CN103417679A - Method for extracting anti-cerebral-ischemia material from roses and application of anti-cerebral ischemia material - Google Patents
Method for extracting anti-cerebral-ischemia material from roses and application of anti-cerebral ischemia material Download PDFInfo
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Abstract
The invention relates to the method for extracting an anti-cerebral-ischemia material from roses and the application of the anti-cerebral-ischemia material. The problem of extraction of the anti-cerebral-ischemia material from the roses can be effectively solved and the application of rose extractive in preparation of anti-cerebral-ischemia medicine is achieved. The method comprises the following steps that the roses are smashed into medicine powder, and reflux extraction is conducted on the medicine powder through petroleum ether for one hour so that degreasing is completed; residue of the degreased rose medicine powder is filtered out, the petroleum ether is volatilized until the rose medicine powder is dry, and therefore rose powder is obtained; the rose powder is soaked with ethyl alcohol, reflux retraction is conducted for two times, extractive liquid is filtered, decompression is conduced so that the ethyl alcohol can be recycled until the smell of the ethyl alcohol does not exist, concentration is conducted, and distilled water is added to concentration liquid so that the concentration liquid can be dispersed into sample liquid; AB-8 type macroporous resin on the sample liquid is eluted through distilled water, and water liquid is removed; elution is conducted through ethyl alcohol, decompression is conducted so that ethyl alcohol can be recycled, concentration is conducted on the sample liquid until dry powder is obtained, and rose general flavone is obtained. The rose general flavone can be effectively used for preparation of the anti-cerebral-ischemia medicine, and the application of the rose extractive in the preparation of the anti-cerebral-ischemia medicine is achieved. According to the method for extracting the anti-cerebral-ischemia material from roses and the application of the anti-cerebral-ischemia material, raw materials are abundant, the anti-cerebral-ischemia material is easy to prepare, the extractive has the anti-cerebral-ischemia function, and the anti-cerebral-ischemia material is innovation of medicine.
Description
Technical field
The present invention relates to medicine, particularly in a kind of Flos Rosae Rugosae, extract method and the application thereof of anti-cerebral ischemia material.
Background technology
Ischemic cerebrovascular belongs to the scope of the traditional Chinese medical science " Ischemic Apoplexy ", it is the focal dysfunction of brain caused by local cerebral blood supply, confession hypoxgia or interruption, common are transient ischemic attack, cerebral embolism, cerebral thrombosis, be the commonly encountered diseases frequently-occurring disease of serious threat human life health, cause especially dead and disabled principal disease.Ischemia reperfusion injury (ischemia reperfusion injury, IRI) refers to be organized in and suffers the certain hour ischemia, after recovering blood supply, and the pathological phenomenon that degree of tissue damage increases the weight of.At present, medicine has become the important subject of society and medical circle concern to the therapeutic intervention of ischemic brain injury, finds effective medicine and prevents and treats the research of cerebral ischemia and more and more paid attention to.
Previously on the understanding basis because of in wind, fire, expectorant, the stasis of blood etc., adopt the traditional Chinese medical science single or many because of determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs, obtained certain curative effect, but further the curative effect raising is really difficult.In recent years, along with the in-depth to the evil understanding of tradition poison, going deep into of the development of apoplexy clinical practice and modern pathology Mechanism Study, propose to control apoplexy from the poison opinion, thereby improve the apoplexy curative effect, oneself becomes new viewpoint and focus in apoplexy etiology and therapeutics research.For the poison in apoplexy is evil, the brain method is protected in the proposition removing toxic substances to Liu Shi etc., in the method for summary, has: Quyujiedu, and blood stasis dispelling is the basis of removing toxic substances, removing toxic substances is conducive to blood stasis dispelling.This method is not only applicable to the treatment of cerebral infarction, and the treatment hemorrhagic apoplexy is clinical that good effect also arranged; Method of dissipating heat and detoxifying, the generation of pyretic toxicity is real relevant with blood stasis, the turbid stagnant heat-transformation of a specified duration of expectorant and positive internal organs in body after apoplexy, because fire is contained removing toxic substances, toxicity is burning hot, therefore still have fiercely apt to changely, easily attack the characteristics of internal organs, especially damage brain matter, even cause death, break for a long time and use the rule for the treatment of treatment primary disease that " blood circulation promoting and blood stasis dispelling " is the master; Thereby propose to lack with method of dissipating heat and detoxifying treatment brain, not only for the treatment cerebral ischemia, increased new content, also enriched the Therapeutic Method of apoplexy simultaneously, for apoplexy has been opened up new content, improved clinical efficacy, innovative significance is arranged very much, not only tcm clinical practice is had to important directive function, and will contribute to improve the Chinese medicine combined therapy effect of apoplexy.
Modern medicine study is shaped with effect, apoptosis of energy metabolism impairment, platelet aggregation, vasoconstriction and microcirculation disturbance, radical damage, toxicity of excitatory amino acid effect, intracellular calcium overload, inflammation damnification, NO and NOS etc. about the generator of cerebral ischemia, cerebral ischemia is due to the brain blood circulation obstacle, cause cellular energy metabolism exhaustion, thus the result of the Cascade of Injury started.This Cascade of Injury relates to the different stages such as excitatory amino acid (EAA) toxicity, infarction depolarization on every side, inflammation and apoptosis.Modern medicine control cerebral ischemia adopts the Drug intervention more, the clinical medicine that can be used for preventing and treating cerebral ischemia mainly contains the aspirin of antiplatelet drug, clopidogrel, ticlopidine and aspirin-dipyridamole, the heparin of anticoagulant, Low molecular heparin, the warfarin of oral anticoagulant, the dicoumarol pin, the urokinase of thrombolytic (UK), streptokinase (SK), the vasodilation nicergoline, the free radical scavenger superoxide dismutase, dexamethasone, mannitol, vitamin E, the calcium antagonist nimodipine, glutamate receptor antagonists, nos inhibitor and statins etc., wherein maximum with the application of aspirin antiplatelet aggregation.A lot of these medicines of bibliographical information have prevention and treatment Cerebral Ischemia, but existing medicine is more expensive and administering mode is limited, side effect is not suitable for repeatedly applying or because administration time is long, dosage is large etc., application is restricted due to price.Because the characteristics of primary disease need the long period medication more, the untoward reaction of long-term prescription, the price of medicine, the restriction of the course for the treatment of etc. all affect the treatment to cerebral ischemia.
In the last few years, Chinese scholars turned one's attention to the exploitation of natural drug (Chinese medicine or Chinese medicine preparation), treatment by Chinese herbs cerebrovascular disease with a long history, and Chinese medicine has the advantages such as side effect is little; Chinese medicine has the characteristics of organic conception, and Chinese medicine has multicomponent, acts on the feature of many target spots, from mulitpath, intervenes cerebral ischemia, the damage that alleviates cerebral ischemia re-pouring.For Imaging in Patients with Cerebral Ischemia Disease, the traditional Chinese medical science thinks that its pathogeny and " stasis of blood " relation are the closest, blood stagnant in cerebral venation syndrome is thought in existing research, blood stasis in body after the generation of accumulateing scorchingly hot poison and the apoplexy of accumulating in the course of time, the turbid stagnant with the passing of time heat-transformation of expectorant and positive internal organs are real relevant, pyretic toxicity undermines the brain network, upsets gods, therefore suggestion use take " blood circulation promoting and blood stasis dispelling, heat-clearing and toxic substances removing " improve cerebral ischemia and treat primary disease as the main rule for the treatment of.Chinese medicine utilizes " blood circulation promoting and blood stasis dispelling, heat-clearing and toxic substances removing " decoction for the treatment cerebral ischemia all to obtain effect preferably, obtained widely certainly, using at present the blood circulation promoting and blood stasis dispelling heat-clearing and detoxifying herb control cerebral ischemia re-pouring of holding concurrently is new viewpoint, the pretreatment measure of clinical practice feasibility is arranged, for the control of ischemic cerebrovascular provides new method, use " blood circulation promoting and blood stasis dispelling hold concurrently heat-clearing and toxic substances removing " square medicine to alleviate the thinking of cerebral ischemia reperfusion injury, there is more wide Research Prospects.
Often adopt clinically " blood circulation promoting and blood stasis dispelling " ruling by law to treat cerebral ischemia, focus on and improve cerebral circulation, the neuroprotective cell, remove free radical and reach the effect for the treatment of cerebral ischemia; Viewpoint based on " pyretic toxicity causes apoplexy ", propose " heat-clearing and toxic substances removing " ruling by law and treat Imaging in Patients with Cerebral Ischemia Disease, and heat and toxic materials clearing away medicine focuses on the reaction that reduces inflammation, and reduces the secondary response of cerebral ischemia.Flos Rosae Rugosae has excellent activity but hypotoxic anticancer plants as a kind of for a long time, receives the concern of Chinese scholars in nearly more than 20 years.But until at present, also do not find any report of Flos Rosae Rugosae to cerebral ischemia, the concrete active component of anti-cerebral ischemia is also indefinite, and in the rhodinol extract, main active is total flavones, how about the Flos Rosae Rugosae total flavones is extracted, and as for there is not yet, is publicly reported.
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the present invention's purpose just is to provide method and the application thereof of extracting the anti-cerebral ischemia material in a kind of Flos Rosae Rugosae, can effectively solve and extract the anti-cerebral ischemia material from Flos Rosae Rugosae, realizes the application of Flos Rosae Rugosae extract in preparing anti-cerebral ischemia drugs.
The technical scheme that the present invention solves is Flos Rosae Rugosas pollen to be broken into to medicated powder, with petroleum ether reflux extraction 1h defat; Flos Rosae Rugosas medicine powder filtering residue after defat, fling to petroleum ether to dry, obtains Flos Rosae Rugosas pollen; Flos Rosae Rugosas pollen soak with ethanol 0.5h, reflux, extract, 2 times, each 1h, obtain extracting solution, filters, and decompression recycling ethanol is to nothing alcohol flavor, and concentrated, the concentrated solution adding distil water is dispersed into sample liquid; On sample liquid, AB-8 type macroporous adsorbent resin, first use the distilled water eluting, discards water liquid; Use ethanol elution, decompression recycling ethanol, be concentrated into dry powder again, obtains Flos Rosae Rugosae total flavones (material); This Flos Rosae Rugosae total flavones (material) is effective to prepare anti-cerebral ischemia drugs, realizes the application of Flos Rosae Rugosae extract in preparing anti-cerebral ischemia drugs.
Abundant raw material of the present invention, easily preparation, extract has treating cerebral ischemia, is effective to prepare anti-cerebral ischemia drugs, has opened up the new purposes of Flos Rosae Rugosae, has improved the medical value of Flos Rosae Rugosae, is the innovation on medicine.
The specific embodiment
Below in conjunction with concrete condition, this bright specific embodiment is elaborated.
The present invention, in concrete enforcement, is realized by following concrete grammar: Flos Rosae Rugosas pollen was broken into to the medicated powder of 60-80 mesh sieve, with the petroleum ether reflux extraction 1h defat of 10 times of medicated powder weight; Flos Rosae Rugosas medicine powder filtering residue after defat, fling to petroleum ether to dry, obtains Flos Rosae Rugosas pollen; Flos Rosae Rugosas pollen adds the soak with ethanol 0.5h that 10 Chinese gall Beihua grain weight amounts, volumetric concentration are 60%, reflux, extract, 2 times, each 1h, obtain extracting solution, filter, decompression recycling ethanol, to without the alcohol flavor, is concentrated into the concentrated solution of relative density 1.1-1.4, and the concentrated solution adding distil water is dispersed to the sample liquid be equivalent to containing crude drug amount 0.3g/ml; AB-8 type macroporous adsorbent resin on sample liquid, first use 2 times of column volume distilled water eluting, discards water liquid; With 3 times of column volumes, ethanol elution that volumetric concentration is 10%, discard eluent; Again with 5 times of column volumes, ethanol elution that volumetric concentration is 80%, the ethanol elution that collected volume concentration is 80%, decompression recycling ethanol, be concentrated into dry powder, obtains Flos Rosae Rugosae total flavones (material), general flavone content >=63%; This Flos Rosae Rugosae total flavones (material) is effective to prepare anti-cerebral ischemia drugs, realizes the application of Flos Rosae Rugosae extract in preparing anti-cerebral ischemia drugs.
Through experimentation, the Flos Rosae Rugosae total flavones can significantly improve mice normal pressure and decompression hypoxia-bearing capability; Mouse brain ischemia, mice blood stasis cerebral ischemic model are all had to good improvement effect, can improve hemorheology index, improve the brain energy metabolism; Cerebral Ischemia-reperfusion in Mice, rat cerebral ischemia are also had to fine improvement effect, can alleviate brain tissue impairment, improve Energy Metabolism of Brain Tissue, improve inflammatory factor level etc.But the concrete active component of anti-cerebral ischemia is also indefinite, in the rhodinol extract, main active is total flavones.By observing the impact of Flos Rosae Rugosae total flavones on Cerebral Ischemia-reperfusion in Mice model, focal cerebral ischemic in mice re-perfusion model, Focal Ischemia-Reperfusion in Rats model, it improves the pharmacological action of cerebral ischemia reperfusion injury further investigated, clear and definite its pharmacological component, to developing a kind of medicine that reduces the cerebral ischemia reperfusion injury effect, also for the good treatment cerebral ischemia syndrome medicament of exploitation curative effect, establish experiment basis, relevant experimental data is as follows:
The impact of Flos Rosae Rugosae total flavones on the Focal Ischemia-Reperfusion in Rats model
1 experiment material
1.1 Experimental agents
Flos Rosae Rugosae total flavones of the present invention; Nimodipine tablet, produced lot number 1105036 by Shandong XinHua Pharmacy stock Co., Ltd; NAOLUOTONG JIAONANG, produced lot number 201109002 by Hayao Group Sanjing Pharmaceutical 4th Factory Co., Ltd..
1.2 experiment reagent
Sodium chloride injection, produced lot number 11072121 by Zhengzhou Yonghe Pharmaceutical Co; Sodium carboxymethyl cellulose, learn reagent Manufacturing Co., Ltd by permanent Xinghua, Tianjin and produce, lot number 20060728; Chloral hydrate, produced lot number 20120401 by upper seamount Pu chemical industry company limited; Formalin, produced lot number 20120406 by Tianjin Kermel Chemical Reagent Co., Ltd.; Ethanol (medical alcohol), produced lot number 20120405 by the big disinfectant preparation company limited in Xinxiang City three; Penicillin, by HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory production, lot number 20120507; Adenosine triphosphate (ATP) enzyme testing cassete, build up bio-engineering research by Nanjing and produced, lot number 20130111; The Coomassie brilliant blue testing cassete, build up bio-engineering research by Nanjing and produced, lot number 20130115; The nitric oxide testing cassete, build up bio-engineering research by Nanjing and produced, lot number 20130111; The NOS testing cassete, build up bio-engineering research by Nanjing and produced, lot number 20130114; Malonaldehyde (MDA) testing cassete, build up bio-engineering research by Nanjing and produced, lot number 20130108; The SOD testing cassete, build up bio-engineering research by Nanjing and produced, lot number 20130114; S-100 β protein ELISA detection kit, by R& D produces, lot number 20130101A; TNF-α ELISA detection kit, by R& D, lot number: 20130101A; The ICAM-1ELISA detection kit, by R& D produces, lot number 20130101A; IL-1 β ELISA detection kit, by R& D produces, lot number 20130101A.
1.3 instrument
The UV-2000 ultraviolet-uisible spectrophotometer, produced by UNICO(Shanghai) Instruments Co., Ltd.; The TGL-16G desk centrifuge, produced by Anting Scientific Instrument Factory, Shanghai; The JA1103N electronic analytical balance, produced by Ao Haosi (Shanghai) company; DZKW-4 type electronic thermostatic water-bath, by Beijing forever bright Medical Instruments factory produce; Adjustable pipette, produced by Shanghai Lei Bo Analytical Instrument Co., Ltd; Ultrasonic cleaner, produced by Shanghai High Kudos Science Instrument Co., Ltd.; The glass refiner, produced by Zhengzhou Glassware Factory.
1.4 laboratory animal
The Wistar rat, male, SPF level, body weight 230-250g, Lukang Medical Co., Ltd., Shandong, this batch of rat quality certification number 0013889.
2 experimental techniques
2.1 modeling and administration
112 of the wistar rats that to get body weight be 260-290g, male, evenly be divided into immediately 7 groups, that is: sham operated rats, model group, nimodipine group, brain ruton group, the large, medium and small dosage group of Flos Rosae Rugosae total flavones, 16 every group.Nimodipine group (positive control drug, dosage is 20mg/kg, is made into drug level 2mg/ml with 0.5%CMC before use, 1ml/100g, 10 times of quantity); NAOLUOTONG JIAONANG group (positive control drug, dosage is 500mg/kg, is made into drug level 50mg/ml with 0.5%CMC before use, 1ml/100g, 10 times of quantity); The large, medium and small dosage group of Flos Rosae Rugosae total flavones (200mg/kg, 100mg/kg, 50mg/kg are made into drug level 20mg/ml, 10mg/ml, 5mg/ml, 1ml/100g with 0.5%CMC before use); Sham operated rats, model group gavage same volume 0.5%CMC, administration every day 1 time, successive administration 7d.
In at 8 in 6d evening in batches fasting can't help water, 7d 8 a.m. batch weighing, after administration 1h, 10% chloral hydrate (0.3ml/100g) intraperitoneal injection of anesthesia rat, cervical region hits exactly the otch of taking back, successively separate and expose left carotid (CCA), external carotid artery (ECA), internal carotid artery (ICA), ligation common carotid artery and external carotid artery, close internal carotid artery with the temporary transient folder of bulldog clamp, cut the 0.2mm osculum in common carotid artery apart from bifurcated 5mm place, the line bolt is inserted, enter internal carotid artery through the common carotid artery furcation, upwards deeply to the above 18-20mm of bifurcated, until resistance is arranged, block the middle cerebral artery porch, ligation internal carotid artery (a blank group exposure left side blood vessel is not made plug wire and processed), extract gently the bolt line after 2h out.All rats thromboembolism left side middle cerebral artery, set up middle cerebral artery occlusion-pour into again (MCAO) animal model.
2.2 detect index and assay method
2.2.1.24h after carry out the neuroethology scoring: adopt the Zealonga point system.Standards of grading: 0 minute: impassivity afunction symptom, movable normal person; 1 minute: can not full extension offside fore paw person; 2 minutes: while creeping, occur to the hemiplegia side person of turn-taking; 3 minutes: during walking, health rolled the person to hemiplegia; 4 minutes: can not spontaneously walk, the loss of consciousness person; 5 minutes: death.Scoring is to reject more than 0 minute and 4 minutes); Pluck eyeball and get blood, after half an hour, centrifugal, get supernatant and survey S-100 β protein content; After the rat broken end, strip rapidly cerebral tissue, half brain prepares 10% brain homogenate for detection of MDA, SOD, NO, NOS, TNF-α, IL-1 β, ICAM-1, ATP enzyme, Coomassie brilliant blue; Second half cerebral tissue, fix by 10% formalin, for HE dyeing, observes cerebral morphology and change.
2.2.2MDA kit measurement method:
The measurement operation sequence list of MDA in brain homogenate
Eddy blending machine mixes, and the test tube mouth is tightened with preservative film, with syringe needle, stings an aperture, 95 ℃ of water-bath 40min, and after taking out, flowing water is cooling, and then 4000 rev/mins, centrifugal 10min, get supernatant, 520nm, the 1cm optical path, the distilled water zeroing, survey and respectively manage absorbance
MDA content (nmol/mgprot) in tissue=(measuring pipe absorbance-mensuration blank tube absorbance) * standard substance concentration ÷ (standard pipe absorbance-standard blank tube absorbance) ÷ protein content (mgprot/ml)
2.2.3NO kit measurement method:
In brain homogenate, NO measures sequence list
NO content in tissue (μ mol/gprot)=(measuring pipe absorbance-mensuration blank tube absorbance) * standard substance concentration ÷ (standard pipe absorbance-standard blank tube absorbance) ÷ protein content (mgprot/ml)
2.2.4NOS kit measurement method:
In brain homogenate, NOS measures sequence list
TNOS vigor (U/ml)=(TNOS measures pipe-TNOS blank tube) ÷ colour generation thing nanomole extinction coefficient * (reactant liquor cumulative volume ÷ gets liquid measure) ÷ (colorimetric optical path * response time) ÷ sample protein content (mggrot/L)
2.2.5SOD kit measurement method:
In brain homogenate, SOD measures sequence list
Mix, room temperature is placed 10min, in wavelength 550nm place, and 1cm optical path cuvette, distilled water zeroing, colorimetric.
Extension rate before the extension rate * sample test of SOD vigor (U/ml)=(control tube absorbance-mensuration pipe absorbance) ÷ control tube absorbance ÷ 50% * reaction system
2.2.6 Rat Interleukin 1 β (IL-1 β) ELISA detection kit assay method
Detect principle: test kit adopts double antibody one step sandwich assay elisa (ELISA).Toward being coated with in the coated micropore of interleukin-1 beta (IL-1 β) antibody in advance, add successively the detection antibody of specimen, standard substance, HRP labelling, through incubation thoroughly washing.With substrate TMB colour developing, TMB changes into blueness under the catalysis of peroxidase, and changes into final yellow under sour effect.The depth of color and the interleukin-1 beta in sample (IL-1 β) are proportionate.Measure absorbance (OD value) by microplate reader, calculation sample concentration under the 450nm wavelength.
Operating procedure: take out required lath from the aluminium foil bag equilibrium at room temperature 20min, the residue lath is put back to 4 ℃ with the valve bag sealing.Standard substance hole and sample aperture are set, and the standard substance hole adds the standard substance 50 μ l of various variable concentrations; Sample aperture first adds sample to be tested 10 μ l, then adds sample diluent 40 μ l; Blank well does not add.Except blank well, in standard substance hole and sample aperture, every hole adds the detection antibody 100 μ l of horseradish peroxidase (HRP) labelling, with the shrouding film, seals reacting hole, 37 ℃ of water-baths or calorstat incubation 60min.Discard liquid, pat dry in absorbent paper, cleaning mixture is filled it up with in every hole, and standing 1min, get rid of cleaning mixture, in absorbent paper, pats dry, and so repeats to wash plate 5 times (the also available plate machine washing plate of washing).Every hole adds substrate A, each 50 μ l of B, and 37 ℃ of lucifuges are hatched 15min.Every hole adds stop buffer 50 μ l, in 15min, measures the OD value in each hole at 450nm wavelength place.
The drawing standard curve: in the Excel worksheet, with standard substance concentration, make abscissa, corresponding OD value is made vertical coordinate, draws out the standard substance linear regression, by curvilinear equation, calculates each sample concentration value.
2.2.7 rat S100 β albumen (S-100 β) ELISA detection kit assay method
Detect principle, operating procedure: same interleukin-1 ' beta '
2.2.8 rat tumor necrosin & (TNF-α) ELISA detection kit assay method
Detect principle, operating procedure: same interleukin-1 ' beta '
2.2.9 adhesion molecule 1(ICAM-1 between rat cell) ELISA detection kit assay method
Detect principle, operating procedure: same interleukin-1 ' beta '
3. statistical procedures method
Data analysis carries out the statistical procedures of data information, average ± standard deviation for measurement data with the medical statistics bag of SPSS17.0for windows
Mean, relatively adopt one factor analysis of variance between each group, the neat person of variance test adopts the Ridit check with least significant difference (LSD) method, heterogeneity of variance person with Games-Howell method check, ranked data.
4. experimental result
4.1 on the impact of evaluating focal brain ischemia in rats function of nervous system scoring, mortality rate in Table 7
The impact of table 7 on the scoring of evaluating focal brain ischemia in rats function of nervous system, mortality rate
*: with model group, compare P<0.05; *: with model group, compare P<0.01
As seen from the above table: rats in sham-operated group does not have the neurological deficit performance, after residue is respectively organized rat ischemia 24h, delayed ischemic neurological deficits in various degree all occurred; With sham operated rats, compare, model group has significant statistical significance (P<0.01), and the modeling success is described; Nimodipine group obvious difference (P<0.05), it can obviously improve nervous function damage; Brain ruton group, the large, medium and small dosage group of Flos Rosae Rugosae total flavones have significant statistical significance (P<0.01), can significantly improve the nervous function damage of rat, significantly reduce the scoring of rat neurological deficit.The mortality rate of model group is the highest, and mortality rate of each group of administration all decreases, and the reduction mortality rate that each group of administration all can be in various degree be described, reduces brain tissue impairment, protects cerebral tissue.
4.2 on the impact of evaluating focal brain ischemia in rats brain homogenate NO and NOS vigor in Table 8
The impact of table 8 on evaluating focal brain ischemia in rats brain homogenate NO and NOS vigor
*: with model group, compare P<0.05; *: with model group, compare P<0.01
As seen from the above table, with sham operated rats, compare, in the model group rat cerebral even slurry, NO content significantly raises (P<0.01), and the NOS vigor significantly strengthens (P<0.01), and the success of Focal Cerebral Ischemia Reperfusion model is described.With model group, compare, nimodipine, brain ruton, the large, medium and small dosage group of Flos Rosae Rugosae total flavones all can significantly reduce NO content (P<0.01), significantly weaken NOS vigor (P<0.01).Show that the Flos Rosae Rugosae total flavones is significantly improved effect to the variation of NO content and NOS vigor in Focal Ischemia-Reperfusion in Rats model brain homogenate.
4.3 on the impact of evaluating focal brain ischemia in rats brain homogenate MDA and SOD vigor in Table 9
The impact of table 9 on evaluating focal brain ischemia in rats brain homogenate MDA and SOD vigor
*: with model group, compare P<0.05; *: with model group, compare P<0.01
As seen from the above table, with sham operated rats, compare, in model group rat ischemia side cerebral tissue, MDA content (P<0.01) significantly raises, and SOD level (P<0.01) significantly reduces, and the success of Focal Cerebral Ischemia Reperfusion model is described; With model group, compare, nimodipine group, brain ruton group, the large, medium and small dosage group of Flos Rosae Rugosae total flavones all can significantly reduce the MDA content (P<0.01) in cerebral tissue, significantly increased SOD level (P<0.01).Show that the Flos Rosae Rugosae total flavones can significantly reduce evaluating focal brain ischemia in rats cerebrum lipid peroxidating degree, and then the protection cerebral tissue.
4.4 on the impact of evaluating focal brain ischemia in rats brain homogenate ATP enzyme activity in Table 10
The impact of table 10 on evaluating focal brain ischemia in rats brain homogenate ATP enzyme activity
*: with model group, compare P<0.05; *: with model group, compare P<0.01
As seen from the above table: compare the Na in the rat cerebral tissue of model group with sham-operation
+K
+-ATP enzyme, Mg
++-ATP enzyme, Ca
++-ATP enzyme activity significantly reduces (P<0.01), and the success of Focal Cerebral Ischemia Reperfusion model is described; Compare Na in nimodipine group, brain ruton group, the large, medium and small dosage group of Flos Rosae Rugosae total flavones cerebral tissue with model group
+K
+-ATP enzyme, Mg
++-ATP enzyme, Ca
++-ATP enzyme activity is significantly rising (P<0.01) all.Show that the Flos Rosae Rugosae total flavones is to Na in the rat brain focal cerebral ischemia/reperfusion injury
+K
+-ATP enzyme, Mg
++-ATP enzyme, Ca
++The variation of-ATP enzyme activity is improved effect, improves energy metabolism impairment in cerebral tissue.
4.5 on evaluating focal brain ischemia in rats brain homogenate TNF-α, IL-1 β content impact in Table 11
The impact of table 11 on evaluating focal brain ischemia in rats brain homogenate TNF-α, IL-1 β content
*: with model group, compare P<0.05; *: with model group, compare P<0.01
As seen from the above table: with sham operated rats, compare, in model group rat ischemia side cerebral tissue, TNF-alpha content (P<0.01), IL-1 β content (P<0.01) significantly raise, and the success of Focal Ischemia-Reperfusion in Rats model is described; With model group, compare, nimodipine group, brain ruton group, the large, medium and small dosage group of Flos Rosae Rugosae total flavones all can significantly reduce TNF-alpha content (P<0.01), the IL-1 β content (P<0.01) in cerebral tissue.Show that the Flos Rosae Rugosae total flavones can significantly reduce evaluating focal brain ischemia in rats cerebral tissue TNF-α, IL-1 β content.
4.6 on the impact of evaluating focal brain ischemia in rats brain homogenate ICAM-1, serum S-100 β content in Table 12
The impact of table 12 on evaluating focal brain ischemia in rats brain homogenate ICAM-1, serum S-100 β content
*: with model group, compare P<0.05; *: with model group, compare P<0.01
As seen from the above table: with sham operated rats, compare, model group rat ischemia side cerebral tissue ICAM-1 content significantly raises (P<0.01), and serum S-100 β content significantly raises (P<0.01), and the success of Focal Cerebral Ischemia Reperfusion model is described; With model group, compare, nimodipine group, brain ruton group, the large, medium and small dosage group of Flos Rosae Rugosae total flavones all can significantly reduce the ICAM-1 content (P<0.01) in cerebral tissue, the S-100 β content (P<0.01) in serum.Show that the Flos Rosae Rugosae total flavones can significantly reduce evaluating focal brain ischemia in rats cerebral tissue ICAM-1 content, the S-100 β content in serum.Especially the heavy dose of best results of Flos Rosae Rugosae total flavones.
4.7 on the impact of evaluating focal brain ischemia in rats cerebral tissue cortical areas pathological change in Table 13
The Flos Rosae Rugosae total flavones is as follows to Focal Ischemia-Reperfusion in Rats model brain cortex Histopathological Studies: sham operated rats brain cortical neural cell normal, neurogliocyte normal; The degeneration of model group brain cortical neural cell, the most of pyknosis of neurogliocyte; Nimodipine group brain cortical neural cell reduces and degeneration, and neurogliocyte increases, most of normal; The brain cortical neural cell degeneration of brain ruton group or atrophy, the neurogliocyte normal; The heavy dose of group of Flos Rosae Rugosae total flavones brain cortical neural cell normal, the neurogliocyte normal; The most of brain cortical neural cell of dosage group normal in the Flos Rosae Rugosae total flavones, the neurogliocyte normal; The degeneration of Flos Rosae Rugosae total flavones small dose group brain cortex minority neurocyte, the pyknosis of neurogliocyte minority.
The pathological change of table 13 pair Focal Ischemia-Reperfusion in Rats cerebral tissue cortex
"-" neurocyte normal, the neurogliocyte normal; The degeneration of "+" minority neurocyte, the neurogliocyte normal; The most of degeneration of " ++ " neurocyte, neurogliocyte normal or part pyknosis; " +++" all neurocyte degeneration, the complete pyknosis of neurogliocyte or disappearance.According to semi-quantitative standards, each group of laboratory animal is measured
Through the Ridit check, with sham operated rats, to compare, ischemia-reperfusion group has significant statistical significance (P<0.01), and the modeling success is described.With model group, compare, brain ruton group has obvious statistical significance (P<0.05), illustrates that it obviously improves the damage of cerebral tissue; The large, medium and small dosage group of Flos Rosae Rugosae total flavones has significant statistical significance (P<0.01); Results suggest: the Flos Rosae Rugosae total flavones significantly improves the degree of injury of Focal Cerebral Ischemia Reperfusion model cerebral tissue cortical areas neurocyte, neurogliocyte.
4.8 on the impact of evaluating focal brain ischemia in rats cerebral tissue hippocampus nerve nucleus pathological change in Table 14
The Flos Rosae Rugosae total flavones is as follows to Focal Ischemia-Reperfusion in Rats model cerebral tissue nerve nucleus pathological observation: sham operated rats cerebral tissue nerve nucleus cell normal, glial cell normal; Model group cerebral tissue nerve nucleus cytopathy, the most of pyknosis of glial cell; Nimodipine group cerebral tissue nerve nucleus cytopathy, the most of pyknosis of glial cell; Brain ruton group cerebral tissue nerve nucleus cytopathy companion edema, the glial cell pyknosis; The heavy dose of group of Flos Rosae Rugosae total flavones cerebral tissue nerve nucleus cell normal, the pyknosis of glial cell part; The indivedual degeneration of dosage group cerebral tissue nerve nucleus cell in the Flos Rosae Rugosae total flavones, the glial cell pyknosis; Flos Rosae Rugosae total flavones small dose group cerebral tissue nerve nucleus cell normal, the glial cell normal.
Table 14 pair evaluating focal brain ischemia in rats cerebral tissue nerve nucleus pathological change semiquantitative determination result
"-" neurocyte normal, the neurogliocyte normal; The degeneration of "+" minority neurocyte, the neurogliocyte normal; " ++ " partial nerve cytopathy, neurogliocyte normal or part pyknosis; " +++" all neurocyte degeneration, the complete pyknosis of neurogliocyte or disappearance
Through the Ridit check, with sham operated rats, to compare, ischemia-reperfusion group has significant statistical significance (P<0.01), and the modeling success is described.With model group, compare, the large, medium and small dosage group of Flos Rosae Rugosae total flavones has significant statistical significance (P<0.01); Results suggest: the Flos Rosae Rugosae total flavones significantly improves the degree of injury of ischemia-reperfusion injury model cerebral tissue nerve nucleus, glial cell.
5 conclusions
S-100 β protein content and the cortical areas of cerebral tissue and the pathological change of nerve nucleus in NO, NOS, MDA, SOD, IL-1 β, ICAM-1, TNF-a, serum in function of nervous system's scoring by observing the Focal Ischemia-Reperfusion in Rats model, mortality rate, brain homogenate, prompting Flos Rosae Rugosae total flavones Flos Rosae Rugosae total flavones can improve the large neural afunction scoring of experiment, mortality rate, improves the pathological changes situation of cerebral cortex and hippocampus; By removing free radical, suppress lipid peroxidation, thereby alleviate the cerebral ischemia reperfusion injury degree; Inflammatory reaction after the inhibition ischemia, and improve a series of cascade reaction media and the cytokine that inflammation causes, thus cerebral ischemia reperfusion injury is played a protective role, best with the heavy dose of group of Flos Rosae Rugosae total flavones.
By above-mentioned data, shown, Flos Rosae Rugosae total flavones prepared by the present invention has the effect of anti-cerebral ischemia, is effective to prepare the medicine of anti-cerebral ischemia, has enlarged the medical value of Flos Rosae Rugosae, is the innovation in the medicine preparation, and good economic and social benefit is arranged.
Claims (2)
1. extract the method for anti-cerebral ischemia material in a Flos Rosae Rugosae, it is characterized in that, Flos Rosae Rugosas pollen was broken into to the medicated powder of 60-80 mesh sieve, with the petroleum ether reflux extraction 1h defat of 10 times of medicated powder weight; Flos Rosae Rugosas medicine powder filtering residue after defat, fling to petroleum ether to dry, obtains Flos Rosae Rugosas pollen; Flos Rosae Rugosas pollen adds the soak with ethanol 0.5h that 10 Chinese gall Beihua grain weight amounts, volumetric concentration are 60%, reflux, extract, 2 times, each 1h, obtain extracting solution, filter, decompression recycling ethanol, to without the alcohol flavor, is concentrated into the concentrated solution of relative density 1.1-1.4, and the concentrated solution adding distil water is dispersed to the sample liquid be equivalent to containing crude drug amount 0.3g/ml; AB-8 type macroporous adsorbent resin on sample liquid, first use 2 times of column volume distilled water eluting, discards water liquid; With 3 times of column volumes, ethanol elution that volumetric concentration is 10%, discard eluent; Again with 5 times of column volumes, ethanol elution that volumetric concentration is 80%, the ethanol elution that collected volume concentration is 80%, decompression recycling ethanol, be concentrated into dry powder, obtains the Flos Rosae Rugosae total flavones.
2. the application of Flos Rosae Rugosae total flavones in preparing anti-cerebral ischemia drugs that the described method of claim 1 is extracted.
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Cited By (5)
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| CN103655802A (en) * | 2013-12-04 | 2014-03-26 | 中国农业大学 | Method for extracting antioxidant active component from rose leaf |
| CN104083476A (en) * | 2014-07-24 | 2014-10-08 | 河南中医学院 | Application of rose extract in preparing anti-depression medicaments |
| CN107441152A (en) * | 2017-08-12 | 2017-12-08 | 河南中医药大学 | A kind of flower of kudzuvine general flavone is preparing the application in treating brain ischemia medicament repeatedly |
| CN108185432A (en) * | 2017-12-31 | 2018-06-22 | 西双版纳天恩生物科技开发有限公司 | The preparation method of Flos Rosae Rugosas pollen is lyophilized |
| CN115137772A (en) * | 2022-05-26 | 2022-10-04 | 苏州大学 | Application of rose extract in preparing medicine for preventing and treating diseases caused by premature senility of alveolar epithelial cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103655802A (en) * | 2013-12-04 | 2014-03-26 | 中国农业大学 | Method for extracting antioxidant active component from rose leaf |
| CN103655802B (en) * | 2013-12-04 | 2016-06-08 | 中国农业大学 | The extracting method of anti-oxidation active substance in a kind of rose leaf |
| CN104083476A (en) * | 2014-07-24 | 2014-10-08 | 河南中医学院 | Application of rose extract in preparing anti-depression medicaments |
| CN107441152A (en) * | 2017-08-12 | 2017-12-08 | 河南中医药大学 | A kind of flower of kudzuvine general flavone is preparing the application in treating brain ischemia medicament repeatedly |
| CN108185432A (en) * | 2017-12-31 | 2018-06-22 | 西双版纳天恩生物科技开发有限公司 | The preparation method of Flos Rosae Rugosas pollen is lyophilized |
| CN115137772A (en) * | 2022-05-26 | 2022-10-04 | 苏州大学 | Application of rose extract in preparing medicine for preventing and treating diseases caused by premature senility of alveolar epithelial cells |
| CN115137772B (en) * | 2022-05-26 | 2024-05-10 | 苏州大学 | Application of rose extract in preparation of medicine for preventing and treating diseases caused by premature aging of alveolar epithelial cells |
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