CN102258581A - Plantain seed extract, preparation method and application thereof - Google Patents
Plantain seed extract, preparation method and application thereof Download PDFInfo
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- CN102258581A CN102258581A CN 201110243504 CN201110243504A CN102258581A CN 102258581 A CN102258581 A CN 102258581A CN 201110243504 CN201110243504 CN 201110243504 CN 201110243504 A CN201110243504 A CN 201110243504A CN 102258581 A CN102258581 A CN 102258581A
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- Prior art keywords
- solvent
- extract
- semen plantaginis
- ethanol
- iridoids
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- 238000002360 preparation method Methods 0.000 title claims abstract description 20
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- 240000008790 Musa x paradisiaca Species 0.000 title 1
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- 238000000034 method Methods 0.000 claims abstract description 65
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 211
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- 210000000582 semen Anatomy 0.000 claims description 96
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 46
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Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the technical field of natural medicines, in particular to plantain seed extract, a preparation method and application thereof. In the plantain seed extract, the main effective ingredients are iridoids and phenylethanoid glycosides which are vegetable extracts, wherein the vegetable extracts are extracted from the vegetable plantain seed with a solvent; and then refined and purified by a chromatographic process. The invention also provides the applications of the plantain seed extract in preparation of the medicines for preventing the vascular dementia.
Description
Technical field
The invention belongs to natural medicine technical field.Be specifically related to a kind of Semen Plantaginis extract and preparation method thereof and purposes.
Background technology
Along with the continuous aggravation of world population aging, numerous disease relevant with aging such as neurodegenerative diseases are just having a strong impact on people's Health and Living quality.Vascular dementia (Vascular Dementia, VD) be by the dementia syndrome due to the vascular lesion, dementia patients is because of the infringement of memory and other cognitive function, social activity ability and basic daily life can weaken or lose, this not only causes great misery to patient's spirit and health, and family and society have also been caused great burden.Modern medicine study is thought, the normal and cortex pathological changes of the generation of vascular dementia, and especially the ischemia of left side cortex ischemia and thalamus, Hippocampus changes closely related.In addition, bilateral, multiple cerebral infarction, the ischemia infraction and large tracts of land cerebral lesion the playing an important role of critical positions to primary disease.Not only the generation of apoplexy development has close cause effect relation with VD, and long-term chronic brain ischemia also can cause the generation of VD.Vascular dementia patient generally can't take care of oneself, and needs the household to take care of constantly, brings white elephant for patient, family, society.
Semen Plantaginis (Semenplantaginis) is one of China tradition medical material, and the beginning sees " Book of Songs ", and the medicinal beginning is stated from Shennong's Herbal and is listed in the medicine top gradely, has another name called: hold sway, cattle is lost, the Oviductus Ranae clothing.In addition, claim Herba Plantaginis dish, Folium Fumicis Dentati, wheel dish, pig Auricled Hedyotis Herb etc. in addition.Semen Plantaginis is Plantaginaceae plant Plantago depressa Willd (Plantago depressa Willd.), claims granule Herba Plantaginis or Herba Plantaginis (Plantago asiatica Linn.) again, and distribution is all arranged throughout the country, and resource is extensive.Effect with relieving stranguria by diuresis, eliminating dampness by diuresis antidiarrheal, liver heat removing and eyesight improving, removing heat-phlegm.Contain a large amount of phlegmatic temperaments (polysaccharide compound) and plantenolic acid, choline, adenine, succinic acid, protein, trace element etc. in the Semen Plantaginis.Contain flavone compound: mainly contain luteolin, Homoplantaginin, plantagin etc.Triterpenoid compound: mainly contain ursolic acid, oleanolic acid etc.Volatile oil: mainly contain 2,6 ditertiary butyl p cresol and 3-tertiary butyl-4-hydroxy methoxybenzene etc.; Cyclenes ether glycosides compound is as aucubin, Geniposidic acid; Phenethyl alcohol glycoside compounds: verbascoside etc.
The active site that wherein has most DEVELOPMENT PROSPECT comprises cyclenes ether glycosides compound: Geniposidic acid, aucubin etc. and phenethyl alcohol glycoside compounds: verbascoside etc.The present report that also from Semen Plantaginis, extracts the method for iridoid constituents and phenethyl alcohol glycoside constituents and be used for the treatment of senile dementia.
Summary of the invention
The invention provides a kind of preparation method of Semen Plantaginis extract, prepared Semen Plantaginis extract mainly contains phenethyl alcohol glycosides and iridoids two active components, and wherein iridoids content is 19-32%, and phenethyl alcohol glycosides content is 32-42%.
A kind of method for preparing Semen Plantaginis extract may further comprise the steps:
Step 1: Semen Plantaginis is pulverized, extract 1-4 time or reflux, extract, or supersound extraction or merceration or percolation extraction with first polar solvent, sucking filtration goes out extracting solution; Centrifugal or the sucking filtration of extracting solution is isolated supernatant; The mixed solution of aqueous solution, acetone, acetone and water that described first polar solvent is the 0-100%C1-C6 alkylol or contain acid or the above-mentioned solvent of alkali;
Step 2: make with extra care or directly supernatant is made with extra care with chromatograph with chromatograph after supernatant is mixed in silica gel or kieselguhr, the eluting solvent eluting, stream part of iridoid and phenethyl alcohol glycoside compounds is rich in collection, and concentrate drying gets Semen Plantaginis extract; Wherein said chromatograph is macroporous adsorbent resin chromatography, polyamide chromatograph, purification on normal-phase silica gel or reverse phase silica gel chromatograph or its combination.
As preferably, the mixed solution of aqueous solution, acetone, acetone and water that described first polar solvent is the 50-95%C1-C6 alkylol or contain acid or the above-mentioned solvent of alkali.Acid or alkali are strong acid, highly basic or weak acid, weak base described in the described above-mentioned solvent that contains acid or alkali, are preferably the Na of 0.5%HCL, 1% acetic acid, 0.5%NaOH or 1%
2CO
3
More preferably, described C1-C6 alkylol is the mixed solvent of methanol, ethanol, isopropyl alcohol or n-butyl alcohol or their compositions.
As preferably, described eluting solvent is the mixed solvent of water, C1-C6 alkylol, halogenated hydrocarbons, ether solvent, ketones solvent, esters solvent or its two or more compositions.
More preferably, described eluting solvent is to be selected from methanol, ethanol, isopropyl alcohol, n-butyl alcohol, dichloromethane, chloroform, ether, methyl tert-butyl ether, acetone, 2-butanone, ethyl acetate, Ethyl formate or is the mixed solvent of chloroform, ethyl acetate, ethanol, first alcohol and water composition.
As preferably, described usefulness first polar solvent of step 1 is extracted as first polar solvent that at every turn adds 4-20BV and extracts to the temperature of solvent refluxing at 0 ℃.
More preferably, carry out concentration before step 1 extracting solution is clear in separation, preferred simmer down to the volume mass of extracting solution and Semen Plantaginis raw material than counting 0.1-1 with ml/g: 10-1.
As preferably, it is the temperature of room temperature to solvent refluxing that described usefulness first polar solvent of step 1 extracts temperature range.
The present invention also provides the Semen Plantaginis extract according to method for preparing, and described Semen Plantaginis extract is rich in iridoids and phenethyl alcohol glycosides, and wherein iridoids content is 19-32%, and phenethyl alcohol glycosides content is 32-42%.Wherein the phenethyl alcohol glycosides main component is verbascoside (Verbascoside), and its structure is as follows:
The iridoids main component is Geniposidic acid (Geniposidic acid), and its structure is as follows:
In the method for preparation Semen Plantaginis extract of the present invention, the stream part of being rich in iridoids and phenethyl alcohol glycosides can be detected by thin layer chromatography (TLC), ultraviolet (UV), high pressure liquid chromatography methods such as (HPLC), preferably detects by ultraviolet (UV) method.
The present invention also provides the purposes of described Semen Plantaginis extract in the medicine of preparation treatment vascular dementia.Modern pharmacodynamic experiment result shows, iridoids content of the present invention is 19-32%, phenethyl alcohol glycosides content is that each dosage group of Semen Plantaginis extract of 32-42% can improve the vascular dementia model learning and memory in rats ability of intending to some extent, alleviate the europathology damage of rat model, illustrate that Semen Plantaginis extract of the present invention has the effect of Fructus Alpiniae Oxyphyllae, hypermnesis, can be applicable to prepare in the medicine for the treatment of vascular dementia.
A kind of preparation provided by the invention contains the method for the Semen Plantaginis extract of iridoids and phenethyl alcohol glycosides, by the extraction of uniqueness, the Semen Plantaginis extract that process for refining can obtain being rich in iridoids and phenethyl alcohol glycosides.Wherein the macroporous adsorbent resin method is made with extra care and is suitable for suitability for industrialized production.The Semen Plantaginis extract that makes has high-load iridoids and phenethyl alcohol glycoside compounds, and iridoids content is 19-32%, and phenethyl alcohol glycosides content is 32-42%, can be used for preparing the medicine for the treatment of vascular dementia, has a good application prospect.
The specific embodiment
The invention discloses a kind of Semen Plantaginis extract and preparation method thereof and purposes, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts ultrasonic extraction, add 70% ethanol with 10 times liquid-solid ratioes, ultrasonic (frequency 80KHz) extracts 30min, repeats to extract sucking filtration 3 times, merge extractive liquid,, reclaim ethanol and be concentrated into 1000ml, be cooled to room temperature, left standstill 24 hours, centrifugally get rid of filter, clear filtrate.
(2) refining 1.: the above-mentioned upper prop solution for the treatment of joins in the HP-20 type macroporous resin column that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, the methanol of water, 10%, 30%, 50% and 75% concentration gradient is successively washed, HPLC detects, stream part of iridoid (washing and 10% part) and phenethyl alcohol glycosides (30% part) is rich in collection, merge, concentrating under reduced pressure, spray drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoid content is 23%, ingredient content of phenylethanoid glycosides 36%.
Or refining 2.: the above-mentioned upper prop solution for the treatment of joins in the HP-20 type macroporous resin column that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, the methanol of water, 30% and 75% concentration gradient is successively washed, HPLC detects, collect stream part (30% part) of containing iridoid and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, spray drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoid content is 22%, ingredient content of phenylethanoid glycosides 35.5%.
Embodiment 2:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and extracts with 70% alcohol heating reflux, adds 70% ethanol with 4 times liquid-solid ratio at every turn, refluxed 2 hours for the first time, the second time with refluxed 1 hour for the third time.Sucking filtration, merge extractive liquid, reclaims ethanol and also is concentrated into 1000ml, is cooled to room temperature, leaves standstill 24 hours, centrifugally gets rid of filter, clear filtrate.
(2) make with extra care 1.: the flow velocity of above-mentioned filtrate with 1BV/h joined in the AB-8 type macroporous resin column that Ethanol Treatment is crossed, the ethanol of water, 10%, 30% and 75% concentration gradient is successively washed, HPLC detects, collection is rich in the washing and the 10% ethanol part of iridoids and is rich in phenethyl alcohol glycosides 30% ethanol stream part, merge, be concentrated into extractum, vacuum drying obtains Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 25%, ingredient content of phenylethanoid glycosides 32%.
Or make with extra care 2.: the flow velocity of above-mentioned filtrate with 0.5-3BV/h joined in the AB-8 type macroporous resin column that Ethanol Treatment is crossed, the ethanol of water, 30% and 75% concentration gradient is successively washed, HPLC detects, collect 30% ethanol stream part of containing iridoids chemical combinationization and two kinds of compositions of phenethyl alcohol glycosides simultaneously, be concentrated into extractum, vacuum drying obtains Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoid content is 24%, ingredient content of phenylethanoid glycosides 32%.
Embodiment 3:
(1) extract: Semen Plantaginis is softening with steam, and oven dry is pulverized, sieve, the Plantago Seed 1000g after bury at the place of taking by weighing adopts decocting to extract, add deionized water with 40 times liquid-solid ratioes, extracted reprocessing 3 times at every turn 2 hours, with extracting liquid filtering, merge, reclaim ethanol and be concentrated into 1000ml, be cooled to room temperature, left standstill 24 hours, and centrifugally got rid of filter, clear filtrate.
(2) make with extra care 1.: the flow velocity with 1BV/h joins above-mentioned filtrate in the post of the polyamide that Ethanol Treatment is crossed, the ethanol of water, 10%, 50% and 75% concentration gradient is successively washed, HPLC detects, the washing and the 10% ethanol elution part of iridoids is rich in collection, be rich in 50% ethanol elution part of phenethyl alcohol glycosides, merge, concentrating under reduced pressure, vacuum drying obtain Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 21%, and the phenethyl alcohol glycoside kind compound content is 38%.
Or make with extra care 2.: the flow velocity with 1BV/h joins above-mentioned filtrate in the post of the polyamide that Ethanol Treatment is crossed, the ethanol of water, 50% and 75% concentration gradient is successively washed, HPLC detects, collect the 50% ethanol elution part that is rich in iridoid and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying obtain Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 19%, and the phenethyl alcohol glycoside kind compound content is 38%.
Embodiment 4:
(1) extracts: Semen Plantaginis is pulverized, cross 20 mesh sieves, take by weighing 1000g and add 60% ethanol room temperature lixiviate 4 times, each 24.0 hours.Merge extractive liquid, reclaims ethanol and also is concentrated into 1000ml, is cooled to room temperature, leaves standstill 24 hours, centrifugally gets rid of filter, clear filtrate.
(2) refining 1.: treat that with above-mentioned upper prop filtrate joins on the ADS-8 post that Ethanol Treatment crosses with the flow velocity of 2BV/h, in order to 1BV/h flow velocity elder generation water eluting, the alcoholic solution eluting of reuse 10%, 30% and 70% concentration, HPLC detects, collection is rich in 10% stream part of iridoids and is rich in 30% eluting part of phenethyl alcohol glycosides, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 21%, and phenethyl alcohol glycosides content is 35%.
Or refining 2.: treat that with above-mentioned upper prop filtrate joins on the ADS-8 post that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, in order to 1BV/h flow velocity elder generation water eluting, the alcoholic solution eluting of reuse 30% and 70% concentration, HPLC detects, collect the 30% eluting part that contains iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 19%, and phenethyl alcohol glycosides content is 35%.
Embodiment 5:
(1) extraction: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts 60% ethanol (containing 0.5%NaOH) heating and refluxing extraction 4 times, and each liquid-solid ratio with 10 times adds 60% ethanol (containing 0.5%NaOH), refluxes 2 hours for the first time, back three backflows 1 hour.Sucking filtration, merge extractive liquid, reclaims ethanol and is concentrated into extractum.
(2) make with extra care 1.: condensed cream and silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) post, earlier with ethyl acetate-methanol (10: 1) eluting, reuse ethyl acetate-methanol (5: 1) and ethyl acetate-methanol (1: 1) eluting, collection is rich in phenethyl alcohol glycosides stream part (ethyl acetate: methanol is 10: 1) and is rich in iridoids stream part (ethyl acetate-methanol is 5: 1 parts), merge, concentrating under reduced pressure, vacuum drying promptly get the Semen Plantaginis iridoids with the phenethyl alcohol glycosides extract.Measure through ultraviolet (UV) method, wherein iridoid content is 32%, and phenethyl alcohol glycosides content is 38%.
Or make with extra care 2.: with condensed cream and silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) post, earlier with ethyl acetate-methanol (5: 1) eluting, reuse ethyl acetate-methanol (1: 1) eluting, collect the stream part (ethyl acetate: methanol is 5: 1) of being rich in iridoids simultaneously and containing two kinds of compositions of phenethyl alcohol glycosides, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoid content is 31%, and phenethyl alcohol glycosides content is 38%.
Embodiment 6:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts ultrasonic extraction, add 60% methanol with 10 times liquid-solid ratioes, 60 ℃ of temperature, supersound extraction 30min when frequency is 80KHz, repeat to extract 4 times, sucking filtration, merge extractive liquid, reclaims ethanol and is concentrated into 1000ml, be cooled to room temperature, left standstill 24 hours, and centrifugally got rid of filter, clear filtrate.
(2) refining 1.: treat that with above-mentioned upper prop filtrate joins on the polyamide column that Ethanol Treatment crosses with the flow velocity of 3BV/h, in order to 1BV/h flow velocity water eluting, the alcoholic solution eluting of reuse 5%, 30% and 70% concentration, HPLC detects, the washing of iridoids and 30% eluting part of 5% ethanol part and phenethyl alcohol glycosides are rich in collection, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content be 20% and the phenethyl alcohol glycoside kind compound content be 35%.
Or refining 2.: treat that with above-mentioned upper prop filtrate joins on the polyamide column that Ethanol Treatment crosses with the flow velocity of 3BV/h, in order to 1BV/h flow velocity water eluting, the alcoholic solution eluting of reuse 30% and 70% concentration, HPLC detects, collect the 30% eluting part that is rich in iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content be 21% and the phenethyl alcohol glycoside kind compound content be 35%.
Embodiment 7:
(1) extracts: Semen Plantaginis is pulverized, cross 20 mesh sieves, take by weighing 1000g and add 95% ethanol room temperature lixiviate 2 times, each 24.0 hours.Merge extractive liquid, reclaims ethanol and is concentrated into extractum.
(2) make with extra care 1.: the extractum silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) chromatographic column, behind chloroform-methanol (15: the 1) eluting, reuse chloroform-methanol (5: 1) and chloroform-methanol (1: 1) eluting, HPLC detects, and collects the stream part (the chloroform-methanol ratio is 15: 1 parts) and the iridoids stream part (the chloroform-methanol ratio is 5: 1 parts) that are rich in the phenethyl alcohol glycoside compounds, merges, concentrating under reduced pressure, the dry Semen Plantaginis extract that gets.Measure through ultraviolet (UV) method, wherein iridoids content is 21%, and the phenethyl alcohol glycoside kind compound content is 36%.
Or make with extra care 2.: the extractum silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) chromatographic column, behind chloroform-methanol (5: the 1) eluting, reuse chloroform-methanol (1: 1) eluting, HPLC detects, collect the stream part (the chloroform-methanol ratio is 5: 1 parts) of being rich in iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, the dry Semen Plantaginis extract that gets.Measure through ultraviolet (UV) method, wherein iridoids content is 22%, and the phenethyl alcohol glycoside kind compound content is 36%.
Embodiment 8:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts ultrasonic extraction, add acetone with 10 times liquid-solid ratioes, 60 ℃ of temperature, supersound extraction 30min when frequency is 80KHz, repeat to extract 2 times, sucking filtration, merge extractive liquid, reclaims ethanol and is concentrated into 1000ml, be cooled to room temperature, left standstill 24 hours, and centrifugally got rid of filter, clear filtrate.
(2) make with extra care 1.: filtrate is used n-butanol extraction, reclaim solvent and get n-butyl alcohol extract; Extract is crossed reverse phase silica gel (C-18) post, water and 10% ethanol, 30% ethanol, 70% alcoholic solution eluting, HPLC detects, and 30% stream part of the 10% ethanol flow point and the phenethyl alcohol glycoside compounds of iridoids is rich in collection, merge concentrating under reduced pressure, the dry Semen Plantaginis extract that gets.Measure through ultraviolet (UV) method, wherein iridoids content be 32% and phenethyl alcohol glycosides content be 36%.
Or make with extra care 2.: filtrate is used n-butanol extraction, reclaim solvent and get n-butyl alcohol extract; Extract is crossed reverse phase silica gel (C-18) post, water, and 30% ethanol, 70% alcoholic solution eluting, HPLC detects, and collects 30% stream part of containing iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, the dry Semen Plantaginis extract that gets.Measure through ultraviolet (UV) method, wherein iridoids content is 31%, and the phenethyl alcohol glycoside kind compound content is 36%.
Embodiment 9:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts 70% isopropyl alcohol heating and refluxing extraction, adds 70% isopropyl alcohol with 20 times liquid-solid ratioes, reflux, extract, 1 time.Sucking filtration, merge extractive liquid, reclaims ethanol and also is concentrated into 800ml, is cooled to room temperature, leaves standstill 24 hours, centrifugally gets rid of filter, clear filtrate.
(2) make with extra care 1.: concentrated solution is passed through reverse phase silica gel (C-18) post, behind water and the 10% methanol solution eluting, the methanol solution eluting of reuse 50% concentration, HPLC detects, the 10 methanol part of iridoids and 50% stream part of phenethyl alcohol glycoside compounds are rich in collection, merge concentrating under reduced pressure, the dry Semen Plantaginis extract that gets.Measure through ultraviolet (UV) method, wherein iridoids content is 28%, and the phenethyl alcohol glycoside kind compound content is 35%.
Or make with extra care 2.: concentrated solution is passed through reverse phase silica gel (C-18) post, water, 50% methanol and 70% methanol solution eluting, HPLC detects, and collects 50% stream part of containing iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, the dry Semen Plantaginis extract that gets.Measure through ultraviolet (UV) method, wherein iridoids content is 26%, and the phenethyl alcohol glycoside kind compound content is 35%.
Embodiment 10:
(1) extracts: Semen Plantaginis is pulverized, cross 20 mesh sieves, take by weighing 1000g and add 50% acetone room temperature lixiviate 3 times, each 24.0 hours.Merge extractive liquid, reclaims ethanol and is concentrated into extractum.
(2) make with extra care 1.: the extractum silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) chromatographic column, behind chloroform-95% ethanol (20: the 1) eluting, reuse chloroform-95% ethanol (5: 1) and chloroform-95% ethanol (1: 1) eluting, HPLC detects, and collects the stream part (chloroform-95% ethanol ratio is 20: 1 parts) and the iridoids (chloroform-95% ethanol is 5: 1 parts) that are rich in the phenethyl alcohol glycoside compounds, merges, concentrating under reduced pressure, the dry Semen Plantaginis extract that gets.Measure through ultraviolet (UV) method, wherein iridoids content is 23%, and the phenethyl alcohol glycoside kind compound content is 32%.
Or make with extra care 2.: the extractum silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) chromatographic column, behind chloroform-95% ethanol (5: the 1) eluting, reuse chloroform-95% ethanol (1: 1) eluting, HPLC detects, collect stream part (chloroform-95% ethanol ratio is 5: 1 parts) of containing iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying gets Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 22%, and the phenethyl alcohol glycoside kind compound content is 36%.
Embodiment 11:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts 50% alcohol heating reflux to extract, and adds 50% ethanol with 10 times liquid-solid ratioes, reflux, extract, 3 times.Sucking filtration, merge extractive liquid, reclaims ethanol and also is concentrated into 1000ml, is cooled to room temperature, leaves standstill 24 hours, centrifugally gets rid of filter, clear filtrate.
(2) refining 1.: treat that with above-mentioned upper prop filtrate joins on the D101 post that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, in order to 1BV/h flow velocity water eluting, the alcoholic solution eluting of reuse 5%, 30% and 70% concentration, HPLC detects, the 5% ethanol part of iridoids and 30% eluting part of phenethyl alcohol glycoside compounds are rich in collection, merge, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content be 23% and the phenethyl alcohol glycoside kind compound content be 35%.
Or refining 2.: treat that with above-mentioned upper prop filtrate joins on the D101 post that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, in order to 1BV/h flow velocity water eluting, the alcoholic solution eluting of reuse 30% and 70% concentration, HPLC detects, collect the 30% eluting part that contains iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 24%, and the phenethyl alcohol glycoside kind compound content is 35%.
Embodiment 12:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts ultrasonic extraction, add 50% ethanol (containing 1% acetic acid) with 10 times liquid-solid ratioes, 60 ℃ of temperature, supersound extraction 30min when frequency is 80KHz, repeat to extract 3 times, sucking filtration, merge extractive liquid, reclaims ethanol and is concentrated into 1000ml, be cooled to room temperature, left standstill 24 hours, and centrifugally got rid of filter, clear filtrate.
(2) make with extra care 1.: the extractum silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) chromatographic column, behind ethyl acetate-95% ethanol (15: the 1) eluting, reuse ethyl acetate-95% ethanol (3: 1) and ethyl acetate-95% ethanol (1: 1) eluting, HPLC detects, and collects the stream part (chloroform-95% ethanol ratio is 15: 1 parts) and the iridoids stream part (ethyl acetate-95% ethanol is 3: 1 parts) that are rich in the phenethyl alcohol glycoside compounds, merges, concentrating under reduced pressure, the dry Semen Plantaginis extract that gets.Measure through ultraviolet (UV) method, wherein iridoids content is 26%, and the phenethyl alcohol glycoside kind compound content is 36%.
Or make with extra care 2.: the extractum silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) chromatographic column, behind ethyl acetate-95% ethanol (5: the 1) eluting, reuse ethyl acetate-95% ethanol (1: 1) eluting, HPLC detects, collect stream part (chloroform-95% ethanol ratio is 5: 1 parts) of containing iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying gets Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 24%, and the phenethyl alcohol glycoside kind compound content is 36%.
Embodiment 13:
(1) extracts: Semen Plantaginis is pulverized, cross 20 mesh sieves, take by weighing 1000g and add 20 times of deionized waters (containing 0.5%HCL) room temperature lixiviate 3 times, each 24.0 hours.Merge extractive liquid, is concentrated into 1000ml, is cooled to room temperature, leaves standstill 24 hours, centrifugally gets rid of filter, clear filtrate.
(2) refining 1.: above-mentioned filtrate is joined on the HPD-100 post that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, in order to 1BV/h flow velocity water eluting, the methanol solution eluting of reuse 5%, 30% and 75% concentration, HPLC detects, collection is rich in iridoids stream part (5% eluting part) and is rich in phenethyl alcohol glycosides stream part (30% eluting part), merge concentrating under reduced pressure, vacuum drying promptly gets Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 23%, and the phenethyl alcohol glycoside kind compound content is 35%.
Or refining 2.: above-mentioned filtrate is joined on the HPD-100 post that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, in order to 1BV/h flow velocity water eluting, the methanol solution eluting of reuse 30% and 75% concentration, HPLC detects, collect stream part (30% eluting part) of containing iridoids simultaneously and being rich in two kinds of compositions of phenethyl alcohol glycosides, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 23%, and the phenethyl alcohol glycoside kind compound content is 35%.
Embodiment 14:
(1) extracts: Semen Plantaginis is pulverized, cross 20 mesh sieves, take by weighing 1000g and adopt decocting to extract, add deionized water with 40 times liquid-solid ratioes, the each extraction 1 hour, reprocessing 3 times is with extracting liquid filtering, merge, reclaim ethanol and be concentrated into 1000ml, be cooled to room temperature, left standstill 24 hours, centrifugally get rid of filter, clear filtrate.
(2) make with extra care 1.: the flow velocity with 1BV/h joins above-mentioned filtrate the AB-8 type macroporous adsorbent resin of crossing through Ethanol Treatment, the methanol of water, 10%, 30% and 70% concentration gradient is successively washed, HPLC detects, 30% methanol-eluted fractions part of iridoids 10% methanol part and phenethyl alcohol glycosides is rich in collection, merge, concentrating under reduced pressure, vacuum drying obtain Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 23%, and the phenethyl alcohol glycoside kind compound content is 36%.
Or make with extra care 2.: the flow velocity with 1BV/h joins above-mentioned filtrate the AB-8 type macroporous adsorbent resin of crossing through Ethanol Treatment, the methanol of water, 30% and 70% concentration gradient is successively washed, HPLC detects, collect stream part (30% methanol-eluted fractions part) of containing iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying obtains Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 22%, and the phenethyl alcohol glycoside kind compound content is 38%.
Embodiment 15:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts the deionized water ultrasonic extraction, add deionized water with 20 times liquid-solid ratioes, 30 ℃ of temperature, supersound extraction 30min when frequency is 80KHz, repeat to extract 3 times, sucking filtration, merge extractive liquid, reclaims ethanol and is concentrated into 1000ml, be cooled to room temperature, left standstill 24 hours, and centrifugally got rid of filter, clear filtrate.
(2) make with extra care 1.: above-mentioned filtrate extracts with Ethyl formate, reclaims solvent and gets the Ethyl formate extract.With Ethyl formate extract water dissolution, with filtrate with the flow velocity of 0.5BV/h by reverse phase silica gel post (C-18), behind water and the 10% methanol solution eluting, the methanol solution eluting of reuse 50% concentration, HPLC detects, iridoids stream part (10% eluting part) and phenethyl alcohol glycosides (50% part) stream part are rich in collection, and concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 28%, and the phenethyl alcohol glycoside kind compound content is 36%.
Or make with extra care 2.: above-mentioned filtrate extracts with Ethyl formate, reclaims solvent and gets the Ethyl formate extract.With Ethyl formate extract water dissolution, with filtrate with the flow velocity of 0.5BV/h by reverse phase silica gel post (C-18), water, 50% methanol and 70% methanol solution eluting, HPLC detects, collection contains stream part (50% part) of iridoids and two kinds of compositions of phenethyl alcohol glycosides, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 24%, and the phenethyl alcohol glycoside kind compound content is 34%.
Embodiment 16:
(1) extracts: Semen Plantaginis is pulverized, cross 20 mesh sieves, take by weighing 1000g and add 20 times 30% ethanol (containing 1%NaCO3) room temperature lixiviate 2 times, each 24.0 hours.Merge extractive liquid, reclaims ethanol and also is concentrated into 1000ml, is cooled to room temperature, leaves standstill 24 hours, centrifugally gets rid of filter, clear filtrate.
(2) refining 1.: treat that with above-mentioned upper prop filtrate joins on the D101 post that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, in order to 1BV/h flow velocity water eluting, the methanol solution eluting of reuse 5%, 30% and 70% concentration, HPLC detects, collection is rich in 5% methanol stream part of iridoids and is contained 30% methanol-eluted fractions part of phenethyl alcohol glycosides, merge, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 23%, and phenethyl alcohol glycosides content is 35%.
Or refining 2.: treat that with above-mentioned upper prop filtrate joins on the D101 post that Ethanol Treatment crosses with the flow velocity of 0.5BV/h, in order to 1BV/h flow velocity water eluting, the methanol solution eluting of reuse 30% and 70% concentration, HPLC detects, collect the 30% methanol-eluted fractions part that contains iridoids simultaneously and contain two kinds of compositions of phenethyl alcohol glycosides, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 23%, and phenethyl alcohol glycosides content is 35%.
Embodiment 17:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts 30% alcohol heating reflux to extract, and adds 30% ethanol with 15 times liquid-solid ratioes, reflux, extract, 3 times.Sucking filtration, merge extractive liquid, reclaims ethanol and also is concentrated into 1000ml, is cooled to room temperature, leaves standstill 24 hours, centrifugally gets rid of filter, clear filtrate.
(2) make with extra care 1.: filtrate is used n-butanol extraction, reclaim the solvent n-butyl alcohol, condensed cream and silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) post, behind Ethyl formate-95% ethanol (10: the 1) eluting, reuse Ethyl formate-95% ethanol (4: 1) and Ethyl formate-95% ethanol (1: 1) gradient elution, phenethyl alcohol glycosides (Ethyl formate-95% ethanol is 10: 1) stream part and iridoids stream part (Ethyl formate-95% ethanol is 4: 1 parts) are rich in collection, merge, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 29%, and the phenethyl alcohol glycoside kind compound content is 36%.
Or make with extra care 2.: filtrate is used n-butanol extraction, reclaim the solvent n-butyl alcohol, condensed cream and silica gel mixed sample, by purification on normal-phase silica gel (100-200 order) post, behind Ethyl formate-95% ethanol (5: the 1) eluting, reuse Ethyl formate-95% ethanol (1: 1) gradient elution is collected stream part (Ethyl formate-95% ethanol is 5: 1) of containing iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 27%, and the phenethyl alcohol glycoside kind compound content is 35%.
Embodiment 18:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts ultrasonic extraction, add 30% ethanol with 15 times liquid-solid ratioes, 60 ℃ of temperature, supersound extraction 30min when frequency is 80KHz, repeat to extract 3 times, sucking filtration, merge extractive liquid, reclaims ethanol and is concentrated into 1000ml, be cooled to room temperature, left standstill 24 hours, and centrifugally got rid of filter, clear filtrate.
(2) make with extra care 1.: the flow velocity of above-mentioned filtrate with 0.5BV/h joined in pretreated AB-8 type macroporous resin column, the ethanol of water, 10%, 30% and 75% concentration gradient is successively washed, HPLC detects, the 10% eluting part of iridoids and 30% ethanol elution part of phenethyl alcohol glycosides are rich in collection, merge, be concentrated into thick paste, vacuum drying obtains Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 22%, and phenethyl alcohol glycosides content is 36%.
Or make with extra care 2.: the flow velocity of above-mentioned filtrate with 0.5BV/h joined in pretreated AB-8 type macroporous resin column, the ethanol of water, 30% and 75% concentration gradient is successively washed, HPLC detects, collect the 30% ethanol elution part that contains iridoids and phenethyl alcohol glycosides simultaneously, be concentrated into thick paste, vacuum drying obtains Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 23%, and phenethyl alcohol glycosides content is 34%.
Embodiment 19:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts 45% alcohol heating reflux to extract, and adds 45% ethanol with 15 times liquid-solid ratioes, reflux, extract, 3 times.Sucking filtration, merge extractive liquid, reclaims ethanol and also is concentrated into 1000ml, is cooled to room temperature, leaves standstill 24 hours, centrifugally gets rid of filter, clear filtrate.
(2) make with extra care 1.: the flow velocity with 1BV/h joins above-mentioned filtrate in the post of the HP-20 that Ethanol Treatment is crossed, the methanol of water, 10%, 30% and 75% concentration gradient is successively washed, the 10% methanol-eluted fractions part of iridoids and 30% methanol-eluted fractions part of phenethyl alcohol glycosides are rich in collection, merge, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 27%, and the phenethyl alcohol glycoside kind compound content is 35%.
Or make with extra care 2.: the flow velocity with 1BV/h joins above-mentioned filtrate in the post of the HP-20 that Ethanol Treatment is crossed, the methanol of water, 30% and 75% concentration gradient is successively washed, collect the 30% methanol-eluted fractions part that contains iridoids and two kinds of stream parts of phenethyl alcohol glycosides simultaneously, merge, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 26%, and the phenethyl alcohol glycoside kind compound content is 36%.
Embodiment 20:
(1) extract: Semen Plantaginis is pulverized, and crosses 20 mesh sieves, takes by weighing 1000g and adopts ultrasonic extraction, add 45% ethanol with 15 times liquid-solid ratioes, 30 ℃ of temperature, supersound extraction 30min when frequency is 80KHz, repeat to extract sucking filtration, merge extractive liquid, 3 times, reclaim ethanol and be concentrated into the extractum shape, the adding distil water ultrasonic dissolution, sucking filtration, filtrate is used n-butanol extraction, reclaim the solvent n-butyl alcohol, get n-butyl alcohol extract.
(2) make with extra care 1.: the n-butyl alcohol extract dissolved in distilled water, filter, filtrate is passed through reverse phase silica gel post (C-18) with the flow velocity of 0.5BV/h, behind water and the 10% methanol solution eluting, the methanol solution eluting of reuse 50% concentration, the HPLC detection is collected and is rich in 50% stream part that 10% of iridoids flows part and phenethyl alcohol glycosides, merge concentrating under reduced pressure, vacuum drying promptly gets Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 31%, and the phenethyl alcohol glycoside kind compound content is 42%.
Or make with extra care 2.: the n-butyl alcohol extract dissolved in distilled water, filter, filtrate is passed through reverse phase silica gel post (C-18) with the flow velocity of 0.5BV/h, water, 50% methanol and 70% methanol solution eluting, HPLC detects, collect 50% methanol stream part of containing iridoids and two kinds of compositions of phenethyl alcohol glycosides simultaneously, concentrating under reduced pressure, vacuum drying promptly get Semen Plantaginis extract.Measure through ultraviolet (UV) method, wherein iridoids content is 32%, and the phenethyl alcohol glycoside kind compound content is 40%.
EXPERIMENTAL EXAMPLE: Semen Plantaginis extract is for the influence of intending vascular dementia rat space learning memory ability
Hereinafter adopt with abbreviation " pdw " representative and contain phenethyl alcohol glycosides and iridoid content greater than 50% Semen Plantaginis extract.
The observation of ability of learning and memory is an indispensable important indicator in the dull-witted clinical experimental research with estimating, this experiment is specially selected the water maze that is usually used in detecting the animal learning memory ability in the world for use in order to estimate the ability of learning and memory of medicine to animal pattern objectively.Its detection be rat in training repeatedly, the hidden platform of fixed position is sought by association, forms stable locus cognition, this spatial cognition be a kind of with different I be reference the cognition of reference point, formed memory is that a kind of georeferencing is remembered.From information Processing and extracting mode, this georeferencing memory enters awareness system, the mechanism of its storage relates generally to limbic system (as Hippocampus) and the relevant brain of cerebral cortex district, should belong to declarative memory, and clinical forgetful and dull-witted patient, declarative memory is at first impaired and more outstanding just.So from the memory attribute that detects, the screening study that utilization Morris water maze is prevented and treated this type of disease active drug is more appropriate.
1. experiment material
The sample (pdw) of experimental drug embodiment 1 preparation.
A laboratory animal cleaning level SD rat, male, body weight 190-210g is supplied with by Beijing Vital River Experimental Animals Technology Co., Ltd., and the quality certification number is scxk (capital) 2010-0011.Nature diet under the experiment condition experimentized after conforming three days.
Instrument Morris water maze DMS-2 system is provided by Chinese Academy of Sciences institute of materia medica
2. experimental technique
2.1 intend the preparation of vascular dementia rat model
Male SD rat, body weight 190-210g, 10% chloral hydrate intraperitoneal injection of anesthesia, it is fixing to lie on the back, and throat portion unhairing sterilization tailing edge medisection is separated bilateral common carotid arteries, cuts off sew up wound after the dual silk thread ligation.The only free bilateral common carotid arteries of sham operated rats, each is organized rat and raises with condition.
2.2 laboratory animal grouping
Animal is divided into dosage group (n=15), positive drug donepezil group (n=15), positive drug nicergoline group (n=15) among sham operated rats (n=10), model group (n=15), pdw small dose group (n=15), the heavy dose of group of pdw (n=15), the pdw at random.
2.3 administration: postoperative administration in 24 hours, pdw small dose group rat are pressed the heavy dose of group of 12.5mg/kg, pdw rat and are pressed among 50mg/kg, the pdw dosage group rat and press the 25mg/kg gastric infusion; Positive drug donepezil group is pressed 0.58mg/kg, the nicergoline group is pressed the 9mg/kg gastric infusion.Sham operated rats and model group animal are irritated stomach and the capacity normal saline such as give.Successive administration 40 days carried out the training of Morris water maze from the 42nd day.
The Morris water maze is mainly by round pool with videotape automatically and the computer for analysis system forms, by the camera system of top, labyrinth the heuristic process of rat in the labyrinth being carried out whole process takes, as the movement locus of rat, movement time, move distance, motion mode etc., avoided artificial factor.Platform is placed on Southwest Quadrant (III quadrant) center apart from pool wall 22cm place, before the test water is injected the pond, depth of water 30cm (be that the water surface exceeds platform surface 1cm, make rat can not see platform), and water temperature is controlled between 20-21 ℃, 25 ℃ of room temperatures.Require various experiment conditions to remain unchanged during experiment.Whole experiment is divided into hides flat acquisition experiment (hidden-platform acquisition training) and space search experiment (probetrial testing) two parts of closing.
EXPERIMENTAL EXAMPLE 1:
To intending the influence of vascular dementia rat orientation navigation test escape latency
The results are shown in Table 1.It is shorter and shorter through the time of swimming instruction searching platform that each organizes rat, 1st, 2 days each treated animal escape latency are not seen notable difference, obviously prolong (P＜0.05) since the 3rd day swimming instruction model group rat escape latency than sham operated rats, illustrate that there are tangible space learning dysmnesia in rat model; Each dosage group rat escape latency of pdw all has minimizing in various degree than the model group rat, and particularly middle dosage group obviously shortened (P＜0.05) than model group in 3,4 days in swimming; The positive drug nicergoline also obviously shortened (P＜0.05) than model group on the 4th day in swimming.
Table 1. medicine is to the influence of intending vascular dementia rat Morris water maze escape latency (x ± s)
Annotate: compare with model group:
*P＜0.05
With reference to above-mentioned experimental technique, Semen Plantaginis extract to embodiment 2-20 preparation positions the test of sea trial escape latency, the result shows that each dosage group rat escape latency of pdw all has minimizing in various degree than the model group rat, compare the Semen Plantaginis extract of embodiment 1 preparation, the difference not statistically significant.
EXPERIMENTAL EXAMPLE 2: to intending the influence in vascular dementia rat orientation navigation test swimming path
Shorter and shorter through the path that each treated animal searching platform of swimming instruction is swum, each is organized the path of rat swimming instruction in the 1st, 2 days and is not seen notable difference, but since swimming instruction in the 3rd day, swimming path of model group rat and the apparent in view prolongation of sham operated rats (P＜0.05); Each dosage group swimming path of pdw all has in various degree than the model group rat reduces, and particularly middle dosage group obviously shortened (P＜0.05) than model group in 3,4 days in swimming and the results are shown in Table 2.
Table 2. medicine is to the influence of intending vascular dementia rat Morris water maze swimming instruction swimming path (x ± s)
Annotate: compare with model group:
*P＜0.05
With reference to above-mentioned experimental technique, Semen Plantaginis extract to embodiment 2-20 preparation positions sea trial swimming path test, the result shows that all there is minimizing in various degree in each dosage group swimming path of pdw than the model group rat, compare the Semen Plantaginis extract of embodiment 1 preparation, the difference not statistically significant.
EXPERIMENTAL EXAMPLE 3: to intending the influence of vascular dementia rat Morris water maze space exploration ability
Space exploration test by record remove respectively organize behind the platform in the labyrinth rat in time, the path of the motion of original platform quadrant and the number of times that accurately passes the original platform position reflect that rat is through the spatial memory ability of preceding 10 swimming instructions to platform.If animal has formed stronger memory to platform in the locus of water maze really, when being placed in the labyrinth once more, can near the original platform in-scope, on purpose seek platform.This experimental result demonstration, after training, each organizes rat does not have notable difference at platform quadrant swimming time and the ratio (tP/tT) of total swimming time and the swimming of platform quadrant apart from the ratio (dP/dT) that accounts for total swimming distance.But the model group rat is compared the number of times that accurately passes through the original platform position and obviously reduces (P＜0.05) with sham operated rats, illustrates that the spatial memory ability suffers damage.Pdw respectively organizes number of times that rat accurately passes through the original platform position all has in various degree raising than model group, particularly in dosage group obvious difference (P＜0.05).
Table 3.pdw is to the influence of intending vascular dementia rat Morris water maze space exploration ability (x ± s)
| Group | n | tP/tT(％) | dP/dT(％) | Wear ring number of times (inferior) |
| Sham operated rats | 9 | 31.20±7.33 | 32.67±3.78 * | 1.46±0.67 * |
| Model | 11 | 26.08±9.52 | 23.78±8.29 | 0.66±0.76 * |
| Pdw is big | 13 | 30.74±10.59 | 28.11±10.08 | 1.02±0.79 |
| Among the pdw | 12 | 29.74±7.28 | 27.57±8.86 | 1.48±1.12 * |
| Pdw is little | 13 | 27.10±8.79 | 26.41±9.32 | 0.99±0.80 |
| Donepezil | 9 | 26.18±8.92 | 25.49±7.99 | 0.96±0.43 |
| Nicergoline | 10 | 29.24±11.47 | 28.55±11.67 | 1.01±0.83 |
Annotate: compare with model group:
*P＜0.05.
With reference to above-mentioned experimental technique, Semen Plantaginis extract to embodiment 2-20 preparation carries out the space exploration ability test, the result shows that pdw respectively organizes number of times that rat accurately passes through the original platform position and than model group in various degree raising arranged all, compare the Semen Plantaginis extract of embodiment 1 preparation, the difference not statistically significant.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (12)
1. method for preparing Semen Plantaginis extract may further comprise the steps:
Step 1: Semen Plantaginis is pulverized, extract 1-4 time or reflux, extract, or supersound extraction or merceration or percolation extraction with first polar solvent, sucking filtration goes out extracting solution; Centrifugal or the sucking filtration of extracting solution is isolated supernatant; The mixed solution of aqueous solution, acetone, acetone and water that described first polar solvent is the 0-100%C1-C6 alkylol or contain acid or the above-mentioned solvent of alkali;
Step 2: make with extra care or directly supernatant is made with extra care with chromatograph with chromatograph after supernatant is mixed in silica gel or kieselguhr, the eluting solvent eluting, stream part of iridoid and phenethyl alcohol glycoside compounds is rich in collection, and concentrate drying gets Semen Plantaginis extract; Wherein said chromatograph is macroporous adsorbent resin chromatography, polyamide chromatograph, purification on normal-phase silica gel or reverse phase silica gel chromatograph or its combination.
2. method according to claim 1 is characterized in that, the mixed solution of aqueous solution, acetone, acetone and water that described first polar solvent is the 50-95%C1-C6 alkylol or contain acid or the above-mentioned solvent of alkali.
3. method according to claim 2 is characterized in that, described C1-C6 alkylol is the mixed solvent of methanol, ethanol, isopropyl alcohol or n-butyl alcohol or their compositions.
4. method according to claim 1 is characterized in that, described eluting solvent is the mixed solvent of water, C1-C6 alkylol, halogenated hydrocarbons, ether solvent, ketones solvent, esters solvent or its two or more compositions.
5. method according to claim 1 is characterized in that, described eluting solvent is for being selected from methanol, ethanol, isopropyl alcohol, n-butyl alcohol, dichloromethane, chloroform, ether, methyl tert-butyl ether, acetone, 2-butanone, ethyl acetate, Ethyl formate.
6. method according to claim 1 is characterized in that, described eluting solvent is the mixed solvent of chloroform, ethyl acetate, ethanol, first alcohol and water composition.
7. method according to claim 1 is characterized in that, described usefulness first polar solvent of step 1 is extracted as first polar solvent that at every turn adds 4-20BV and extracts to the temperature of solvent refluxing at 0 ℃.
8. method according to claim 1 is characterized in that, carries out concentration before step 1 extracting solution is clear in separation, preferred simmer down to the volume mass of extracting solution and Semen Plantaginis raw material than counting 0.1-1 with ml/g: 10-1.
9. method according to claim 1 is characterized in that, it is the temperature of room temperature to solvent refluxing that described usefulness first polar solvent of step 1 extracts temperature range.
10. method according to claim 1 is characterized in that, acid or alkali are strong acid, highly basic or weak acid, weak base described in the described above-mentioned solvent that contains acid or alkali, is preferably the Na of 0.5%HCL, 1% acetic acid, 0.5%NaOH or 1%
2CO
3
11. the Semen Plantaginis extract according to each described method preparation of claim 1-10 is characterized in that iridoids content is 19-32%, phenethyl alcohol glycosides content is 32-42%.
12. according to the purposes of the described Semen Plantaginis extract of claim 11 in the medicine of preparation treatment vascular dementia.
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