CN102935100A - Preparation method and applications of queen lagerstroemia folium apocyni veneti general flavones - Google Patents
Preparation method and applications of queen lagerstroemia folium apocyni veneti general flavones Download PDFInfo
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Abstract
The invention relates to a preparation method and applications of queen lagerstroemia folium apocyni veneti general flavones. The method adopts a membrane separation technology to extract and separate extract of queen lagerstroemia folium apocyni veneti so as to obtain queen lagerstroemia folium apocyni veneti general flavones; and animal test proves that the queen lagerstroemia folium apocyni veneti general flavones obtained by the method have good preventive and therapeutic effect on Alzheimer's disease, thereby effectively solving the problem of medicament for preventing and treating Alzheimer's disease, realizing prevention and treatment on Alzheimer's disease, and developing new medical value of queen lagerstroemia folium apocyni veneti general flavones.
Description
Technical field
The present invention relates to a kind of preparation method and purposes of aprocynum henderson hook leaf flavonoids, particularly from poacynum hendersonii woodson leaf, extract and separate the application of total flavones in preparation control alzheimer disease drug that obtains.
Background technology
Alzheimer's disease (Alzheimer
,S disease, AD) also claim that senile dementia is a kind of gradual central nervous system degenerative disease of being more common in middle-aged and elderly people, take progressive cognitive disorder, the hypophrenia and personality changes as clinical main manifestations.In elderly population, become at present the 4th cause of death that is only second to heart disease, malignant tumor and apoplexy.At present not yet clear and definite about the pathogenesis of AD; also there is no effective treatment means; doctor trained in Western medicine mainly adopts the Drug therapys such as cholinesterase inhibitor, glutamate receptor antagonists, antioxidant, neuroprotective; but these medicines can only play the effect of alleviating some symptom; can't replenish refreshing eventually unit, nerve synapse and the neurotransmitter lost in a large number in the cerebral tissue; can not effectively delay, termination or reverse disease progress, and the poisonous side effect of medicine that has is larger.
A large amount of clinical report and clinical practices show: Chinese medicine demonstrates preferably curative effect and potential applicability in clinical practice in the AD treatment, particularly delaying to have potential advantage aspect the disease progression.Experimental data shows that many Chinese medicines have the effect of antagonism AD cell and animal model neuronal damage and apoptosis.Along with research deeply and the accumulation of clinical application experience we research emphasis is placed on the peculiar Chinese medicine aprocynum henderson hook in Xinjiang.The domestic ground such as Xinjiang Yili of China, Kuerle, Altay that mainly are distributed in of wild Flos Caryophylli hemp, be grown in saline and alkaline, the sandy wasteland is regional and Gobi desert on the perennial root plant resources, unique growing environment makes it have very special medicinal health effect.The Xinjiang aprocynum henderson hook is used as medicine and has history in thousand, its Pharmacological be it is found that very early and is used for clinical, according to Compendium of Material Medica, herbal for Relief of Famines record, the effect such as its Folium Apocyni Veneti has clearing away heat and promoting diuresis, suppressing the hyperactive liver, calm the nerves, the clinical diseases such as hypertension, coronary heart disease, cervical spondylosis, nephritis edema, neurasthenia, climacteric syndrome, formal typing Pharmacopoeia of the People's Republic of China in 1977 of being used for the treatment of.Modern pharmacology studies have shown that poacynum hendersonii woodson leaf is nutritious, is rich in the compositions such as lupeol, hyperin, Trifolin, several amino acids, polysaccharide, vitamin.Wherein Flavonoid substances is the main effective site of Folium Apocyni Veneti, studies show that in recent years that the aprocynum henderson hook leaf flavonoids had the effects such as antioxidation, defying age, antithrombotic, enhancing human body immunity, but the application of aprocynum henderson hook leaf flavonoids in the control of alzheimer's disease is there are no report, and medicinal application also has no report to this total flavones as preparation control alzheimer's disease simultaneously.
The extraction of relevant Folium Apocyni Veneti total flavones separates currently reported, mainly contains 1. water extract-alcohol precipitation, resin separation purification; 2. organic solvent extraction, the macroporous resin separation and purification.These methods all exist extraction ratio lower to some extent, and consumption of organic solvent is large, the processing time is long, environmental pollution is serious, be unfavorable for the shortcomings such as health.Therefore study environmental protection, the Chinese medicine ingredients extraction separation method has become one of advanced subject of current biological medicine simply and easily.Membrane separation technique is according to the size of molecule relative molecular mass, and the effects such as the screening by fenestra, absorption realize optionally separating to the different component in the system, reach separation and purification on the molecular level, and it belongs to a kind of physical separation method.Membrane separation technique especially has great advantage to the separation of Chinese medicine, and it does not need to add chemical reagent, non-secondary pollution, and separation temperature is less demanding, and is little to active component destruction in the Chinese medicine, has again certain decolorization simultaneously.Membrance separation can effectively be removed microgranule, pyrogen, microorganism and other invalid components, can significantly improve the quality of product and stability thereof, safety.This technology has that loss of activity is little, separating step is few, operating condition is gentle, is easy to that technique is amplified and the advantage such as continued operation.
Summary of the invention
The preparation method and the purposes that the purpose of this invention is to provide a kind of aprocynum henderson hook leaf flavonoids, the method adopts membrane separation technique that poacynum hendersonii woodson leaf extract has been carried out extracting separation, obtain the aprocynum henderson hook leaf flavonoids, the aprocynum henderson hook leaf flavonoids that obtains by the method has confirmed that through animal experiment the aprocynum henderson hook leaf flavonoids has good preventive and therapeutic effect to alzheimer's disease, effectively solve the medicine problem of control alzheimer's disease, opened up the new medical value of aprocynum henderson hook leaf flavonoids.
The preparation method of a kind of aprocynum henderson hook leaf flavonoids of the present invention follows these steps to carry out:
A, the poacynum hendersonii woodson leaf of drying was pulverized the 20-50 mesh sieve became medicated powder, the concentration of doubly measuring with 8-10 is alcohol heating reflux 1-2 hour of 60-80%, extract 2-3 time, merge extractive liquid,, filter, Recycled ethanol, and be concentrated into when temperature 50 C that to measure relative density be 1.10-1.20, obtain concentrated solution;
B, with the solution left standstill of by volume 1:1 dilution of step a concentrated solution water, filter, filtrate is with 15000 rev/mins of centrifugalize, get centrifugal rear gained supernatant, micro-filtration membrane with aperture 0.02-0.1 μ m is filtered except heavy, transmembrane pressure is 0.5Mpa-1.0Mpa, feed temperature 30-50 ℃, obtains permeate;
C, the permeate of step b is carried out hollow fiber ultrafiltration membrane separate, transmembrane pressure 0.5Mpa-1.0Mpa, obtains ultrafiltrate by feed temperature 30-50 ℃;
D, the ultrafiltrate of step c is carried out hollow fiber nanofiltration membrane again separate, transmembrane pressure 1.0Mpa-2.0Mpa, gets concentrated solution by feed temperature 30-50 ℃;
E, with steps d gained concentrated solution concentrating under reduced pressure, be drying to obtain aprocynum henderson hook effective ingredient total flavones.
The molecular cut off of step b hollow fiber ultrafiltration membrane is 2000-8000.
The molecular cut off of step c hollow fiber nanofiltration membrane is 200-600.
A kind of purposes of aprocynum henderson hook leaf flavonoids, this aprocynum henderson hook leaf flavonoids are used for the medicine at preparation control alzheimer's disease.
The preparation method of a kind of aprocynum henderson hook leaf flavonoids of the present invention and purposes, its advantage is: the present invention has overcome the deficiency of existing extraction and separation technology, adopting membrane separation technique that poacynum hendersonii woodson leaf extract has been carried out extracting separates, membrane separation technique belongs to a kind of physical separation method, select different apertures to hold back according to molecular weight of material, neither destroy material composition, selective again, a kind of low energy consumption, oligosaprobic separation method.The method has improved the content of target component-total flavones, has removed most of invalid components, and the active component of product has obtained effective enrichment.The present invention is because have higher selectivity to separate targets, thus can change micro-filtration membrane, ultrafilter membrane and NF membrane specification and operating condition according to the needs of reality to product, thus control easily the composition of gained target product.With method provided by the invention separation and purification total flavones from poacynum hendersonii woodson leaf, the effective ingredient accumulation rate is high, loss is few, general flavone content reaches more than 90% in the product, under the prerequisite that does not change the effective ingredient structure, shortened the production cycle, reduced energy consumption, product quality is greatly improved.Method provided by the present invention has that separating step is few, operating condition is gentle is easy to that technique is amplified and the advantages such as continued operation, is a kind of environmental type abstraction technique.
Further specify the present invention below in conjunction with the specific embodiment, but the scope of protection of present invention is not limited to following embodiment.
The specific embodiment
Embodiment 1
Get dry poacynum hendersonii woodson leaf pulverizing medicinal materials, cross 20 mesh sieves, get 2kg medicated powder, adding 10 times of amount concentration is that 60% alcoholic solution reflux was extracted 2 times in 1 hour, and merge extractive liquid, filters, Recycled ethanol, and be concentrated in the time of 50 ℃ that to measure relative density be 1.10-1.20, obtain concentrated solution;
Be medical material with the concentrated solution dilute with water
:Medicinal liquid=1
:1 solution (refers to concentrated solution is diluted with water to the solution that equates with the medical material bulking value, it is the 100g medical material, concentrated solution is diluted to 100ml), leave standstill, filter, filtrate is with 15000 rev/mins of centrifugalize, get centrifugal rear gained supernatant, adopt the micro-filtration membrane of aperture 0.02 μ m to filter except heavy, transmembrane pressure is 0.5Mpa, 30 ℃ of feed temperatures obtain permeate;
Be 2000 ultrafilter membrane ultrafiltration with gained permeate molecular cut off, transmembrane pressure 0.5Mpa, 30 ℃ of feed temperatures obtain ultrafiltrate;
It is that 200 hollow fiber nanofiltration membranes separate that ultrafiltrate is held back molecule again, transmembrane pressure 1.0Mpa, and 30 ℃ of feed temperatures obtain concentrated solution;
With gained concentrated solution concentrating under reduced pressure, be drying to obtain poacynum hendersonii woodson leaf effective ingredient total flavones 256g, content detection adopts high effective liquid chromatography for measuring (operation is referring to 2010 editions P196 of the Pharmacopoeia of the People's Republic of China in detail), and this product contains total flavones (take the hyperin note) as 92.6%.
Embodiment 2
Get dry poacynum hendersonii woodson leaf pulverizing medicinal materials, cross 30 mesh sieves, get 2kg medicated powder, adding 8 times of amount concentration is that 70% alcoholic solution reflux was extracted 2 times in 2 hours, and merge extractive liquid, filters, Recycled ethanol, and be concentrated in the time of 50 ℃ that to measure relative density be 1.10-1.20, obtain concentrated solution;
Be medical material with the concentrated solution dilute with water
:Medicinal liquid=1
:1 solution leaves standstill, and filters, and filtrate is got centrifugal rear gained supernatant with 15000 rev/mins of centrifugalize, filters except heavy with the micro-filtration membrane of aperture 0.04 μ m, and transmembrane pressure is 1.0Mpa, and 40 ℃ of feed temperatures obtain permeate;
Be 4000 ultrafilter membrane ultrafiltration with gained permeate molecular cut off, transmembrane pressure 1.0Mpa, 40 ℃ of feed temperatures obtain ultrafiltrate;
It is that 400 hollow fiber nanofiltration membrane separates that ultrafiltrate is carried out molecular cut off again, transmembrane pressure 2.0Mpa, and 40 ℃ of feed temperatures get concentrated solution;
Get gained concentrated solution concentrating under reduced pressure, be drying to obtain poacynum hendersonii woodson leaf effective ingredient total flavones 279g, content detection, this product contain total flavones (take the hyperin note) as 93.4%.
Embodiment 3
Get dry poacynum hendersonii woodson leaf pulverizing medicinal materials, cross 40 mesh sieves, get 2kg medicated powder, adding 9 times of amount concentration is that 70% alcoholic solution reflux was extracted 3 times in 1 hour, and merge extractive liquid, filters, Recycled ethanol, and be concentrated in the time of 50 ℃ that to measure relative density be 1.10～1.20, obtain concentrated solution;
Be medical material with the concentrated solution dilute with water
:Medicinal liquid=1
:1 solution leaves standstill, and filters, and filtrate is got centrifugal rear gained supernatant with 15000 rev/mins of centrifugalize, filters except heavy with the micro-filtration membrane of aperture 0.08 μ m, and transmembrane pressure is 1.0Mpa, and 40 ℃ of feed temperatures obtain permeate;
Be 8000 ultrafilter membrane ultrafiltration with gained permeate molecular cut off, transmembrane pressure 1.0Mpa, 40 ℃ of feed temperatures obtain ultrafiltrate;
It is that 600 hollow fiber nanofiltration membrane separates that ultrafiltrate is carried out molecular cut off again, transmembrane pressure 1.0Mpa, and 40 ℃ of feed temperatures get concentrated solution;
Get gained concentrated solution concentrating under reduced pressure, be drying to obtain poacynum hendersonii woodson leaf effective ingredient total flavones 304g, content detection, this product contain total flavones (take the hyperin note) as 94.9%.
Embodiment 4
Get dry poacynum hendersonii woodson leaf pulverizing medicinal materials, cross 50 mesh sieves, get 2kg medicated powder, adding 8 times of amount concentration is that 80% alcoholic solution reflux was extracted 2 times in 2 hours, and merge extractive liquid, filters, Recycled ethanol, and be concentrated in the time of 50 ℃ that to measure relative density be 1.10～1.20, obtain concentrated solution;
Be medical material with the concentrated solution dilute with water
:Medicinal liquid=1
:1 solution leaves standstill, and filters, and filtrate is got centrifugal rear gained supernatant with 15000 rev/mins of centrifugalize, filters except heavy with the micro-filtration membrane of aperture 0.1 μ m, and transmembrane pressure is 0.5Mpa, and 50 ℃ of feed temperatures obtain permeate;
Be 8000 ultrafilter membrane ultrafiltration with gained permeate molecular cut off, transmembrane pressure 0.5Mpa, 50 ℃ of feed temperatures obtain ultrafiltrate;
It is that 600 hollow fiber nanofiltration membrane separates that ultrafiltrate is carried out molecular cut off again, transmembrane pressure 2.0Mpa, and 50 ℃ of feed temperatures get concentrated solution;
Get gained concentrated solution concentrating under reduced pressure, be drying to obtain poacynum hendersonii woodson leaf effective ingredient total flavones 293g, content detection, this product contain total flavones (take the hyperin note) as 93.3%.
Embodiment 5
The purposes of aprocynum henderson hook leaf flavonoids in the medicine of preparation control alzheimer's disease by the method for the invention acquisition, through animal experiment, proof aprocynum henderson hook leaf flavonoids has good prevention effect to alzheimer disease, and relevant experimental data is as follows:
1 material:
1.1 laboratory animal: select 60 of healthy SD rats, male and female half and half, body weight 250 ± 20g, animal card number: SCXK (army) 2002006 is provided the cleaning level by The Fourth Military Medical University's Experimental Animal Center.Be divided at random operative control group, model group, total flavones group (high, in and low dosage) and Western medicine group, every group respectively 10;
1.2 reagent: A β
25-35Available from Sigma company, HPLC 〉=98%, the self-control of aprocynum henderson hook leaf flavonoids (purity〉90.0%), other reagent is analytical pure.(French PFIZER PGM company makes donepezil hydrochloride, batch number: 5136903);
1.3 key instrument: rat stereotaxic instrument (Xibei Photoelectric Instruments Factory, Xi'an production); Morris water maze system (anti-former teaching and research room of The Fourth Military Medical University provides): the Morris water maze is the cylindrical tank of the rustless steel of inwall black (d=100 cm, h=50 cm), a built-in height position adjustable is the black round platform movably, the round platform top diameter is that 10 cm. are at equidistant four gauge points in all directions that arrange of pond upper limb, with the subpoint of these four place of entry in the water surface and bucket bottom, the water surface and pond are divided into four quadrants, round platform is arranged in the middle of a certain quadrant. during experiment, make the depth of water flood round platform top 2 cm, to regulate between water temperature to 22～24 ℃. the indoor article ornaments are relative with the experimenter standing place fixing within whole experimental period, and strictly control noise jamming.Morris water maze system is take image pick-up card, video camera, image monitor etc. as main development hardware, each level built-in function data acquisition and statistical disposition of utilizing the image software bag to provide;
2 methods:
2.1 the preparation of reagent: A β
25-351.0 mg is dissolved in 500 μ l physiological saline solution (2g/L) ,-20 ℃ of preservations place 37 ℃ to hatch 7 days one-tenth gel states before use, and the aprocynum henderson hook leaf flavonoids is dissolved in the distilled water, and matching while using is used preposition room temperature, shakes up;
2.2 animal processing method and modeling: operative control group and model control group gavage give 10 ml/kg normal saline, day once, and continuous 20 days, the high, normal, basic dosage group of total flavones is gavage 80mg/kg respectively, 40mg/kg, 20mg/kg, day is once, continuous 20 days, Western medicine group rat gavage every day gives donepezil hydrochloride 0.33 mg/kg, once a day, and 20 days, respectively inject the physiological saline solution of 5 μ l in the 8th day operative control group bilateral hippocampus of gavage, other five groups of bilateral hippocampus are respectively injected A β
25-355 μ l(10 μ g), continue gavage, all animals are in gavage the 15th day, i.e. hippocampal injection A β
25-35Do water maze laboratory after 7 days, surveyed 6 days continuously, the orientation navigation experiment of row rat was namely respectively organized rat from four different directions quadrants observations respectively and is swum over to needed time of platform and distance, got the pathologic finding (conventional H E stained preparation) that tissue carries out the rat hippocampus position in the 20th day;
3 results:
3.1 behavioristics's experimental result: compare with operative control group, all prolong the incubation period of model control group rat each quadrant in the Morris water maze laboratory (referring to that rat does not move about in water, i.e. its time that disorients and consume) (
P＜0.05), the swimming distance second, fourth quadrant obviously shorten (
P＜0.05), illustrate that the stationkeeping ability of model control group rat descends.Total flavones gavage group (high, middle dosage), Western medicine group and model control group relatively all obviously shortenings of most quadrant intrinsic incubations (
P＜0.05), the swimming distance second, fourth quadrant obviously shorten (
P＜0.05), illustrate that control has certain effect; Total flavones gavage group (high, middle dosage) and Western medicine group rat no matter be incubation period or the distance of swimming all without obvious variation (
P 〉0.05), see Table 1, table 2:
3.2 histopathology: check with operative control group and compare that the model control group neurons of cerebral cortex in rats is painted more shallow, the neuron vacuolation, the karyopycnosis that have, pyramidal cell reduces, and the Neurons of Cerebral Cortex number slightly reduces, and arranges inhomogeneous; The hippocampal neurons number reduces, and is segmental and loses, and neuron is misaligned, lack unity and coherence visible a large amount of karyopycnosis non-viable non-apoptotic cell.Total flavones gavage group (high, middle dosage) neurons of cerebral cortex in rats number has no obvious minimizing, and hippocampal neurons is methodically arranged, marshalling, and cell number has no obvious minimizing.Western medicine group neurocyte form is intact, arranges more neat;
4 conclusions: although the AD pathogenesis is also very not clear and definite at present, oneself is extensively approved A β to amyloid (amyloidprotein, A β) as the initiation factor of AD morbidity
25-35Be the functional fragment of A β, experiment in vivo and vitro studies show that A β
25-35Can cause degradation AD sample symptom under injury of neurons in hippocampus, the learning and remembering ability.Confirm by test, the aprocynum henderson hook leaf flavonoids can obviously improve the learning and memory function of A amyloid-beta dementia rats, and can alleviate the pathological change of dementia rats cortex and Hippocampus, and has dose dependent, shown the aprocynum henderson hook leaf flavonoids the prevention and the treatment alzheimer's disease in good application prospect, for it is developed as the control alzheimer's disease drug provision experimental basis.
Claims (4)
1. the preparation method of an aprocynum henderson hook leaf flavonoids is characterized in that following these steps to carrying out:
A, the poacynum hendersonii woodson leaf of drying was pulverized the 20-50 mesh sieve became medicated powder, the concentration of doubly measuring with 8-10 is alcohol heating reflux 1-2 hour of 60-80%, extract 2-3 time, merge extractive liquid,, filter, Recycled ethanol, and be concentrated into when temperature 50 C that to measure relative density be 1.10-1.20, obtain concentrated solution;
B, with the solution left standstill of by volume 1:1 dilution of step a concentrated solution water, filter, filtrate is with 15000 rev/mins of centrifugalize, get centrifugal rear gained supernatant, micro-filtration membrane with aperture 0.02-0.1 μ m is filtered except heavy, transmembrane pressure is 0.5Mpa-1.0Mpa, feed temperature 30-50 ℃, obtains permeate;
C, the permeate of step b is carried out hollow fiber ultrafiltration membrane separate, transmembrane pressure 0.5Mpa-1.0Mpa, obtains ultrafiltrate by feed temperature 30-50 ℃;
D, the ultrafiltrate of step c is carried out hollow fiber nanofiltration membrane again separate, transmembrane pressure 1.0Mpa-2.0Mpa, gets concentrated solution by feed temperature 30-50 ℃;
E, with steps d gained concentrated solution concentrating under reduced pressure, be drying to obtain aprocynum henderson hook effective ingredient total flavones.
2. method according to claim 1, the molecular cut off that it is characterized in that step b hollow fiber ultrafiltration membrane is 2000-8000.
3. method according to claim 1, the molecular cut off that it is characterized in that step c hollow fiber nanofiltration membrane is 200-600.
4. the purposes of an aprocynum henderson hook leaf flavonoids is characterized in that this aprocynum henderson hook leaf flavonoids is used for the medicine at preparation control alzheimer's disease.
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| CN103393735A (en) * | 2013-08-09 | 2013-11-20 | 中国人民解放军兰州军区乌鲁木齐总医院 | Poacynum hendersonii woodson leaf total flavones dispersible tablet and preparation method thereof |
| CN104208112A (en) * | 2014-09-18 | 2014-12-17 | 乌鲁木齐华新分析测试高科技开发公司 | Method for extracting general flavone from folium apocyni veneti |
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| JP2017145266A (en) * | 2017-06-06 | 2017-08-24 | ネイチャーパワー株式会社 | Anti-dementia composition |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103083379A (en) * | 2013-02-28 | 2013-05-08 | 中国人民解放军兰州军区乌鲁木齐总医院 | Preparation method and application of snow chrysanthemum flavonoids |
| CN103393735A (en) * | 2013-08-09 | 2013-11-20 | 中国人民解放军兰州军区乌鲁木齐总医院 | Poacynum hendersonii woodson leaf total flavones dispersible tablet and preparation method thereof |
| CN104208112A (en) * | 2014-09-18 | 2014-12-17 | 乌鲁木齐华新分析测试高科技开发公司 | Method for extracting general flavone from folium apocyni veneti |
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| JP2017145266A (en) * | 2017-06-06 | 2017-08-24 | ネイチャーパワー株式会社 | Anti-dementia composition |
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Application publication date: 20130220 |