CN106265783A - The medical usage of gold Fructus Chebulae's inhibiting activity of acetylcholinesterase effective extract and combinations thereof thing and preparation method - Google Patents
The medical usage of gold Fructus Chebulae's inhibiting activity of acetylcholinesterase effective extract and combinations thereof thing and preparation method Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology field, be specifically related to the preparation method of a kind of gold Fructus Chebulae's inhibiting activity of acetylcholinesterase extract and combinations thereof thing and it is at the medical usage treating the aspects such as Alzheimer (AD).Extract of the present invention obtains by the following method: gold Fructus Chebulae extracts through solvent supersonic, after extracting solution concentrates, separates with silicagel column, and the silicagel column after loading, first with the methylene chloride-methanol mixed solvent eluting that volume ratio is 100:3, continuing by volume ratio is 100；The methylene chloride-methanol mixed solvent eluting of 5, with the recovered solvent of eluent of the methylene chloride-methanol mixed solvent eluting of 100:5, is dried, obtains this extract；Or first with the methylene chloride-methanol mixed solvent eluting that volume ratio is 100:25, continue with the methylene chloride-methanol mixed solvent eluting that volume ratio is 100:50, with the recovered solvent of eluent of the methylene chloride-methanol mixed solvent eluting of 100:50, it is dried, obtains this extract.
Description
Technical field
The invention belongs to pharmaceutical technology field, be specifically related to the preparation method of a kind of gold Fructus Chebulae's 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
Gold Fructus Chebulae is the dry golden yellow mature fruit of Combretum Racemosum plant Fructus Chebulae TerminALia chebULa Retz.When autumn, fruit maturation in winter, the full person of yellow is adopted in choosing, removes impurity, dries in the shade.Clear uncommon draw, adjust unit, detoxify, help digestion, wound healing, granulation promoting.Depend on for conspicuous, Xi La, Ba Da, wrap disease as dry in Ba Da, wound, fracture.
The present invention by a large scale, systematically screening active ingredients, it is surprised to find that Uigurs medicine gold Fructus Chebulae extract and part chemical constituent thereof, having acetylcholinesterase significantly and do inhibitory activity, 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 gold Fructus Chebulae extract.
Present invention also offers with the gold Fructus Chebulae extract compositions as main active.
Invention also provides the preparation of gold Fructus Chebulae 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 gold Fructus Chebulae is through methanol supersound extraction, after extracting solution concentrates, with silica gel (SiO2) post separation, silicagel column after loading, with methylene chloride-methanol mixed solvent gradient elution (methylene chloride-methanol, ratio is shown in Table 1), each methylene chloride-methanol 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 methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) the methylene chloride-methanol mixed solvent (methylene chloride-methanol that affords, 100:5, volume ratio) the recovered organic solvent of eluent, it is dried, it is inhibiting activity of acetylcholinesterase extract.And the eluting position of other ratios, there is no the effect of inhibiting activity of acetylcholinesterase.Optimum is first with 100:3 10 times of column volumes of methylene chloride-methanol mixed solvent eluting, continues and is the extract of inhibiting activity of acetylcholinesterase of the present invention with the eluent of methylene chloride-methanol 30 times of column volumes of mixed solvent eluting of 100:5 after drying.The extract of inhibiting activity of acetylcholinesterase, can be enriched with by above method, thus separates with other impurities compositions.This gold Fructus Chebulae's 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: dichloromethane: ethyl acetate: methanol (6:1:1) coloration method: vanillin-sulfuric acid develops the color；
5. or Uigurs medicine gold Fructus Chebulae is through methanol supersound extraction, after extracting solution concentrates, with silica gel (SiO2) post separation, silicagel column after loading, with methylene chloride-methanol mixed solvent gradient elution (methylene chloride-methanol, ratio is shown in Table 1), each methylene chloride-methanol 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 methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) the methylene chloride-methanol mixed solvent (methylene chloride-methanol that affords, 100:50, volume ratio) the recovered organic solvent of eluent, it is dried, it is inhibiting activity of acetylcholinesterase extract.And the eluting position of other ratios, there is no the effect of inhibiting activity of acetylcholinesterase.Optimum is first with 10 times of column volumes of methylene chloride-methanol mixed solvent eluting of 100:25, continues and is the extract of inhibiting activity of acetylcholinesterase of the present invention with the eluent of methylene chloride-methanol 30 times of column volumes of mixed solvent eluting of 100:50 after drying.The extract of inhibiting activity of acetylcholinesterase, can be enriched with by above method, thus separates with other impurities compositions.This gold Fructus Chebulae's 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: dichloromethane: ethyl acetate: methanol (1:1:1) coloration method: vanillin-sulfuric acid develops the color.
Specifically, discovery procedure is as follows:
1, gold Fructus Chebulae is through the preparation of methanol sonicated extract
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML methanol supersound extraction 15 min, after extracting solution concentrates, obtains extract SN0211A, sample 2.1882 g, and keep sample 0.1892g, applied sample amount 1.9990g, mixes sample silica gel 2.0 g, blank silica gel 10 g, methylene chloride-methanol system elutions.
2, methylene chloride-methanol mixed solvent gradient elution method and result
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML methanol supersound extraction 15 min, after extracting solution concentrates, obtains extract SN0245A, 1.1856g, upper prop, and methanol dissolves, and shares methanol 15 mL.Upper silica gel column chromatography, mix sample silica gel 3.0 g, blank silica gel 10 g, glass column internal diameter 2.0 cm, methylene chloride-methanol system gradient elution (condition is shown in Table 1), column volume 22 mL, each gradient elution 200mL, obtain, each concentration extracts the eluate of eluent, recycling design, obtain, methylene chloride-methanol gradient elution extract, TLC analysis result is shown in Fig. 3, Fig. 4, TLC analysis condition: GF254 silica gel plate, development system: dichloromethane: ethyl acetate: methanol (6:1:1), dichloromethane: ethyl acetate: methanol (1:1:1) (6ml adds 3d formic acid), 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. in 96 hole ELISA Plate
0), 25 μ L
AChE (0. 033 U/mL, pH 8. 0 PB dissolved dilution), 25 μ L sample solution (40 μ g mL- 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 %=［ A criterion group-(A sample-component A blank group) ］/(A criterion group-A blank group) × 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 gold Fructus Chebulae is through organic solvent supersound extraction, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, continues with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, the recovered organic solvent of eluent with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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 gold Fructus Chebulae is through organic solvent supersound extraction, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, continues with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, the recovered organic solvent of eluent with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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
Therefore, the preparation method of gold Fructus Chebulae's inhibiting activity of acetylcholinesterase extract is:
Uigurs medicine gold Fructus Chebulae is through organic solvent supersound extraction, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 2-50 times of column volume of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 2-50 times of column volume of eluting, the recovered organic solvent of eluent with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, it is dried, is this activity extract.Conditions above optimum is first with petroleum ether: ethyl acetate mixed solvent (petroleum ether: ethyl acetate, 100:3, volume ratio) 10 times of column volumes of eluting, continue with petroleum ether: ethyl acetate mixed solvent (petroleum ether: ethyl acetate, 100:5, volume ratio) 30 times of column volumes of eluting.Wherein,
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.Activity extract extraction consumption of organic solvent 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.
Uigurs medicine gold Fructus Chebulae is through organic solvent supersound extraction, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 2-50 times of column volume of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 2-50 times of column volume of eluting, the recovered organic solvent of eluent with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, it is dried, is this activity extract.Conditions above optimum is first with 10 times of column volumes of methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, continues with 30 times of column volumes of methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting.Wherein,
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.Activity extract extraction consumption of organic solvent 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.
5. the medical usage research of gold Fructus Chebulae acetylcholine esterase inhibition activity extract
The activity extract prepared by the golden Fructus Chebulae's inhibiting activity of acetylcholinesterase method for preparing extractive optimized, after tested, its inhibiting activity of acetylcholinesterase is IC50=17.7μg/mL。
There is the medical usage of the aspects such as treatment Alzheimer due to acetylcholinesteraseinhibitors inhibitors.Therefore, it has been found that golden Fructus Chebulae's inhibiting activity of acetylcholinesterase extract there is medical usage widely.
6, compositions of gold Fructus Chebulae's acetylcholine esterase inhibition activity extract and preparation method thereof research
1) solid dispersion
Prescription
Gold Fructus Chebulae extract
20 g
Vc
1 g
Polyvinylpyrrolidone
79 g
Solid dispersion 100g
Preparation method:
The mass ratio weighing gold Fructus Chebulae 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 gold Fructus Chebulae 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 golden Fructus Chebulae's effective extract PVP clathrate.
2) cyclodextrin clathrate
Prescription:
Gold Fructus Chebulae 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 gold Fructus Chebulae's 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:
Gold Fructus Chebulae 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 gold Fructus Chebulae effective extract dispersion, precision weighs gold Fructus Chebulae extract, Vc, add 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, pulverize, cross 100 mesh sieves；
2. by the polyvinylpolypyrrolidone of the golden Fructus Chebulae's 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, ambient temperatare puts 15min
After, 60 DEG C of oven for drying 45min, 16 mesh sieve granulate, add the Pulvis Talci of recipe quantity and differential silica gel, after mix homogeneously, tabletting and get final product.
7, the detection method research of gold Fructus Chebulae's acetylcholine esterase inhibition activity extract
It was found that the feature of gold Fructus Chebulae's 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: dichloromethane: ethyl acetate: methanol (6:1:1), dichloromethane: ethyl acetate: methanol (1:1:1) (6ml adds 3d formic acid), coloration method: vanillin-sulfuric acid develops the color.
Accompanying drawing illustrates:
The TLC chromatogram of Fig. 1 gold Fructus Chebulae's inhibiting activity of acetylcholinesterase extract；
The TLC chromatogram of Fig. 2 gold Fructus Chebulae's inhibiting activity of acetylcholinesterase extract；
The TLC chromatogram of Fig. 3 gold Fructus Chebulae's solvent extractable matter；
The TLC chromatogram of Fig. 4 gold Fructus Chebulae's 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 gold Fructus Chebulae 20 g, through 100
ML methanol supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=72.0
μg/mL。
Embodiment 2:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML ethanol ultrasonic extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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 3:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML petroleum ether supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) the recovered organic solvent of eluent of eluting, rotation is steamed seasoning, is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=70.0
μg/mL。
Embodiment 4:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML acetone supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=69.0
μg/mL。
Embodiment 5:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML chloroform supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=67.0
μg/mL。
Embodiment 6:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML ethyl acetate supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=65.0
μg/mL。
Embodiment 7:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML methanol-water mixed solvent (methanol: water=1:1) supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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 8:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML ethanol water mixed solvent (ethanol: water=1:1) supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=52.0
μg/mL。
Embodiment 9:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML ethanol-water mixed solvent (ethanol: water=90:10) supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (2.0 cm internal diameter pillar) after loading, first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=71.0
μg/mL。
Embodiment 10:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML methanol supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=81.0
μg/mL。
Embodiment 11:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML ethanol ultrasonic extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=86.0
μg/mL。
Embodiment 12:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML petroleum ether supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) the recovered organic solvent of eluent of eluting, rotation is steamed seasoning, is this activity extract.After tested, its inhibiting activity of acetylcholinesterase is IC50=77.0
μg/mL。
Embodiment 13:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML acetone supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=82.0
μg/mL。
Embodiment 14:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML chloroform supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=78.0
μg/mL。
Embodiment 15:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML ethyl acetate supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=71.0
μg/mL。
Embodiment 16:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML methanol-water mixed solvent (methanol: water=1:1) supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=84.0
μg/mL。
Embodiment 17:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML ethanol water mixed solvent (ethanol: water=1:1) supersound extraction 15 min, after extracting solution concentrates, separates with silicagel column, the silicagel column (2.0 after loading
Cm internal diameter pillar), first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=55.0
μg/mL。
Embodiment 18:
Take Uigurs medicine gold Fructus Chebulae 20 g, through 100
ML ethanol-water mixed solvent (ethanol: water=90:10) supersound extraction 15 min, after extracting solution concentrates, separate with silicagel column, silicagel column (2.0 cm internal diameter pillar) after loading, first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, 10 times of column volumes of eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, 30 times of column volumes of eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, 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=73.0
μg/mL。
Embodiment 19: solid dispersion
The mass ratio weighing gold Fructus Chebulae 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 gold Fructus Chebulae 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 golden Fructus Chebulae's effective extract PVP clathrate.
Embodiment 20: cyclodextrin clathrate
Being ground well with 1-5 times of water by beta-schardinger dextrin-, add gold Fructus Chebulae's 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 gold Fructus Chebulae effective extract dispersion, precision weighs gold Fructus Chebulae extract, Vc, add 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, pulverize, cross 100 mesh sieves；
2. by the polyvinylpolypyrrolidone of the golden Fructus Chebulae's 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, ambient temperatare puts 15min
After, 60 DEG C of oven for drying 45min, 16 mesh sieve granulate, add the Pulvis Talci of recipe quantity and differential silica gel, after mix homogeneously, tabletting and get final product.
Claims (11)
1. a golden Fructus Chebulae extract, is characterized by that gold Fructus Chebulae extracts through solvent supersonic, after extracting solution concentrates, separates with silicagel column, and the silicagel column after loading, first with the methylene chloride-methanol mixed solvent eluting that volume ratio is 100:3, continuing by volume ratio is 100；The methylene chloride-methanol mixed solvent eluting of 5, with the recovered solvent of eluent of the methylene chloride-methanol mixed solvent eluting of 100:5, is dried, is this extract；Or first with the methylene chloride-methanol mixed solvent eluting that volume ratio is 100:25, continue with the methylene chloride-methanol mixed solvent eluting that volume ratio is 100:50, with the recovered solvent of eluent of the methylene chloride-methanol mixed solvent eluting of 100:50, it is dried, is this extract.
2. golden Fructus Chebulae extract, 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 optimal conditions is methanol and 95% ethanol.
3. golden Fructus Chebulae extract, it is characterized by that gold Fructus Chebulae extracts through solvent supersonic, after extracting solution concentrates, separate with silicagel column, silicagel column after loading, first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:3, volume ratio) eluting, continue with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) eluting, with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:5, volume ratio) the recovered organic solvent of eluent of eluting, it is dried, is this extract；Or first with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:25, volume ratio) eluting, continues with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, the recovered organic solvent of eluent with methylene chloride-methanol mixed solvent (methylene chloride-methanol, 100:50, volume ratio) eluting, it is dried, is this extract.
4. the Uigurs medicine gold Fructus Chebulae 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 100:3 10 times of column volumes of methylene chloride-methanol mixed solvent eluting, continuing with 30 times of column volumes of methylene chloride-methanol mixed solvent eluting of 100:5；Or optimum is first with 10 times of column volumes of methylene chloride-methanol mixed solvent eluting of 100:25, continue with 30 times of column volumes of methylene chloride-methanol mixed solvent eluting of 100:50.
6. a pharmaceutical composition, it is characterised in that comprise the Uigurs medicine gold Fructus Chebulae extract described in claim 1-5 any one and pharmaceutically acceptable carrier.
7. a pharmaceutical preparation, it is characterised in that comprise the golden Fructus Chebulae 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).
11. such as claim 1 one kinds of golden Fructus Chebulae extracts, it is characterised in that detecting by TLC method, its chromatographic condition is: GF254Silica gel plate, development system: dichloromethane: ethyl acetate: methanol=6:1:1, dichloromethane: ethyl acetate: methanol=1:1:1(6ml adds 3d formic acid), coloration method: vanillin concentrated sulphuric acid develops the color；The TLC detection method of another kind of gold Fructus Chebulae extract, its chromatographic condition is: GF254Silica gel plate, development system: dichloromethane: ethyl acetate: methanol=1:1:1(6ml adds 3d formic acid), coloration method: vanillin concentrated sulphuric acid develops the color.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110613721A (en) * | 2019-09-23 | 2019-12-27 | 中国人民解放军第四军医大学 | New application of corilagin in preparing medicine for resisting sleep deprivation injury |
| CN111721870A (en) * | 2020-06-24 | 2020-09-29 | 兰州大学 | Method for high-throughput screening of acetylcholinesterase inhibitor |
-
2015
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110613721A (en) * | 2019-09-23 | 2019-12-27 | 中国人民解放军第四军医大学 | New application of corilagin in preparing medicine for resisting sleep deprivation injury |
| CN110613721B (en) * | 2019-09-23 | 2021-11-16 | 中国人民解放军第四军医大学 | New application of corilagin in preparing medicine for resisting sleep deprivation injury |
| CN111721870A (en) * | 2020-06-24 | 2020-09-29 | 兰州大学 | Method for high-throughput screening of acetylcholinesterase inhibitor |
| CN111721870B (en) * | 2020-06-24 | 2022-08-02 | 兰州大学 | Method for high-throughput screening of acetylcholinesterase inhibitor |
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