CN101366738B - Application of ilex pubescens total flavone in preparing medicament for preventing and controlling transient ischemic attack syndrome - Google Patents
Application of ilex pubescens total flavone in preparing medicament for preventing and controlling transient ischemic attack syndrome Download PDFInfo
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Abstract
The invention relates to an application of Pubescent Holly Root total flavone in preparing medicine for preventing and treating transient cerebral ischemia syndromes, which effectively solves the problem about the medicine for preventing and treating transient cerebral ischemia syndromes. The invention has the following technical proposal: a Pubescent Holly Root medicinal material is ground and extracted twice after heating reflux by use of ethanol; extract solutions are merged and filtered; the ethanol is reclaimed; the merged extract solutions are concentrated to the relative density between 1.10 and 1.20; a concentrated solution is dispersed by use of water and turned into a solution in which the proportion of liquid medicine and the medicinal material is 1:1; the solution is kept to stand and filtered; a filtrate is centrifuged; centrifugal liquid is ultrafiltered, passed through a polyamide column, eluted by use of water and then eluted by use of an ethanol solution after water liquid is discarded; eluent is collected on standby; the eluent in two steps is merged and then eluted by use of sodium carbonate aqueous solution; and the eluent is collected, has the pH regulated to be neutral by use of hydrochloric acid, decompressed, concentrated, dried and ground, so as to obtain the Pubescent Holly Root total flavone. The Pubescent Holly Root total flavone is effectively used for preparing the medicine for preventing and treating transient cerebral ischemia syndromes, realizes the treatment for cerebral ischemia syndromes, and benefits mankind.
Description
One, technical field
The present invention relates to field of medicaments, particularly a kind of Radix Ilicis Pubescentis total flavones is in the application of preparation control transient ischemic attack (TIA) medicine.
Two, background technology
Radix Ilicis Pubescentis is the root of holly plant Radix Ilicis Pubescentis, contain multiple flavonoid, phenolic constituent, sterol, aminoacid, saccharide, tannin and three terpene components, has heat-clearing and toxic substances removing, the function of blood circulation and channel invigorating, be mainly used in and control anemopyretic cold, dyspnea and cough due to lung-heat, laryngeal edema, tonsillitis, dysentery, coronary heart disease, hemiplegia due to the cerebrovascular accident, thromboangiitis obliterans, erysipelas, scald, central serous chorioretinopathy, uveitis, and skin acute suppurative inflammation, modern medicine study shows that also Radix Ilicis Pubescentis is influential to cardiovascular, be applied to blood pressure lowering clinically, slowly and lastingly, along with going deep into of medical research, the medical of Radix Ilicis Pubescentis is worth to have had newly and begins, but Radix Ilicis Pubescentis sees that report is arranged in the end in the application of control transient ischemic attack, particularly Radix Ilicis Pubescentis effective ingredient total flavones as preparation treatment transient ischemic attack medicinal application do not have report arranged.
Three, summary of the invention
At above-mentioned situation, the present invention's purpose just provides a kind of Radix Ilicis Pubescentis total flavones that is extracted by Radix Ilicis Pubescentis is prevented and treated the transient ischemic attack medicine in preparation application, effectively solve the medicine problem of control transient ischemic attack, the technical scheme of its solution is, get the Radix Ilicis Pubescentis medical material, pulverize, 70% alcohol heating reflux with 8 times of amounts extracts twice, merge extractive liquid,, filter, reclaim ethanol, and be concentrated in the time of 50 ℃ that to measure relative density be 1.10-1.20, the concentrated solution water is separated into medicinal liquid: the solution of medical material=1: 1, leave standstill, filter, filtrate is centrifugal, the centrifugal liquid ultrafiltration, by polyamide column, first water elution with 2 times of column volumes, discard water liquid, 8 times of column volumes of reuse, 50% alcoholic solution eluting, it is standby to collect eluent, merges twice eluent, other changes the aqueous sodium carbonate eluting that pH value is 9-10, collect eluent, it is neutral transferring pH with hydrochloric acid, concentrating under reduced pressure, 60 ℃ of drying and crushing, get Radix Ilicis Pubescentis total flavones, wherein, total flavones is no less than 54.0% in rutin, total saponins must not surpass 5.0% in the ginsenoside Re, this Radix Ilicis Pubescentis total flavones is effective to prepare the medicine of treatment control transient ischemic attack, realizes the treatment to the cerebral ischemia syndrome, brings benefit to the mankind.
Four, the specific embodiment
Below in conjunction with concrete condition the specific embodiment of the present invention is elaborated.
When the preparation Radix Ilicis Pubescentis total flavones, the present invention adopts orthogonal experiment design method, chooses principal element such as concentration of alcohol to influencing extraction effect, alcohol adding amount, extraction time, extraction time is the investigation factor, content with total flavones and total saponins is testing index, determine that optimum extraction process is for getting the Radix Ilicis Pubescentis coarse powder, so get the Radix Ilicis Pubescentis medical material, pulverizing, 70% alcohol heating reflux with 8 times of amounts extracts twice, merge extractive liquid, filters, and reclaims ethanol, and be concentrated in the time of 50 ℃ that to measure relative density be 1.10～1.20, the concentrated solution water is separated into medicinal liquid: the solution of medical material=1: 1, leave standstill, and filter, filtrate is centrifugal, the centrifugal liquid ultrafiltration, by polyamide column, first water elution with 2 times of column volumes, discard water liquid, 8 times of column volumes of reuse, 50% alcoholic solution eluting, it is standby to collect eluent, merges twice eluent, other changes pH value is 9～10 aqueous sodium carbonate eluting, collect eluent, it is neutral transferring pH with hydrochloric acid, concentrating under reduced pressure, 60 ℃ of drying and crushing get Radix Ilicis Pubescentis total flavones.Said medicinal liquid: the solution of medical material=1: 1 is meant the concentrated solution water is dispersed into the solution that equates with the medical material bulking value, as the total drug material of 100g, concentrated solution is dispersed into (being diluted to) 100ml.
Radix Ilicis Pubescentis total flavones of the present invention effectively solves and is used to prepare the medicine of preventing and treating transient ischemic attack, and through animal experiment, Radix Ilicis Pubescentis total flavones has significant curative effect to the control transient ischemic attack, and relevant test situation is as follows:
Experiment 1, Radix Ilicis Pubescentis total flavones are to the influence of chmice acute cerebral ischemic model
1. experiment material
1.1 medicine and reagent
Radix Ilicis Pubescentis total flavones, Henan College Of Traditional Chinese Medicine chemistry chamber, content〉60%; HUATUO ZAIZAO WAN, Qixing Pharmaceutical Co., Ltd., Guangzhou, lot number 060629; Nimodipine tablet, Shandong XinHua Pharmacy stock Co., Ltd, lot number 0609118; Normal saline, Zhengzhou chemistry pharmaceutcal corporation, Ltd, lot number 060712.
1.2 instrument
Electronic balance is produced by Shanghai Min Qiao precision instrument company limited.
1.3 animal
Mice: KM, cleaning level, body weight 18～20g; Provide the animal quality certification number by Hebei province's Experimental Animal Center: DK0508068.
1.4 data analysis
Carry out the statistical procedures of data information with the medical statistical packages of SPSS11.5for windows, (x ± s) expression relatively adopts variance analysis to measurement data between group with average ± standard deviation.
2. experimental technique and result
Get 60 of 18～20g male mices, be divided into 6 groups at random, i.e. Radix Ilicis Pubescentis total flavones (0.4g/kg, 0.2g/kg, 0.1g/kg, being made into concentration is 20mg/ml, 10mg/ml, 5mg/ml), (be 10 times of clinical consumption, 20mg/kg, liquor strength are 1mg/ml to the nimodipine group, administration volume 0.2ml/10g), and the HUATUO ZAIZAO WAN group (be 10 times of clinical consumption, 2.67g/kg, liquor strength 0.134g/ml, administration volume 0.2ml/10g), blank group (irritating the normal saline of same volume every day).Administration every day 1 time, successive administration 7 days.1h after last administration, 2mm place behind ear breaks end rapidly with shears, causes the acute cerebral ischemia model, the frequency of respiration and the breathing of writing down mice then hold time (it is standard that all mices all dehisce to pant with nothing).Laboratory temperature when the result breaks end is 21 ℃, and the time is points in the mornings 8, the results are shown in Table 1.
Table 1 Radix Ilicis Pubescentis total flavones is to the influence of acute cerebral ischemia mouse breathing number of times and breathing time (x ± s)
Annotate: with blank group ratio, * P＜0.05; * P＜0.01
As can be seen from the above table: compare with the blank group, large, medium and small dosage Radix Ilicis Pubescentis total flavones group and HUATUO ZAIZAO WAN group all can obviously prolong the breathing persistent period (P＜0.05) behind the mice broken end, but the nimodipine group significant prolongation mouse breathing persistent period (P＜0.01); Compare with the blank group, big or middle dosage Radix Ilicis Pubescentis total flavones group, nimodipine group all can obviously increase mice broken end back frequency of respiration (P＜0.05).Illustrate that Radix Ilicis Pubescentis total flavones can significantly increase the frequency of respiration and the breathing time of decapitated mice.
4. conclusion
The experimental result prompting, Radix Ilicis Pubescentis total flavones can significantly increase the frequency of respiration and the breathing time of decapitated mice.
Experiment 2, Radix Ilicis Pubescentis total flavones are to the influence of mice transient ischemic attack re-perfusion model
1. experiment material
1.1 medicine and reagent
Radix Ilicis Pubescentis total flavones, Henan College Of Traditional Chinese Medicine chemistry chamber, content〉60%; HUATUO ZAIZAO WAN, Qixing Pharmaceutical Co., Ltd., Guangzhou, lot number 060629; Nimodipine tablet, Shandong XinHua Pharmacy stock Co., Ltd, lot number 0609118; Normal saline, Zhengzhou chemistry pharmaceutcal corporation, Ltd, lot number 060712; Chloral hydrate is produced lot number 20060517 by last seamount Pu chemical industry company limited; Formalin is by Laiyang City company limited production in pairs, lot number 20060801; The ATP detection kit is built up bio-engineering research by Nanjing and is produced, lot number 20070713; The LD detection kit is built up bio-engineering research by Nanjing and is produced, lot number 20070725; The Coomassie brilliant blue protein determination kit builds up bio-engineering research by Nanjing and is produced, lot number 20070725
1.2 instrument
UV-2000 spectrophotometer is produced by UNICO(Shanghai) Instruments Co., Ltd.; FA (N)/JA (N) series electronic balance is produced by Shanghai Min Qiao precision instrument company limited; The TGL-16G High speed refrigerated centrifuge is produced by Anting Scientific Instrument Factory, Shanghai; DZKW-4 type electronic thermostatic water-bath is produced by the bright forever Medical Instruments in Beijing factory; Adjustable pipette is produced by Shanghai Lei Bo Analytical Instrument Co., Ltd.
1.3 animal
Mice: KM, the cleaning level.Body weight 18～20g; Provide the mice quality certification number by Hebei province's Experimental Animal Center: DK0508068.
1.4 data analysis
Carry out the statistical procedures of data information with the medical statistical packages of SPSS11.5for windows, (x ± s) expression relatively uses variance analysis to measurement data between group with average ± standard deviation; Ranked data adopt the Ridit check.
2. experimental technique and result
2.1 animal grouping and administering mode
Get body weight and be 70 of mices about 18～20g, male, evenly be divided into 7 groups at random, wherein make the cerebral ischemia re-pouring model for 6 groups, other establishes 1 group of sham operated rats.The modeling type is irritated Radix Ilicis Pubescentis total flavones (0.4g/kg, 0.2g/kg, 0.1g/kg respectively for 6 groups, being made into concentration is 20mg/ml, 10mg/ml, 5mg/ml,), the positive controls nimodipine (be 10 times of clinical people, 20mg/kg, liquor strength 1mg/ml, administration volume 0.2ml/10g) and the HUATUO ZAIZAO WAN group (be 10 times of clinical consumption, 2.67g/kg, liquor strength 0.134g/ml, administration volume 0.2ml/10g), model group (irritating the normal saline with volume), the blank group is as sham operated rats.Administration every day 1 time, successive administration 7 days.
2.2 modeling method and drawing materials
1h after administration in the 10th day, 12h after the fasting, 4% chloral hydrate 0.1ml/10g intraperitoneal injection of anesthesia (righting reflex loss of mice), use the hair of shears clip cervical region then, with the clean cervical region of alcohol wipe, make the cervical region median incision with scalpel, muscle around separating gradually is up to seeing bilateral common carotid arteries (CCA), reuse elbow pincet is peeled off peripheral nerve, separation of C CA respectively puts the surgical thread of a 5cm length under each tremulous pulse, the acupuncture needle that diameter is about 0.3mm is with the blood vessel ligation, unclamp the rope of four lower limbs of mice then, it is reclined in cage, and this process is noted its insulation, and room temperature remains on 25 ℃.Extract acupuncture needle behind the 10min gently out, finish again perfusion, its empty group surgical procedure of playing tricks, promptly except not ligation CCA, other operation is with each group of modeling.Quick broken end is got brain after irritating 15min again, measures clotting time with the blood capillary draw blood immediately; Simultaneously separate brain immediately in ice pan, brain is separated into symmetric brain from sagittal line, half takes out hippocampus, is put in rapidly to soak in the bottle in 10% formalin solution to fix; Half is placed on and blots clean top bloodstain on the filter paper in addition, its weight then weighs with scale, the ice-cold saline volume that should add according to this re-computation is with cerebral tissue (g): ratio homogenate in filling the glass refiner of ice cube of normal saline (ml)=1: 9.4 ℃, the centrifugal 20min of 3000r/min.Get supernatant in cryopreservation below-20 ℃.Make 10% brain homogenate by the testing cassete description operation, measure LD, Na respectively
+-K
+-ATPase, Ca
++-ATPase, Mg
++-ATPase and Ca
++-Mg
++-ATPase vigor.
2.3 result
Radix Ilicis Pubescentis total flavones the results are shown in Table 2 to the influence of mice transient ischemic attack re-perfusion model blood coagulation time, to the influence of brain energy metabolism, the results are shown in Table 3, table 4.
Table 2 Radix Ilicis Pubescentis total flavones is to the influence of mice transient ischemic attack re-perfusion model clotting time (x ± s)
Annotate: compare * * P＜0.01 with model group
Can find out that from last table with the sham operated rats ratio, the model group clotting time significantly shortens (P＜0.01), illustrate make the cerebral ischemia re-pouring model after clotting time significantly shorten.With the model group ratio, low dose of Radix Ilicis Pubescentis total flavones group and HUATUO ZAIZAO WAN group be the obvious clotting time of lengthening model animal (P＜0.05) all, and other dosage groups have prolongation trend.
Table 3 Radix Ilicis Pubescentis total flavones is to mice transient ischemic attack re-perfusion model brain homogenate Na
+-K
+-ATP and Ca
++-Mg
++The influence of-ATP vigor (x ± s)
Annotate: compare * P＜0.05 with model group; * P＜0.01
As can be seen from the above table: relatively, the ATP enzyme activity significantly reduces (P＜0.01) in the homogenate of model group mouse brain, and the Na of cell membrane is described with sham operated rats (blank group)
+-K
+-ATP, Ca
++-Mg
++-atpase activity reduces, and ATP exhausts, brain cell energy metabolism serious hindrance, damage body brain function.With the model group ratio, large, medium and small dosage Radix Ilicis Pubescentis total flavones group is the Na in the homogenate of elevation model animal brain obviously
+-K
+-ATP enzyme activity (P＜0.05), nimodipine group and the HUATUO ZAIZAO WAN group Na in the brain tissue homogenate that can significantly raise
+-K
+-ATP enzyme activity (P＜0.01); Heavy dose of Radix Ilicis Pubescentis total flavones group and the HUATUO ZAIZAO WAN group Ca in the brain tissue homogenate that can significantly raise
++-Mg
++-ATP enzyme activity significantly raises (P＜0.01), middle dosage Radix Ilicis Pubescentis total flavones group and the nimodipine group Ca in the brain tissue homogenate that can obviously raise
++-Mg
++-ATP enzyme activity (P＜0.05).Show the Na of Radix Ilicis Pubescentis total flavones to mice transient ischemic attack re-perfusion model
+-K
+-ATP, Ca
++-Mg
++-ATP vigor has the rising effect.
Table 4 Radix Ilicis Pubescentis total flavones is to mice transient ischemic attack re-perfusion model brain homogenate Ca
++-ATP and Mg
++The influence of-ATP vigor (x ± s)
Annotate: compare * P＜0.05 with model group; * P＜0.01
As can be seen from the above table: relatively, the ATP enzyme activity significantly reduces (P＜0.01) in the homogenate of model group mouse brain, and the Ca of cell membrane is described with sham operated rats (blank group)
++-ATP and Mg
++-atpase activity reduces, and ATP exhausts, brain cell energy metabolism serious hindrance, damage body brain function.With the model group ratio, heavy dose of Radix Ilicis Pubescentis total flavones group and HUATUO ZAIZAO WAN group be the Ca in the homogenate of elevation model animal brain significantly
++-ATP enzyme activity (P＜0.01), middle dosage Radix Ilicis Pubescentis total flavones group and the nimodipine group Ca in the brain tissue homogenate that can obviously raise
++-ATP enzyme activity (P＜0.05); Heavy dose of Radix Ilicis Pubescentis total flavones group, nimodipine group and the HUATUO ZAIZAO WAN group Mg in the brain tissue homogenate that can significantly raise
++-ATP enzyme activity (P＜0.01), the middle dosage Radix Ilicis Pubescentis total flavones group Mg in the brain tissue homogenate that can obviously raise
++-ATP enzyme activity (P＜0.05).Show the Ca of Radix Ilicis Pubescentis total flavones to mice transient ischemic attack re-perfusion model
++-ATP, Mg
++-ATP vigor has the rising effect.
Radix Ilicis Pubescentis total flavones the results are shown in Table 5 to the influence of lactic acid content in the mice transient ischemic attack re-perfusion model brain homogenate.
Table 5 Radix Ilicis Pubescentis total flavones is to the influence of mice transient ischemic attack re-perfusion model brain homogenate lactic acid content (x ± s)
Annotate: compare * P＜0.05 with model group; * P＜0.01
As can be seen from Table 5: with sham operated rats (blank group) ratio, the model group lactic acid content obviously raises (P＜0.05), illustrates that the lactic acid that cerebral ischemia produces sugared anaerobic metabolism increases, and can cause acidosis; With the model group ratio, large, medium and small Radix Ilicis Pubescentis total flavones dosage group lactic acid content significantly reduces (P＜0.01); Nimodipine group and HUATUO ZAIZAO WAN group lactic acid content obviously reduce (P＜0.05).Illustrate that the lactic acid when Radix Ilicis Pubescentis total flavones can reduce the mice transient ischemic attack and pours into piles up.
The influence that Radix Ilicis Pubescentis total flavones is observed mice transient ischemic attack re-perfusion model brain hippocampus tectology the results are shown in Table 6.
Sham operated rats (blank group) mouse brain neurocyte is all normal, and the neurocyte endochylema is abundant, and nuclear and kernel are all high-visible, is normal.Model group animal brain neurocyte major part is atrophy and changes, and cell volume dwindles, and endochylema obviously reduces, and examines pathological changes such as obvious pyknosis, and the edema pathological change appears in the partial nerve cell, and cell cytosol increases and transparent.The most of cell cytosol of heavy dose of Radix Ilicis Pubescentis total flavones treated animal cranial nerve cell is abundant, obviously recovers; Middle dosage Radix Ilicis Pubescentis total flavones group part animal brain neurocyte is significantly recovered, and cell volume increases, and endochylema is abundant; The cranial nerve cell of part animal still is atrophing state, karyopycnosis.The cranial nerve cell of most of animal of low dose of Radix Ilicis Pubescentis total flavones group presents atrophy phenomenon in various degree, endochylema minimizing in various degree, karyopyknosis.The most of cell cytosol of HUATUO ZAIZAO WAN treated animal cranial nerve cell is abundant, part cell generation atrophy phenomenon, and cell volume dwindles, karyopycnosis.Nimodipine group most animals cranial nerve cell volume obviously dwindles karyopycnosis; The fraction cell cytosol is abundant, and stereomutation is little, and nucleus is all normal.
Table 6 Radix Ilicis Pubescentis total flavones is to the influence (only) of rat cerebral ischemia model cerebral tissue pathological change
"-" animal brain neurocyte is all normal, and cell cytosol is abundant, and nuclear and kernel are high-visible; "+" animal brain neurocyte 20% presents the atrophy phenomenon, and other 80% cell is normal; " ++ " animal brain neurocyte 50% is the atrophy phenomenon, and other cell is recovery in various degree; " +++" animal brain neurocyte 80% presents the atrophy phenomenon, and other cell obtains recovery in various degree.Measure testing each treated animal cranial nerve cell according to the sxemiquantitative index.
As can be seen from the above table: check through Ridit, sham operated rats and model group relatively have significant statistical significance, the sham operated rats cranial nerve cell is all normal, model group cranial nerve cell major part is atrophy and changes, cell volume dwindles, endochylema reduces, and karyopycnosis illustrates that significant cranial nerve cell pathological change appears in the modeling mice.Compare with model group, big or middle dosage Radix Ilicis Pubescentis total flavones, nimodipine and HUATUO ZAIZAO WAN group all can significantly alleviate ischemia-reperfusion induced mice brain tissue injury (P＜0.01), and most of cranial nerve cell endochylema is abundant, and nuclear and kernel are high-visible; Low dose of Radix Ilicis Pubescentis total flavones group all can obviously alleviate ischemia-reperfusion induced mice brain tissue injury (P＜0.05).
4. conclusion
Radix Ilicis Pubescentis total flavones can improve the brain cell energy metabolism of rat transient ischemic attack-re-perfusion model, prevents acidosic generation; Can alleviate the pathological changes of model brain hippocampus tectology; The prompting Radix Ilicis Pubescentis total flavones has better action to the transient ischemic attack animal model.
Experiment 3, Radix Ilicis Pubescentis total flavones are to the influence of rat cerebral ischemia model
1. experiment material
1.1 reagent and medicine
Radix Ilicis Pubescentis total flavones, Henan College Of Traditional Chinese Medicine chemistry chamber, content〉60%; HUATUO ZAIZAO WAN, Qixing Pharmaceutical Co., Ltd., Guangzhou, lot number 060629; Nimodipine tablet, Shandong XinHua Pharmacy stock Co., Ltd, lot number 0609118; Normal saline, Zhengzhou chemistry pharmaceutcal corporation, Ltd, lot number 060712; MDA detection kit, Nanjing are built up bio-engineering research institute, lot number 20070405; ATP detection kit, lot number Nanjing are built up bio-engineering research institute, 20070311; The good blue protein determination kit of coomassie, bio-engineering research institute, lot number 20070418 are built up in Nanjing.
1.2 instrument
The 75-2 spectrophotometer, Shanghai the 3rd analytical tool factory; Glass homogenizer, Shanghai glass apparatus factory; Thermostat water bath, the bright instrument plant in Beijing.
1.3 animal
Rat: Wistar, male, body weight: 180～200g, medical animal experiment center, effluent south provides SCXK (Henan) 20060231.
2. experimental technique and result
Get 70 of rats, wistar, body weight 180-200g, male, evenly be divided into 7 groups at random, gavage big respectively, in, low dose of Radix Ilicis Pubescentis total flavones (0.2g/kg, 0.1g/kg, 0.05g/kg being made into concentration is 10mg/ml, 5mg/ml, 2.5mg/ml,), nimodipine (0.01g/kg is made into 0.5mg/ml), HUATUO ZAIZAO WAN (0.67g/kg, be made into 33.25mg/ml) and the normal saline (2 groups) of same volume (irritate body of stomach long-pending be 2ml/100g), administration every day 1 time, successive administration 7 days, 1h after administration in the 7th day, 10h after the fasting, chloral hydrate anesthesia, broken end is got blood behind separation and the ligation bilateral common carotid arteries 30min, gets brain and makes pathology section and homogenate survey ATP, LD, LDH, indexs such as MDA.
Influence to brain energy metabolism: get rat cerebral even slurry (brain is heavy: normal saline volume=1:9 or redilution become 2% brain homogenate) and measure Na respectively by the testing cassete description operation
+K
+-ATPase, Mg
++-ATPase, Ca
++-ATPase, Ca
++-Mg
++-ATPase vigor the results are shown in Table 1, and LD content, LDH vigor the results are shown in Table 2.
Table 1 Radix Ilicis Pubescentis total flavones merges the influence (x ± s) of cerebral ischemic model brain homogenate energy metabolism to the rat blood stasis
*: the expression with model group than P＜0.01; *: the expression with model group than P＜0.05
As can be seen from the above table, with sham operated rats ratio, model group Na
+K
+-ATPase, Mg
++-ATPase, Ca
++-ATPase vigor all obviously reduces (P＜0.05), Ca
++-Mg
++-ATP vigor all significantly reduces (P＜0.01), illustrates to make the cerebral ischemic model success.With the model group ratio, large, medium and small dosage Radix Ilicis Pubescentis total flavones group can significantly improve brain homogenate Na
+K
+-ATPase vigor (P＜0.01), the HUATUO ZAIZAO WAN group can obviously improve brain homogenate Na
+K
+-ATPase vigor (P＜0.05); In, low dose of Radix Ilicis Pubescentis total flavones group can significantly improve brain homogenate Ca
++-ATPase vigor (P＜0.01), heavy dose of Radix Ilicis Pubescentis total flavones group and HUATUO ZAIZAO WAN group can obviously improve brain homogenate Ca
++-ATPase vigor (P＜0.05); Dosage Radix Ilicis Pubescentis total flavones group big or middle can significantly improve brain homogenate Mg
++-ATPase vigor (P＜0.01), low dose of Radix Ilicis Pubescentis total flavones group and HUATUO ZAIZAO WAN group can obviously improve brain homogenate Mg
++-ATPase vigor (P＜0.05); In, low dose of Radix Ilicis Pubescentis total flavones group can significantly improve brain homogenate Ca
++-Mg
++-ATPase vigor (P＜0.01), heavy dose of Radix Ilicis Pubescentis total flavones group can obviously improve brain homogenate Ca
++-Mg
++-ATPase vigor (P＜0.05).Show that Radix Ilicis Pubescentis total flavones has the improvement effect to the various ATP vigor of rat cerebral ischemia model.
Table 2 Radix Ilicis Pubescentis total flavones merges the influence (x ± s) of cerebral ischemic model brain homogenate lactic acid content and lactic acid dehydrogenase activity to the rat blood stasis
*: the expression with model group than P＜0.01; *: the expression with model group than P＜0.05
As can be seen from the above table, with the sham-operation ratio, the model group lactic acid content significantly increases (P＜0.01), illustrates to make the cerebral ischemic model success.With the model group ratio, in, low dose of Radix Ilicis Pubescentis total flavones group and nimodipine group and HUATUO ZAIZAO WAN group all can obviously reduce brain homogenate lactic acid content (P＜0.05), heavy dose of Radix Ilicis Pubescentis total flavones group, nimodipine group and HUATUO ZAIZAO WAN group all can significantly reduce brain homogenate lactic dehydrogenase enzyme activity (P＜0.01).Show that Radix Ilicis Pubescentis total flavones has the improvement effect to the lactic acid content and the lactic dehydrogenase enzyme activity of rat blood stasis syncerebrum ischemia model.
Influence to antioxidant in the cerebral tissue and oxygen-derived free radicals: (brain is heavy: normal saline volume=1:9) press the testing cassete explanation to measure MDA the results are shown in Table 3 to get rat cerebral even slurry.
Table 3 Radix Ilicis Pubescentis total flavones is to the influence of rat cerebral ischemia model brain homogenate MDA level (x ± s)
*: the expression with model group than P＜0.01; *: the expression with model group than P＜0.05
As can be seen from the above table, with the sham operated rats ratio, model group brain homogenate MDA content obviously raises (P＜0.05), illustrates to make the cerebral ischemic model success.With the model group ratio, large, medium and small dosage Radix Ilicis Pubescentis total flavones group can significantly reduce model M DA level (P＜0.01), and nimodipine group and HUATUO ZAIZAO WAN brain homogenate MDA content obviously reduce (P＜0.01).Show that Radix Ilicis Pubescentis total flavones has obvious reduction effect to the MDA of rat cerebral ischemia model.
Pathological observation result to rat cerebral ischemia model brain tissue: the group of doing evil through another person laboratory animal cranial nerve cell is all normal, and cell volume is big, and endochylema is abundant, and nuclear and kernel are high-visible; The laboratory animal cranial nerve cell volume-diminished of model group, cell cytosol and nucleus have been demarcated smudgy, present tangible atrophy and autolysis; Heavy dose of Radix Ilicis Pubescentis total flavones treated animal cranial nerve cell majority is restored, and cell volume increases, and endochylema is abundant; Middle dosage Radix Ilicis Pubescentis total flavones group part cranial nerve cell obtains recovery in various degree, but a part of cranial nerve cell still is atrophing state; The most of animal brain neurocyte of low dose of Radix Ilicis Pubescentis total flavones group still is atrophy and edema state, and cell volume obviously dwindles, and nucleus is smudgy.HUATUO ZAIZAO WAN group part cranial nerve cell obtains certain recovery; Nimodipine treated animal part cranial nerve cell is atrophing state, and cell volume obviously dwindles, the karyopycnosis that has, and the nuclear that has is smudgy.The results are shown in Table 4.
The total flurried ketone of table 4 Radix Ilicis Pubescentis is to the influence (only) of rat cerebral ischemia model cerebral tissue pathological change
"-" animal brain neurocyte is all normal, and cell cytosol is abundant, and nuclear and kernel are high-visible.The atrophy phenomenon appears in "+" animal brain neurocyte 20%, and cell cytosol reduces, volume-diminished, and karyopycnosis or self-dissolving are examined smudgy.The atrophy phenomenon appears in " ++ " animal brain neurocyte 50%, and cell cytosol reduces, volume-diminished, and karyopycnosis or self-dissolving are examined smudgy.The atrophy phenomenon appears in " +++" animal brain neurocyte 80%, and cell cytosol reduces, volume-diminished, karyopycnosis or autolysis, examine smudgy, the appearance edema that has, nuclear disappearance.Measure testing each treated animal cranial nerve pathological change according to semiquantitative standard.
Through the Ridit check, with the sham operated rats ratio, significant convenient change (P＜0.01) appears in the model group cranial nerve cell, the neurocyte volume dwindles, cell cytosol and nucleus have been demarcated smudgy, present tangible atrophy and autolysis, illustrate that the damage of cranial nerve cell appears in the modeling rat.Compare with model group, big or middle dosage Radix Ilicis Pubescentis total flavones group, nimodipine group and HUATUO ZAIZAO WAN group all can significantly alleviate the brain tissue impairment (P＜0.01) due to the ischemia, and cranial nerve cell is partly significantly recovered, and cell volume increases, and endochylema is abundant; Low dose of Radix Ilicis Pubescentis total flavones group can obviously alleviate the brain tissue impairment (P＜0.05) due to the ischemia.The pathological change of Radix Ilicis Pubescentis total flavones energy antagonism cerebral ischemia is restored most of neurocyte, along with the increase effect enhancing of dosage.
3. conclusion
Radix Ilicis Pubescentis total flavones can improve the energy metabolism of rat cerebral ischemia brain cell, prevents acidosic generation; Alleviate the damage of the cranial nerve cell that ischemia causes; Reduce the generation of oxygen-derived free radicals, increase the generation of antioxidant, have antioxidation; Can improve the energy metabolism of cerebral tissue; Can significantly improve the brain tissue pathology change due to the cerebral ischemia.
Claims (2)
1. a Radix Ilicis Pubescentis total flavones is characterized in that in the application for preparing control transient ischemic attack medicine this Radix Ilicis Pubescentis total flavones is by getting the Radix Ilicis Pubescentis medical material, pulverize, 70% alcohol heating reflux with 8 times of amounts extracts twice, and merge extractive liquid, filters, reclaim ethanol, and being concentrated in the time of 50 ℃ that to measure relative density be 1.10-1.20, the concentrated solution water is separated into medicinal liquid: the solution of medical material=1: 1, leave standstill, filter, filtrate is centrifugal, and polyamide column is passed through in the centrifugal liquid ultrafiltration, first water elution with 2 times of column volumes, discard water liquid, 8 times of column volumes of reuse, 50% alcoholic solution eluting, it is standby to collect eluent, merge twice eluent, other changes the aqueous sodium carbonate eluting that pH value is 9-10, collects eluent, and it is neutral transferring pH with hydrochloric acid, concentrating under reduced pressure, 60 ℃ of drying and crushing get Radix Ilicis Pubescentis total flavones, wherein, total flavones is no less than 54.0% in rutin, and total saponins must not surpass 5.0% in the ginsenoside Re.
2. Radix Ilicis Pubescentis total flavones as claimed in claim 1.
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| CN102258550B (en) * | 2011-08-30 | 2012-10-03 | 河南中医学院 | New application of ilex pubescens extract in preparing medicines for enhancing cerebral ischemia resistance |
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