3, ICAM-1, ATP enzyme,
The influence of SOD, MDA level, NGF and NF-KBp65 expression and cerebral tissue pathology injury's change, points out Patrinia scaniosaefolia general flavone can be with
The scoring of animal nerve afunction, the death rate, brain infarction area percentage are reduced, there is protection to make brain tissue and neuron
With IL-6, IL-1 β, ICAM-1, TNF-α level in reduction brain tissue suppress inflammatory reaction after ischemia-reperfusion, reduce inflammation
The inflammatory mediator and cell factor of a series of caused cascade reaction releases；Mitigate peroxidatic reaction of lipid, reduction brain MDA contains
Amount, raises brain SOD contents, protects brain tissue；ATPase in Brain Tissue level is raised, is improved because energy metabolism impairment is to ischemic
The damage of brain tissue；Bax, Caspase-3 level in brain tissue, significantly rise brain tissue Bcl-2 levels are reduced, suppression cell withers
Die, protect neuronal cell；Promote brain tissue NGF positive expressions, suppress brain tissue NF-KBp65 positive expressions, protection nerve
Member, brain cell self-protective mechanism is activated, and mitigates cerebral ischemia re-pouring injured, is optimal using heavy dose of Patrinia scaniosaefolia general flavone group.
On the basis of animal experiment ensures safety, contrast test is clinically carried out, contrast groups use clopidogrel,
Test group adds clopidogrel using field pennycress general flavone, and relevant test situation is as follows：
Field pennycress general flavone adds Effect of Clopidogrel in Treating transient cerebral ischemia 60
Selection meets the transient cerebral ischemia diagnostic criteria that the 4th cerebrovascular disease academic conference in the whole nation is worked out:It is as of short duration
, reversible, local brain blood circulation disorder, can recurrent exerbation, few person 1~2 time, up to tens of times, breaking-out every time continues
Time, sings and symptoms should be wholly absent in 24h generally at several minutes to 1h or so.
ABCD2 standards of grading:Age ＞ 60 years old (1 point)；Blood pressure is systolic pressure ＞ 140mmHg or diastolic pressure ＞ 90mmHg (1
Point)；Clinical symptoms are single-sided weakness (2 points), not with powerless disfluency (1 point)；Symptom duration ＞ 60min (2 points),
10~59min (1 point)；With diabetes (1 point).Risk stratification:0~3 point is low danger group；4~5 points are middle danger group；6~7 points
For high-risk group.
Select ABCD2 standards of grading in the people of patient 60 of low danger layer, be randomly divided into 2 groups, every group of 30 people, the oral chlorine of control group
Pyrrole Gray (trade name Plavix, 75mg/ pieces, match Norfin, Inc of France), each 75mg, 1d 1 time, first dose of 300mg.Treatment
(capsule, every is made in field pennycress general flavone to group in addition to clopidogrel is taken by control group method, plus with field pennycress general flavone
0.3g, 2 every time, 2 times a day.Two groups of the above treats conventional control blood pressure and blood glucose during 30d, treatment, disables other energy
Enough influence the medicine of coagulation function.
Clopidogrel group adds the number of cases that TIA is terminated in field pennycress general flavone group 7d to distinguish with clopidogrel
For 24,27, time when TIA is terminated in 7d is respectively (9.66 ± 1.21) d, (0.50 ± 1.06) d,
The number of cases for still having breaking-out after 7d is respectively 6,3, and the number of cases that transient cerebral ischemia is aggravated in 30d is 0.Field pennycress general flavone
Plus clopidogrel is better than conventional Effect of Clopidogrel in Treating group to the general curative effect of transient cerebral ischemia patient.
It should be apparent that the field pennycress general flavone that the present invention is extracted from field pennycress has anti-brain from above-mentioned experiment
The effect of ischemic, the medicine of focal cerebral ischemia is treated repeatedly effective for preparing, realize that field pennycress general flavone is preparing treatment
Application in focal cerebral ischemia medicine, its preparation method is simple, easy to operate, has opened up the new medical value of field pennycress and business
Value, is to treat the innovation on focal cerebral ischemia medicine, economic and social benefit is notable.
Claims (1)
1. a kind of application of field pennycress general flavone as sole active agent in treatment focal cerebral ischemia medicine is prepared, described
Field pennycress general flavone be that field pennycress was ground into the coarse powder of 20-30 mesh sieves, first plus the aether backflow of 10 times of weight is extracted
1h, degreasing discards ether；The field pennycress dregs of a decoction volatilize ether, plus 10 times of weight of field pennycress, the immersion of the ethanol of mass concentration 80%
0.5h, refluxing extraction 1h, filtering, obtain first time filtrate；The dregs of a decoction add 10 times of weight of field pennycress, the ethanol of mass concentration 80% again, return
Stream extracts 1h, and filtering obtains second of filtrate, merges filtrate twice, and ethanol is recovered under reduced pressure and is extremely dispersed to phase without alcohol taste, plus distilled water
When in AB-8 type macroporous absorbent resins on the dispersion liquid of the 0.3g/mL containing crude drug amount, dispersion liquid, first washed with 2 times of column volume distillations
It is de-, discard aqueous；After with 4 times of column volumes, the ethanol elution of mass concentration 10%, eluent is discarded；Again with 6 times of column volumes, matter
The ethanol elution of concentration 80% is measured, 80% ethanol eluate is collected, ethanol is recovered under reduced pressure to without alcohol taste, is freeze-dried, crushes, obtain Patrinia scaniosaefolia
Careless general flavone.
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| CN110302282A (en) * | 2019-06-06 | 2019-10-08 | 中国人民解放军南部战区总医院 | Herba Patriniae extract is preparing the application in overriding resistance Acinetobacter bauamnnii drug |
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