CN106031735A - Medical uses and preparing method of dill seed effective extract product with acetylcholinesterase inhibiting activity and composition of the extract product - Google Patents
Medical uses and preparing method of dill seed effective extract product with acetylcholinesterase inhibiting activity and composition of the extract product Download PDFInfo
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Abstract
The invention relates to the technical field of medicines, and particularly relates to a preparing method of a dill seed effective extract product with acetylcholinesterase inhibiting activity and a composition of the extract product, and medical uses of the extract product in the field of treating Alzheimer disease (AD), and the like. The extract product is prepared by subjecting dill seeds to ultrasonic extraction with a solvent, concentrating the extract liquid, and separating with a silica gel column, wherein the silica gel column to which a sample is added with is eluted with a petroleum ether-acetone mixed solvent in a volume ratio of 100:2 and then is eluted with a petroleum ether-acetone mixed solvent in a volume ratio of 100:3, and the eluate obtained by eluting with the petroleum ether-acetone mixed solvent in the volume ratio of 100:3 is subjected to solvent recovering and dried to obtain the extract product; or the silica gel column is eluted with a petroleum ether-acetone mixed solvent in a volume ratio of 100:12 and then is eluted with a petroleum ether-acetone mixed solvent in a volume ratio of 100:17, and the eluate obtained by eluting with the petroleum ether-acetone mixed solvent in the volume ratio of 100:17 is subjected to solvent recovering and dried to obtain the extract product.
Description
Technical field
The invention belongs to pharmaceutical technology field, be specifically related to the preparation method of a kind of Fructus anethi inhibiting activity of acetylcholinesterase extract and combinations thereof thing and it is at the medical usage treating the aspects such as Alzheimer (AD).
Background technology
Fructus anethi be Umbelliferae (Umbelliferae) plant Fructus anethi (Anethum graveolensL.) dry mature fruit.Dry fruit majority is cleaved into schizocarp, in flat oval, long 3～4 millimeters, wide 2～3 millimeters, thickness about 1 millimeter.Appearance brown, is backed with 3 the most significantly rib lines, and two side rib lines extend and make aliform, and minority unsegregated diachenium base portion has remaining fruit stem.Feeble QI is fragrant.Produce the ground such as Jiangsu, Anhui.Chemical composition is mainly Eucarvone, limonene, dill-apiol, bergapton, umbelliprenin i.e. 7-hydroxycoumarin Acacia farnesiana Willd. alcohol ether, wax, clionasterol etc..Have antibacterial, antitussive, relieving asthma, dispel the wind, the effect such as diuresis function of gallbladder promoting.
The present invention by a large scale, systematically screening active ingredients, it is surprised to find that Uigurs medicine Fructus anethi extract, having inhibiting activity of acetylcholinesterase significantly, this activity extract and combinations thereof thing is expected to the medical usage being applied to treat the aspects such as Alzheimer (AD).
Summary of the invention
Technical problem solved by the invention is to provide a kind of Fructus anethi extract.
Present invention also offers the compositions with Fructus anethi extract as main active.
Invention also provides the preparation of Fructus anethi extract and combinations thereof thing and the application in preparing inhibiting activity of acetylcholinesterase medicine.
The present invention is achieved through the following technical solutions:
Uigurs medicine Fructus anethi is through methanol supersound extraction, after extracting solution concentrates, with silica gel (SiO2) post separation, silicagel column after loading, with petroleum ether-acetone mixed solvent gradient elution (petroleum ether-acetone, ratio is shown in Table 1), each petroleum ether-acetone ratio mixed solvent eluent, concentrate, after drying, in-vitro screening system is utilized to test its inhibiting activity of acetylcholinesterase, it is surprised to find that first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered solvent of eluent of eluting, it is dried, it is this extract.And the eluting position of other ratios, there is no the effect of inhibiting activity of acetylcholinesterase.Optimum is first with the petroleum ether-acetone mixed solvent eluting 10 times column volume of 100:2, continues and is the extract of inhibiting activity of acetylcholinesterase after drying with the eluent of the petroleum ether of 100:3-20 times of column volumes of acetone mixed solvent eluting.The extract of inhibiting activity of acetylcholinesterase, can be enriched with by above method, thus separates with other impurities compositions.This Fructus anethi inhibiting activity of acetylcholinesterase extract is from having no that document is reported, its thin layer spectrum color spectrum (TLC) analysis and characterization such as Fig. 1, TLC analysis condition: GF254 silica gel plate, development system: petroleum ether: ethyl acetate: acetone (6:1:1) coloration method: vanillin-sulfuric acid develops the color；
Or Uigurs medicine Fructus anethi is through methanol supersound extraction, after extracting solution concentrates, with silica gel (SiO2) post separation, silicagel column after loading, with petroleum ether-acetone mixed solvent gradient elution (petroleum ether-acetone, ratio is shown in Table 1), each petroleum ether-acetone ratio mixed solvent eluent, concentrate, after drying, in-vitro screening system is utilized to test its inhibiting activity of acetylcholinesterase, it is surprised to find that first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered solvent of eluent of eluting, it is dried, it is this extract.And the eluting position of other ratios, there is no the effect of inhibiting activity of acetylcholinesterase.Or optimum is first with 100；The petroleum ether of 12-acetone mixed solvent eluting 25 times column volume, continues and is the extract of inhibiting activity of acetylcholinesterase of the present invention with the eluent of the petroleum ether of 100:17-35 times of column volumes of acetone mixed solvent eluting.The extract of inhibiting activity of acetylcholinesterase, can be enriched with by above method, thus separates with other impurities compositions.This Fructus anethi inhibiting activity of acetylcholinesterase extract is from having no that document is reported, its thin layer spectrum color spectrum (TLC) analysis and characterization such as Fig. 2, TLC analysis condition: GF254 silica gel plate, development system: petroleum ether: ethyl acetate: acetone=6:1:1, coloration method: vanillin-sulfuric acid develops the color.
Specifically, discovery procedure is as follows:
1, Fructus anethi is through the preparation of methanol sonicated extract
Take Uigurs medicine Fructus anethi 20 g, through 100 mL methanol supersound extraction 15 min, after extracting solution concentrates, obtain extract SN0096A, sample 0.6927 g, keep sample 0.0353g, applied sample amount 0.6574g, mix sample silica gel 0.7 g, blank silica gel 5 g, petroleum ether-acetone system elutions.
2, petroleum ether-acetone mixed solvent gradient elution method and result
nullTake Uigurs medicine Fructus anethi 20 g，Through 100 mL methanol supersound extraction 15 min，After extracting solution concentrates，Obtain extract SN0096A，Sample 0.6927 g，Keep sample 0.0353g，Applied sample amount 0.6574g，Mix sample silica gel 0.7 g，Blank silica gel 5 g，Glass column internal diameter 1.5 cm，Petroleum ether-acetone system elutions (condition is shown in Table 1)，Column volume 12.5 mL，Each gradient elution 100mL，Obtain，Each concentration extracts the eluate of eluent，Recycling design，Obtain，Petroleum ether: acetone gradient elution extract，TLC analysis result is shown in Fig. 3，TLC analysis condition: GF254 silica gel plate，Development system: petroleum ether: ethyl acetate: acetone=6:1:1，Coloration method: vanillin-sulfuric acid develops the color.
Table 1
3, acetylcholine esterase inhibition activity screening technique and result
1)
Experiment condition
A, material: DMSO (dimethyl sulfoxide) (Ke Miou company)；KH2PO4、K2HPO4·3H2O (Xi Long chemical plant, Shantou, Guangdong city)；AchE enzyme (Sigma Co., USA)；PNPG (Sigma Co., USA)
B, experimental apparatus: multi-functional microplate reader Bio-Rad Model 680 type (Bio-Rad Laboratories
Inc.)
2)
Experimental technique and process
Sample treatment: 1 mg testing sample is dissolved in 20μL dimethyl sulfoxide (DMSO), as mother solution, takes 1μL mother solution adds 249μL distilled water is made into 200μg·mL-1Solution.
Inhibiting activity of acetylcholinesterase assay method:
96 hole ELISA Plate measure the AchE inhibitory activity of sample.Concrete operations are as follows: be sequentially added into 20 μ L PB (100mM, pH 8. 0), 25 μ L AChE (0. 033 U/mL, pH 8. 0 PB dissolved dilution), 25 μ L sample solution (40 μ g mL in 96 hole ELISA Plate- 1).After ice bath 5min, add 15 μ L ATCI(0.6mM) and 15 μ L DTNB(1.5mM).After 37 DEG C hatch 60min, under 412 nm, measure its absorbance by microplate reader.According to following formula calculating suppression ratio:
Inhibition of enzyme activity rate %=[ABlank- (ASample- ABackground)] / ABlank× 100
A in formulaBlank: it is not added with the absorption value after example reaction；
ASample: add the absorption value after example reaction；
ABackground: only add the absorption value of sample.
1) experimental result
Table 2
Found that:
The more excellent scheme of the present invention is: Uigurs medicine Fructus anethi is through organic solvent supersound extraction, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) eluting, continues with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) eluting, the recovered solvent of eluent with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) eluting, it is dried, is this inhibiting activity of acetylcholinesterase extract.This inhibiting activity of acetylcholinesterase extract from having no that document is reported, its TLC analysis and characterization such as Fig. 1；
Or Uigurs medicine Fructus anethi is through organic solvent supersound extraction, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) eluting, continues with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) eluting, the recovered solvent of eluent with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) eluting, it is dried, is this inhibiting activity of acetylcholinesterase extract.This inhibiting activity of acetylcholinesterase extract from having no that document is reported, its TLC analysis and characterization such as Fig. 2.
4, the Study on Extraction Method of inhibiting activity of acetylcholinesterase effective extract
1) Research on kinds of Extraction solvent
Respectively with organic solvent extraction such as methanol, acetone, petroleum ether, chloroform, ethyl acetate, methanol-water mixed solvent, ethanol water mixed solvents, and the inhibiting activity of acetylcholinesterase of Test extraction thing, experimental result is as follows:
Table 3
Result of study shows: extraction organic solvent can be methanol, petroleum ether, acetone, chloroform, ethyl acetate, methanol-water mixed solvent, ethanol water mixed solvent.
2) Extraction solvent use quantity research
Respectively with 1,2,5,10,20,30,40,50 times of (weight/volume) organic solvent extraction, and the inhibiting activity of acetylcholinesterase of Test extraction thing, result is as follows:
Table 4
2-50 times (weight/volume) that extraction consumption of organic solvent used is medical material weight of activity extract.
3) research of drying means
The methods such as seasoning are steamed respectively with boulton process, freeze-drying, forced air drying, centrifugal drying, rotation, the acetylcholine esterase inhibition activity extract obtained is dried, with water content, TLC, active testing as index, find that boulton process, freeze-drying, forced air drying, centrifugal drying, rotation are steamed seasoning and be applicable to be dried inhibiting activity of acetylcholinesterase extract, wherein, optimum is boulton process, freeze-drying.
Table 5
Preferably, the preparation method of Fructus anethi inhibiting activity of acetylcholinesterase extract is:
Fructus anethi extracts through solvent supersonic, after extracting solution concentrates, separates with silicagel column, silicagel column after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 2-50 times of column volume of eluting, continues with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 2-50 times of column volume of eluting, the recovered organic solvent of eluent with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) eluting, it is dried, is this extract.Conditions above optimum is first with 10 times of column volumes of petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) eluting, continues with 20 times of column volumes of petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) eluting.Wherein, concrete experimental condition is as follows:
1)
Extract extraction solvent can be methanol, petroleum ether, water, acetone, chloroform, ethyl acetate, methanol-water mixed solvent, ethanol water mixed solvent, and optimal conditions is methanol and 95% ethanol.Extract extraction solvent load is 2-50 times (weight/volume) of medical material weight.
2) extract drying means can be boulton process, freeze-drying, forced air drying, centrifugal drying, rotation steaming seasoning etc., and optimum is boulton process, freeze-drying.
Or Fructus anethi extracts through solvent supersonic, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 2-50 times of column volume of eluting, continues with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 2-50 times of column volume of eluting, the recovered organic solvent of eluent with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) eluting, it is dried, is this extract.Conditions above optimum is first with 25 times of column volumes of petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) eluting, continues with 35 times of column volumes of petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) eluting.Wherein, concrete experimental condition is as follows:
1)
Extract extraction solvent can be methanol, petroleum ether, water, acetone, chloroform, ethyl acetate, methanol-water mixed solvent, ethanol water mixed solvent, and optimal conditions is methanol and 95% ethanol.Extract extraction solvent load is 2-50 times (weight/volume) of medical material weight.
Extract drying means can be boulton process, freeze-drying, forced air drying, centrifugal drying, rotation steaming seasoning etc., and optimum is boulton process, freeze-drying.
5. the medical usage research of Fructus anethi acetylcholine esterase inhibition activity extract (with the recovered solvent of eluent of the petroleum ether that volume ratio is 100:3 or 100:17-acetone mixed solvent eluting, be dried, be this extract)
The activity extract prepared by the Fructus anethi inhibiting activity of acetylcholinesterase method for preparing extractive optimized, after tested, its inhibiting activity of acetylcholinesterase is IC50=23.2 μg/mL。
There is the medical usage of the aspects such as treatment Alzheimer due to acetylcholinesteraseinhibitors inhibitors.Therefore, it has been found that Fructus anethi inhibiting activity of acetylcholinesterase extract there is medical usage widely.
6, compositions of Fructus anethi acetylcholine esterase inhibition activity extract and preparation method thereof research
1) solid dispersion
Prescription
Fructus anethi extract
20 g
Vc
1 g
Polyvinylpyrrolidone
79 g
Solid dispersion 100g
Preparation method:
The mass ratio weighing Fructus anethi extract and carrier polyvinylpyrrolidone (PVP) 1:2,1:4,1:6 in proportion is respectively put in beaker, add appropriate dehydrated alcohol and Vc, it is completely dissolved to Fructus anethi extract and carrier by magnetic stirrer, proceed to rotation after being sufficiently mixed uniformly and steam removing organic solvent in instrument, it is dried, pulverize 80 mesh sieves, obtain Fructus anethi effective extract PVP clathrate.
2) cyclodextrin clathrate
Prescription:
Fructus anethi extract
20 g
Vc
1 g
Beta-schardinger dextrin-
79 g
Clathrate 100g
Preparation method:
Being ground well with 1-5 times of water by beta-schardinger dextrin-, add Fructus anethi effective extract (should being first dissolved in a small amount of organic solvent of slightly water-soluble) and continue to be fully ground to becoming pastel, cold drying i.e. obtains cyclodextrin clathrate.
3) dispersible tablet prescription:
Fructus anethi extract
100
Sodium lauryl sulphate
1
Vc
1
Pregelatinized Starch
10
Soluble starch
100
Polyvinylpolypyrrolidone
10
Microcrystalline Cellulose
9
Differential silica gel
0.3
Pulvis Talci
1
100
Preparation method:
1.
The preparation of Fructus anethi effective extract dispersion, precision weighs Fructus anethi extract, Vc, adds the sodium lauryl sulphate of recipe quantity, it is after 70% ethanol dissolves and adds the soluble starch mix homogeneously of equal proportion by appropriate concentration, it is evaporated at a temperature of 70 DEG C, pulverizes, cross 100 mesh sieves；
2.
Polyvinylpolypyrrolidone by the Fructus anethi effective extract starch dispersions in the first step Yu recipe quantity, pregelatinized Starch, it is that 70% ethanol makees wetting agent by appropriate concentration, stirs while adding, make wet granular and cross 14 mesh sieves, after ambient temperatare puts 15min, 60 DEG C of oven for drying 45min, 16 mesh sieve granulate, add Pulvis Talci and the differential silica gel of recipe quantity, after mix homogeneously, tabletting and get final product.
7, the detection method research of Fructus anethi acetylcholine esterase inhibition activity extract
It was found that the feature of Fructus anethi inhibiting activity of acetylcholinesterase extract can be detected and indicate by the method for thin layer chromatography well.See Fig. 1 and Fig. 2
Actual conditions is: GF254Silica gel plate, development system: petroleum ether: ethyl acetate: acetone=6:1:1, coloration method: vanillin-sulfuric acid develops the color.
Accompanying drawing illustrates:
The TLC chromatogram of Fig. 1 Fructus anethi inhibiting activity of acetylcholinesterase extract；
The TLC chromatogram of Fig. 2 Fructus anethi inhibiting activity of acetylcholinesterase extract；
The TLC chromatogram of Fig. 3 Fructus anethi solvent extractable matter.
Detailed description of the invention:
Following example represent the practicality of the present invention, and the present invention is not limited.
Embodiment 1:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL methanol supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, vacuum drying, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=79.0 μg/mL。
Embodiment 2:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL ethanol ultrasonic extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=83.0 μg/mL。
Embodiment 3:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL petroleum ether supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, seasoning is steamed in rotation, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=77.0 μg/mL。
Embodiment 4:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL acetone supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, forced air drying, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=76.0 μg/mL。
Embodiment 5:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL chloroform supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, centrifugal drying, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=74.0 μg/mL。
Embodiment 6:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL ethyl acetate supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=67.0 μg/mL。
Embodiment 7:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL methanol-water mixed solvents (methanol: water=1:1) supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=83.0 μg/mL。
Embodiment 8:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL ethanol water mixed solvents (ethanol: water=1:1) supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=62.0 μg/mL。
Embodiment 9:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL ethanol-water mixed solvents (ethanol: water=90:10) supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) 20 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=70.0 μg/mL。
Embodiment 10:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL methanol supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, vacuum drying, it is this extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=73.0 μg/mL。
Embodiment 11:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL ethanol ultrasonic extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=77.0 μg/mL。
Embodiment 12:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL petroleum ether supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, seasoning is steamed in rotation, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=70.0 μg/mL。
Embodiment 13:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL acetone supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, forced air drying, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=73.0 μg/mL。
Embodiment 14:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL chloroform supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, centrifugal drying, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=68.0 μg/mL。
Embodiment 15:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL ethyl acetate supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=58.0 μg/mL。
Embodiment 16:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL methanol-water mixed solvents (methanol: water=1:1) supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=76.0 μg/mL。
Embodiment 17:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL ethanol water mixed solvents (ethanol: water=1:1) supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=51.0 μg/mL。
Embodiment 18:
Take Uigurs medicine Fructus anethi 20 g, through 100 mL ethanol-water mixed solvents (ethanol: water=90:10) supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (1.5 cm internal diameter pillar) after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) 25 times of column volumes of eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) 35 times of column volumes of eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) the recovered organic solvent of eluent of eluting, lyophilization, it is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=66.0 μg/mL。
Embodiment 19: solid dispersion
The mass ratio weighing Fructus anethi extract and carrier polyvinylpyrrolidone (PVP) 1:2,1:4,1:6 in proportion is respectively put in beaker, add appropriate dehydrated alcohol and Vc, it is completely dissolved to Fructus anethi extract and carrier by magnetic stirrer, proceed to rotation after being sufficiently mixed uniformly and steam removing organic solvent in instrument, it is dried, pulverize 80 mesh sieves, obtain Fructus anethi effective extract PVP clathrate.
Embodiment 20: cyclodextrin clathrate
Being ground well with 1-5 times of water by beta-schardinger dextrin-, add Fructus anethi effective extract (should being first dissolved in a small amount of organic solvent of slightly water-soluble) and continue to be fully ground to becoming pastel, cold drying i.e. obtains cyclodextrin clathrate.
Embodiment 21: dispersible tablet
1.
The preparation of Fructus anethi effective extract dispersion, precision weighs Fructus anethi extract, Vc, adds the sodium lauryl sulphate of recipe quantity, it is after 70% ethanol dissolves and adds the soluble starch mix homogeneously of equal proportion by appropriate concentration, it is evaporated at a temperature of 70 DEG C, pulverizes, cross 100 mesh sieves；
2.
Polyvinylpolypyrrolidone by the Fructus anethi effective extract starch dispersions in the first step Yu recipe quantity, pregelatinized Starch, it is that 70% ethanol makees wetting agent by appropriate concentration, stirs while adding, make wet granular and cross 14 mesh sieves, after ambient temperatare puts 15min, 60 DEG C of oven for drying 45min, 16 mesh sieve granulate, add Pulvis Talci and the differential silica gel of recipe quantity, after mix homogeneously, tabletting and get final product.
Claims (11)
1. a Fructus anethi extract, it is characterized by that Fructus anethi extracts through solvent supersonic, after extracting solution concentrates, separating with silicagel column, the silicagel column after loading, first with the petroleum ether that volume ratio is 100:2-acetone mixed solvent eluting, continue with the petroleum ether that volume ratio is 100:3-acetone mixed solvent eluting, with the recovered solvent of eluent of the petroleum ether of 100:3-acetone mixed solvent eluting, it is dried, is this extract；
Or first with the petroleum ether that volume ratio is 100:12-acetone mixed solvent eluting, continue with the petroleum ether that volume ratio is 100:17-acetone mixed solvent eluting, with the recovered solvent of eluent of the petroleum ether of 100:17-acetone mixed solvent eluting, it is dried, is this extract.
2. Fructus anethi extract as claimed in claim 1, is characterized by, extracting solvent for use can be methanol, petroleum ether, water, acetone, chloroform, ethyl acetate, methanol-water mixed solvent or ethanol water mixed solvent, and optimum is methanol or 95% ethanol.
3. Fructus anethi extract as claimed in claim 1, it is characterized by that Fructus anethi extracts through solvent supersonic, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:2, volume ratio) eluting, continue with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) eluting, with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:3, volume ratio) the recovered organic solvent of eluent of eluting, it is dried, is this extract；Or first with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:12, volume ratio) eluting, continues with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) eluting, the recovered organic solvent of eluent with petroleum ether-acetone mixed solvent (petroleum ether-acetone, 100:17, volume ratio) eluting, it is dried, is this extract.
4. the Uigurs medicine Fructus anethi extract of dimension as described in claim 1-3, is characterized by, the consumption of eluent is 2-50 times of column volume.
5. the preparation method of extract as described in claim 1-3, is characterized by: optimum for first with the petroleum ether-acetone mixed solvent eluting 10 times column volume of 100:2, continuing with the petroleum ether-acetone mixed solvent eluting 20 times column volume of 100:3；Or optimum is first with 100；The petroleum ether of 12-acetone mixed solvent eluting 25 times column volume, continues with the petroleum ether-acetone mixed solvent eluting 35 times column volume of 100:17.
6. a pharmaceutical composition, it is characterised in that comprise the Uigurs medicine Fructus anethi extract described in claim 1-5 any one and pharmaceutically acceptable carrier.
7. a pharmaceutical preparation, it is characterised in that comprise the Fructus anethi extract described in claim 1-5 any one or the pharmaceutical composition described in claim 6.
8. pharmaceutical preparation as claimed in claim 7, it is characterised in that described preparation is solid dispersion, cyclodextrin clathrate, dispersible tablet.
9. extract described in claim 1-5 any one or the compositions described in claim 6 or the application in preparing inhibiting activity of acetylcholinesterase medicine of the pharmaceutical preparation described in claim 7.
Apply the most as claimed in claim 9, it is characterised in that described inhibiting activity of acetylcholinesterase, can be used for treating the medical usage of the aspects such as Alzheimer (AD).
The 11. Uigurs medicine Fructus anethi extracts as described in claim 1-5 any one, it is characterised in that detecting by TLC method, its chromatographic condition is: GF254Silica gel plate, development system: petroleum ether: ethyl acetate: acetone=6:1:1, coloration method: vanillin concentrated sulphuric acid develops the color.
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| CN201510109413.8A CN106031735A (en) | 2015-03-12 | 2015-03-12 | Medical uses and preparing method of dill seed effective extract product with acetylcholinesterase inhibiting activity and composition of the extract product |
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| CN201510109413.8A CN106031735A (en) | 2015-03-12 | 2015-03-12 | Medical uses and preparing method of dill seed effective extract product with acetylcholinesterase inhibiting activity and composition of the extract product |
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Application publication date: 20161019 |