CN101297863A - Mulberry bark extract with glycosidase inhibiting function and preparation thereof - Google Patents
Mulberry bark extract with glycosidase inhibiting function and preparation thereof Download PDFInfo
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Abstract
The invention discloses a white mulberry root-bark exact with the glucosidase inhibition function, a preparation method thereof and a quality control method. During the preparation of the white mulberry root-bark extract, extraction, concentration and centrifugalization are adopted, and the methods of three different types of anion-cation exchange resin columns, drying and so on are carried out to fully extract and highly concentrate the effective drugs; at the same time, the invention further provides the quality control method for carrying out the content measurement of the extract.
Description
Technical field
The present invention relates to a kind of Chinese medicine extract and preparation method thereof and method of quality control, particularly a kind of Chinese medicine extract and preparation method thereof and method of quality control with glycosidase inhibiting function.
Background technology
Cortex Mori is removed the dry root bark of cork for the moraceae plants Mulberry, falls leaves in autumn end usually and excavates root before germinateing to the spring, scrapes off yellow cork, vertically cuts open, strips root bark, dries and is used as medicine.Multiple alkaloids substances such as 1-deoxynojirimycin (DNJ) that contains in the Cortex Mori and derivant are the clear and definite alpha-glucosidase inhibitor of effect.
At application number is 01113191.8, patent name discloses a kind of method for preparing the Cortex Mori total alkaloids in the patent documentation of " the active Chinese medicine extract of inhibition, Preparation Method And The Use with alpha-glucosidase ", its technology is that medical material is through after extracting, by steps such as concentrated, precipitate with ethanol, flocculations, remove partial impurities, supernatant is collected and is obtained total alkaloids again by ion-exchange resin purification; At application number is 03116817.5, patent name discloses a kind of compound Chinese medicinal preparation with blood sugar reducing function in the patent documentation of " a kind of compound Chinese medicinal preparation that contains Cortex Mori, Radix Et Rhizoma Fagopyri Tatarici with blood sugar reducing function and preparation method thereof ", the preparation technology of wherein related Cortex Mori extract for medical material through behind the ethanol extraction, direct spray drying; At application number is 200410018677.4, and patent name is for disclosing a kind of pharmaceutical composition of being made up of total alkaloids and total flavones from Folium Mori, Cortex Mori preparation in the patent documentation of " the active medical composition and its use of inhibition with alpha-glucosidase ".All do not relate to the method for using weak-type sun resin to come DNJ in the purification Cortex Mori among the preparation technology of the Cortex Mori extract that above-mentioned several parts of patents are related, its quality control index is total alkaloids, not relating to 1-deoxynojirimycin (DNJ) is quality control index, and does not see that 1-deoxynojirimycin (DNJ) content is greater than 50% report.
Summary of the invention
The purpose of this invention is to provide a kind of inhibiting Cortex Mori extract of alpha-glucosidase that has; Another object of the present invention provides a kind of preparation method with the inhibiting Cortex Mori extract of alpha-glucosidase; Another object of the present invention provides a kind of method of quality control with the inhibiting Cortex Mori extract of alpha-glucosidase.
The present invention seeks to be achieved through the following technical solutions:
The content of 1-deoxynojirimycin is 500mg/g～900mg/g in the extract of the present invention.
Preparation method of extract of the present invention is:
The raw medicinal material Cortex Mori is through after the cutting, and as solvent extraction 2～4 times, each 1～3 hour, the extraction temperature was room temperature～90 ℃ with the alcoholic solution of water or 10%～30%, and the total solvent amount is 15～30 times of Cortex Mori raw medicinal material weight; Extracting solution is centrifugal behind 50 ℃～80 ℃ following concentrating under reduced pressure, get supernatant, remove pigment or directly go up the strongly acidic cation-exchange post by carbon column, being eluted to pH value with deionized water is 5～6, the concentration of 2～10 times of column volumes of reuse is the ammonia eluting of 0.2～1.0M, ammonia eluent concentrating under reduced pressure is waved most ammonia, pH value is 8～10, strong basic type anion-exchange resin post on the concentrated solution, and be washed to neutrality, pH value is 11～14 upper prop effluent and the last weak-type cation exchange resin column of water lotion series connection, the pH value that collection flows down from the weak-type cation exchange resin column is 8～9 effluent, washing also is collected into 2-3 column volume water lotion, merges upper prop effluent and water lotion, normal pressure, drying under reduced pressure, lyophilization or spray drying get Cortex Mori extract.
Wherein, the strongly acidic cation-exchange in the invention described above preparation method of extract can be selected 001*1,001*2,001*3,001*4,001*7 isogel type cation exchange resin or large pores cation exchange resins such as D072, D061, D001CC for use; Strong basic type anion-exchange resin can be selected 201*2,201*4,202*7 isogel type anion exchange resin or D090, D096, macroporous type anion exchange resin such as D201, D261 for use; The weak-type cation exchange resin can select 110 for use, D151, D152, D113, WK40, WK20 isogel type or large pores cation exchange resin.
The preferred for preparation method of extract of the present invention is:
The raw medicinal material Cortex Mori is through after the cutting, with water as solvent extraction 3 times, extraction time was respectively 2 hours, 1 hour, 1 hour, extracting temperature is 50 ℃, quantity of solvent is respectively 7 times of Cortex Mori raw medicinal material weight, 5 times, 5 times, extracting solution merges, centrifugal behind 60 ℃ of following concentrating under reduced pressure, get supernatant, last 001*7 gel type cation exchange resin column, (pH value of upper prop liquid is 4～5, the effluent pH value drops to 1～2, illustrate that effective ingredient exchanges on the resin), being eluted to pH value with deionized water is 5～6, the 0.5M ammonia eluting of 4～6 times of column volumes of reuse, ammonia eluent concentrating under reduced pressure is waved most ammonia, pH value is 8～10,201*4 gel strong basic type anion-exchange resin post on the concentrated solution, and be washed to neutrality, pH value is 13～14 upper prop effluent and the last D151 macropore weak-type cation exchange resin column of water lotion series connection, washing also is collected into 2 column volume water lotions, and the pH value that collection is got off from the weak-type cation exchange column is 8～9 effluent and water lotion, and drying under reduced pressure obtains Cortex Mori extract.
The preferred for preparation method of extract of the present invention is:
The raw medicinal material Cortex Mori is through after the cutting, alcoholic solution with 15% is as solvent extraction 4 times, extraction time was respectively 3 hours, 2 hours, 1 hour, 1 hour, and extracting temperature is 80 ℃, and quantity of solvent is respectively 8 times, 6 times, 5 times, 5 times of Cortex Mori raw medicinal material weight; Extracting solution merges, centrifugal behind 55 ℃ of following concentrating under reduced pressure, get supernatant, last 001*7 gel type cation exchange resin column, the pH value of upper prop liquid is 4～5, the effluent pH value drops to 1～2, being eluted to pH value with deionized water is 5～6, the 1.0M ammonia eluting of 2～4 times of column volumes of reuse, ammonia eluent concentrating under reduced pressure is waved most ammonia, pH value is 9, D201 macropore strong basic type anion-exchange resin post on the concentrated solution, and be washed to neutrality, pH value is 13～14 upper prop effluent and the last 110 gel weak-type cation exchange resin columns of water lotion series connection, washing also is collected into 2 column volume water lotions, and the pH value that collection is got off from the weak-type cation exchange resin column is 8～9 effluent and water lotion, and spray drying obtains Cortex Mori extract.
The preferred for preparation method of extract of the present invention is:
The raw medicinal material Cortex Mori is through after the cutting, and as solvent extraction 2 times, extraction time was respectively 3 hours, 2 hours with water, and extracting temperature is 30 ℃, and quantity of solvent is respectively 10 times, 8 times of Cortex Mori raw medicinal material weight; Extracting solution merges, centrifugal behind 70 ℃ of following concentrating under reduced pressure, get supernatant, pass through carbon column, remove pigment, ethanol elution with 15%, 001*4 type cation exchange resin column on 15% the ethanol elution, the pH value of upper prop liquid is 6, the effluent pH value drops to 1～2, being eluted to pH value with deionized water is 5～6, the ammonia eluting of the 0.2M of 6～8 times of column volumes of reuse is waved most ammonia with ammonia eluent concentrating under reduced pressure, and pH value is 9,201*4 gel strong basic type anion-exchange resin post on the concentrated solution, and being washed to neutrality, pH value is 13～14 upper prop effluent and the last WK40 macropore weak-type cation exchange resin column of water lotion series connection, washes and be collected into 2 column volume water lotions, the pH value that collection is got off from the weak-type cation exchange column is 8～9 effluent and water lotion, and lyophilization obtains Cortex Mori extract.
The method of quality control of extract of the present invention is as follows:
According to high effective liquid chromatography for measuring:
The a chromatographic condition: closing silica gel with octadecane base silane Key is filler; With acetonitrile-0.1% acetic acid solution=40-60: 60-40 is mobile phase, flow velocity 1.0ml/min, 25 ℃ of column temperatures; Fluorescence detector excitation wavelength 254nm, emission wavelength 322nm, sample size 10 μ l;
The preparation of b reference substance solution: precision takes by weighing 1-deoxynojirimycin 2-4mg, puts in the 10ml volumetric flask, adds redistilled water to dissolving and being diluted to scale, shakes up; Precision is measured 0.30-0.50ml, puts in the 5ml volumetric flask to add redistilled water to scale, shakes up; With redistilled water mother solution is diluted to the standard solution series of above-mentioned variable concentrations, precision is measured the 10ul standard solution, carries out derivatization and liquid-phase chromatographic analysis by the following step, with concentration of standard solution chromatographic peak area is carried out linear regression, the drawing standard curve.
The preparation of c sample solution: precision takes by weighing extract 0.03-0.06g, puts in the 10ml volumetric flask, adds the hydrochloric acid solution of 0.05-0.15mol/L, ultrasonicly make its dissolving and be diluted to scale, shake up, precision is measured 0.30-0.60ml, put in the 100ml volumetric flask and add deionized water, shake up to scale, standby;
D derivatization process: the standard solution 10 μ l that precision is measured said extracted liquid or variable concentrations put in the 1.5ml tool plug centrifuge tube, and accurate adding 10 μ l pH values are the sodium borate buffer liquid of 8-9, shake up; The accurate 20 μ l chloro-carbonic acids-9-fluorenes methyl ester (9-fluorenylmethyl chloroformate is FMOC-Cl) that adds shakes up; Put into 20-30 ℃ of circulator bath constant temperature 10-20 minute, and took out; Add 10ul 0.1M glycine, shake up; Add 0.1% acetic acid of 0.95ml, shake up, filter, get 10 μ l sample introduction analyses with the disposable aspiration needle filter; By external standard method calculation sample concentration, the content of 1-deoxynojirimycin is between 500mg/g～900mg/g in the extract.
The method of quality control of extract of the present invention is preferably as follows:
According to high effective liquid chromatography for measuring:
The a chromatographic condition: with octadecane base silane Key close silica gel be filler (C18 post 250mm*4.6mm, 5um); With acetonitrile-0.1% acetic acid solution=55: 45 was mobile phase, flow velocity 1.0ml/min, 25 ℃ of column temperatures; Fluorescence detector excitation wavelength 254nm, emission wavelength 322nm, sample size 10 μ l;
The preparation of b reference substance solution: precision takes by weighing 1-deoxynojirimycin 2.29mg, puts in the 10ml volumetric flask, adds redistilled water to dissolving and being diluted to scale, shakes up; Precision is measured 0.40ml, puts in the 5ml volumetric flask to add redistilled water to scale, shakes up; With redistilled water mother solution is diluted to the standard solution series of variable concentrations, precision is measured the 10ul standard solution, carries out derivatization and liquid-phase chromatographic analysis by the following step;
The preparation of c sample solution: precision takes by weighing extract 0.05g, puts in the 10ml volumetric flask, adds the hydrochloric acid solution of 0.05mol/L, ultrasonicly make its dissolving and be diluted to scale, shake up, precision is measured 0.50ml, put in the 100ml volumetric flask and add deionized water, shake up to scale, standby;
D derivatization process: precision measures said extracted liquid or different standard solution 10 μ l with concentration put in the 1.5ml tool plug centrifuge tube, and the accurate 10 μ l pH values that add are 8.5 sodium borate buffer liquid, shake up; The accurate 20 μ l chloro-carbonic acids-9-fluorenes methyl ester (9-fluorenylmethyl chloroformate) that adds shakes up; Put into 30 ℃ of circulator bath constant temperature 10 minutes, and took out; Add 10ul 0.1M glycine, shake up; Add 0.95ml 0.1% acetic acid, shake up, filter with the disposable aspiration needle filter, get 10 μ l sample introduction analyses, the record chromatogram, with the calculated by peak area sample concentration, the content of 1-deoxynojirimycin is between 500mg/g～900mg/g in the extract of the present invention by external standard method.
Description of drawings
Fig. 1: Cortex Mori extract is to the influence of the oral starch tolerance experiment of ICR mice.
The preparation method of extract of the present invention selects three kinds of ion exchange resin that extract of the present invention is carried out Purifying, the content of main active 1-DNJ DNJ increases substantially; Extract of the present invention Has the Inhibiting α-glucosidase active function.
Following experimental example and embodiment are used for further specifying but are not limited to the present invention.
The paste-forming rate of experimental example 1 different preparation method's gained mulberry bark extracts and the comparative experiments of 1-DNJ (DNJ) content
This experimental example relatively adopts following several method to prepare the height of 1-DNJ in the mulberry bark extract (DNJ) content, the results are shown in Table 1:
A, water extraction method: the 100 gram root barks of white mulberry add the water of 7 times of medicinal material weight 70 ℃ of lower extractions twice, Extraction time was respectively 2 hours, 1 hour, the extract concentrate drying;
B, alcohol extracting are followed the example of: the 100 gram root barks of white mulberry add 30% ethanol of 7 times of medicinal material weight 70 ℃ of lower heating Refluxing extraction twice, extraction time was respectively 2 hours, 1 hour, the extract concentrate drying;
C, water extraction and alcohol precipitation method: the 100 gram root barks of white mulberry add the water of 7 times of medicinal material weight 70 ℃ of lower extractions twice, Extraction time was respectively 2 hours, 1 hour, extract is concentrated into 300ml, added 95% of 900ml The ethanol alcohol precipitation, placement is spent the night, and is centrifugal, the supernatant concentrate drying;
D, ethanol extract from water precipitation: 30% the alcohol reflux that the 100 gram root barks of white mulberry add 7 times of medicinal material weight extracts two Inferior, extraction time was respectively 2 hours, 1 hour, and extract is concentrated into 150ml, adds 1200ml's Water sedimentation, placement is spent the night, and is centrifugal, the supernatant concentrate drying;
E, extract are crossed cation exchange resin processes: the water that the 100 gram root barks of white mulberry add 7 times of medicinal material weight exists 70 ℃ of lower extractions twice, extraction time was respectively 2 hours, 1 hour, and extract concentrates, and is centrifugal, gets The upper strongly acidic cation-exchange post of supernatant 200ml is such as the 001*7 type, dense with 300ml 0.5M Degree ammoniacal liquor wash-out, the eluent concentrate drying;
F, extract are crossed the cation and anion exchange resin method: the water that the 100 gram root barks of white mulberry add 7 times of medicinal material weight exists 70 ℃ of lower extractions twice, extraction time was respectively 2 hours, 1 hour, and extract concentrates, and is centrifugal, gets The upper strongly acidic cation-exchange post of supernatant 200ml is such as the 001*7 type, dense with 300ml 0.5M Upper 201*4 gel strong base anion exchange was set after degree ammoniacal liquor wash-out, ammoniacal liquor eluent concentrated and wave most ammoniacal liquor The fat post is collected upper prop efflux and water lotion (being washed to neutrality), and drying obtains mulberry bark extract;
G, water alcohol extract are crossed the carbon column method: the 100 gram root barks of white mulberry add the water of 7 times of medicinal material weight 70 Extract twice under ℃, extraction time was respectively 2 hours, 1 hour, and extract concentrates, and is centrifugal, gets Clear liquid 300ml crosses carbon column, with the ethanol elution of 1000ml 15%, collects the eluent concentrate drying;
H, preparation method of the present invention: the 100 gram root barks of white mulberry add the water of 7 times of medicinal material weight 70 ℃ of lower extractions Twice, extraction time was respectively 2 hours, 1 hour, and extract concentrates, and is centrifugal, gets supernatant 200ml Upper cationic ion-exchange resin such as the 001*7 type, with 300ml 0.5M concentration ammoniacal liquor wash-out, is washed ammoniacal liquor After taking off the liquid reduced pressure concentration and flinging to ammoniacal liquor, PH to 8,201*4 gel strong base anion is handed on the concentrate Change resin column, upper prop efflux and the upper D151 of water lotion series connection or D113 macropore weak-type cation exchange Post, the PH that collection is got off from the weak-type cation exchange column are 8～9 efflux and water lotion, by Drying under reduced pressure obtains extract of the present invention;
The paste-forming rate of the different preparation method's gained of table 1 extract of the present invention and DNJ content are relatively
From above-mentioned interpretation, different preparation method's gained mulberry bark extract paste-forming rates and DNJ contain Amount all has bigger difference, and the method applied in the present invention is compared with other preparation method, and paste-forming rate is big The big minimizing, the 1-DNJ DNJ content in the extract is higher than other preparation method.
The extract of the present invention of experimental example 2 usefulness embodiment 1 preparation is to the oral starch tolerance test of ICR mouse
1, the preparation of experiment reagent
0.15g/ml the preparation of soluble starch: take by weighing soluble starch 22.5g, with drinking water 120ml, Fully mixing is placed in 100 ℃ the boiling water bath and heats, and heats while stirring, and is settled to while hot 150ml, Water is supplied 150ml when treating drop in temperature to room temperature;
The preparation of Glucobay: get Glucobay a slice, be dissolved in the 0.15g/ml soluble starch of 100ml, Abundant mixing;
Extract reagent preparation method of the present invention with embodiment 1 preparation is as follows:
The compound method of the heavy dose of group of extract (dosage 15mg/kg): get extract 27mg, molten In the 0.15g/ml of 20ml soluble starch, abundant mixing;
Extract small dose group (dosage 10mg/kg) compound method: get heavy dose of solution 5ml, Add in the 0.15g/ml soluble starch of 5ml, fully mixing;
2, experimental technique
Normal ICR mouse, body weight 30 ± 2g, fasting be can't help water 12 hours, be divided into 4 groups, every group 10: two dosage groups of control group, Glucobay group and extract of the present invention, with positive drug and be subjected to reagent respectively in certain proportion with gelatinization after starch solution fully mix gastric infusion after (dosage of starch solution is 3g/kg), and clock immediately, measure blood sugar level at 0 minute, 30 minutes, 60 minutes, 90 minutes tail venous blood samplings respectively, experimental result sees Table 2 and Fig. 1:
Table 2 extract of the present invention is to the impact of the oral starch tolerance test of ICR mouse
From above-mentioned interpretation, extract of the present invention has good inhibitory effect to the post-prandial glycemia of normal ICR mice, compare with matched group from 0～120min, two dosage groups of the present invention and tangible glycemic peaks all not occur, illustrate that extract of the present invention has comparatively significantly glycosidase inhibiting function, the effect that heavy dose of group suppresses blood glucose obviously is better than small dose group and glucobay (acarbose) group.
Following embodiment all can realize the effect of above-mentioned experimental example.
The specific embodiment
Embodiment 1: preparation method of extract of the present invention
Raw medicinal material 10kg Cortex Mori is through after the cutting, and as solvent extraction 3 times, extraction time was respectively 2 hours, 1 hour, 1 hour with water, and extracting temperature is 50 ℃, and 70L, 50L, 50L that amount of water is respectively Cortex Mori raw medicinal material weight are doubly; Extracting solution merges, centrifugal behind 60 ℃ of following concentrating under reduced pressure, get the about 40L of supernatant, last 001*7 gel type cation exchange resin column, column volume is 5L, the pH value of upper prop liquid is 4～5, effluent PH drops to 1～2, being eluted to pH value with deionized water is 5～6, and reuse 25L concentration is the ammonia eluting of 0.5M, and ammonia eluent concentrating under reduced pressure is waved most ammonia, PH is 8～9,201*4 gel strong basic type anion-exchange resin post on the concentrated solution, column volume is 4L, is washed to neutrality, D151 macropore weak-type cation exchange resin column is gone up in the upper prop effluent of PH to 13～14 and water lotion series connection, column volume is 5L, is washed to about 2 column volumes, and the PH that collection is got off from the weak-type cation exchange column is 8～9 effluent and water lotion, spray drying, obtain Cortex Mori extract 35g, detect through HPLC, 1-deoxynojirimycin content is 750mg/g in the extract.
Embodiment 2: preparation method of extract of the present invention
Raw medicinal material Cortex Mori 1kg is through after the cutting, alcoholic solution with 15% is as solvent extraction 4 times, extraction time was respectively 3 hours, 2 hours, 1 hour, 1 hour, and extracting temperature is 80 ℃, and quantity of solvent is respectively 8L, 6L, 5L, the 5L of Cortex Mori raw medicinal material weight; Extracting solution merges, centrifugal behind 55 ℃ of following concentrating under reduced pressure, get the 3L supernatant, last 001*7 gel type cation exchange resin column, column volume is 0.4L, the pH value of upper prop liquid is 4～5, effluent PH drops to 1～2, being eluted to PH with deionized water is 5～6, and reuse 1.2L concentration is the ammonia eluting of 1.0M, and ammonia eluent concentrating under reduced pressure is waved most ammonia, pH value is 9, D201 macroporous type strong basic type anion-exchange resin post on the concentrated solution, column volume is 0.3L, is washed to neutrality, the upper prop effluent of PH to 13～14 and the last 110 gel-type weak-type cation exchange resin columns of water lotion series connection, column volume is 0.4L, is washed to about 3 column volumes, and the PH that collection is got off from the weak-type cation exchange resin column is 8～9 effluent and water lotion, drying under reduced pressure, obtain Cortex Mori extract 4g, detect through HPLC, 1-deoxynojirimycin content is 720mg/g in the extract.
Embodiment 3: preparation method of extract of the present invention
Raw medicinal material Cortex Mori 1kg is through after the cutting, and as solvent extraction 2 times, extraction time was respectively 3 hours, 2 hours with water, and extracting temperature is 30 ℃, and quantity of solvent is respectively 10 times, 8 times of Cortex Mori raw medicinal material weight; Extracting solution merges, centrifugal behind 70 ℃ of following concentrating under reduced pressure, get supernatant 3L, pass through carbon column, remove most of pigment, 15% ethanol elution with 8L, 001*4 type strong-acid cation-exchange resin post on 15% the ethanol elution, column volume is 0.5L, and the pH value of upper prop liquid is 6, and effluent PH drops to 1～2, being eluted to PH with deionized water is 5～6, reuse 4.0L concentration is the ammonia eluting of 0.2M, and ammonia eluent concentrating under reduced pressure is waved most ammonia, and PH is 9,201*4 gel type strong base type anion-exchange resin column on the concentrated solution, be washed to neutrality, pH value is 13～14 upper prop effluent and the last WK40 macroporous type weak-type cation exchange column of water lotion series connection, is washed to about 3 column volumes, the PH that collection is got off from the weak-type cation exchange column is 8～9 effluent and water lotion, lyophilization obtains Cortex Mori extract 2.8g, detects through HPLC, and 1-deoxynojirimycin content is 900mg/g in the extract.
Embodiment 4: the method for quality control of the extract of the present invention of embodiment 1 preparation
According to high effective liquid chromatography for measuring:
The a chromatographic condition: with octadecane base silane Key close silica gel be filler (C18 post 250mm*4.6mm, 5um); With acetonitrile-0.1% acetic acid solution=55: 45 was mobile phase, flow velocity 1.0ml/min, 25 ℃ of column temperatures; Fluorescence detector excitation wavelength 254nm, emission wavelength 322nm, sample size 10 μ l;
The preparation of b reference substance solution: precision takes by weighing 1-deoxynojirimycin 2.29mg, puts in the 10ml volumetric flask, adds redistilled water to dissolving and being diluted to scale, shakes up; Precision is measured 0.40ml, puts in the 5ml volumetric flask to add redistilled water to scale, shakes up; With redistilled water mother solution is diluted to the standard solution series of variable concentrations, precision is measured the 10ul standard solution, carries out derivatization and liquid-phase chromatographic analysis by the following step;
The preparation of c sample solution: precision takes by weighing extract 0.05g, puts in the 10ml volumetric flask, adds the hydrochloric acid solution of 0.05mol/L, ultrasonicly make its dissolving and be diluted to scale, shake up, precision is measured 0.50ml, put in the 100ml volumetric flask and add deionized water, shake up to scale, standby;
D derivatization process: precision measures said extracted liquid or different standard solution 10 μ l with concentration put in the 1.5ml tool plug centrifuge tube, and the accurate 10 μ l pH values that add are 8.5 sodium borate buffer liquid, shake up; The accurate 20 μ l chloro-carbonic acids-9-fluorenes methyl ester (9-fluorenylmethyl chloroformate) that adds shakes up; Put into 30 ℃ of circulator bath constant temperature 10 minutes, and took out; Add 10 μ l 0.1M glycine, shake up; Add 0.95ml 0.1% acetic acid, shake up, filter with the disposable aspiration needle filter, get 10 μ l sample introduction analyses, the record chromatogram, with the calculated by peak area sample concentration, with the calculated by peak area sample concentration, detect the content of 1-deoxynojirimycin in the embodiment 1 gained Cortex Mori extract at 750mg/g by external standard method by this method by external standard method.
Claims (8)
1, a kind of preparation method with Cortex Mori extract of glycosidase inhibiting function is characterized in that this preparation method of extract is as follows:
The raw medicinal material Cortex Mori is through after the cutting, and as solvent extraction 2～4 times, each 1～3 hour, the extraction temperature was room temperature～90 ℃ with the alcoholic solution of water or 10%～30%, and the total solvent amount is 15～30 times of Cortex Mori raw medicinal material weight; Extracting solution is centrifugal behind 50 ℃～80 ℃ following concentrating under reduced pressure, get supernatant, remove pigment or directly go up the strongly acidic cation-exchange post by carbon column, being eluted to pH value with deionized water is 5～6, the concentration of 2～10 times of column volumes of reuse is the ammonia eluting of 0.2～1.0M, ammonia eluent concentrating under reduced pressure is waved most ammonia, pH value is 8～10, strong basic type anion-exchange resin post on the concentrated solution, and be washed to neutrality, pH value is 11～14 upper prop effluent and the last weak-type cation exchange resin column of water lotion series connection, the pH value that collection flows down from the weak-type cation exchange resin column is 8～9 effluent, washing also is collected into 2-3 column volume water lotion, merges upper prop effluent and water lotion, normal pressure, drying under reduced pressure, lyophilization or spray drying get Cortex Mori extract.
2, the preparation method of Cortex Mori extract as claimed in claim 1 is characterized in that: strongly acidic cation-exchange is wherein selected 001*1,001*2,001*3,001*4 or 001*7 gel type cation exchanger resin or D072, D061 or D001CC large pores cation exchange resin for use; Strong basic type anion-exchange resin is selected 201*2,201*4 or 202*7 gel-type anion exchange resin or D090, D096, D201 or D261 macroporous type anion exchange resin for use; The weak-type cation exchange resin selects 110 for use, D151, D152, D113, WK40 or WK20 gel-type or large pores cation exchange resin.
3, the preparation method of Cortex Mori extract as claimed in claim 1 is characterized in that this preparation method of extract is as follows:
The raw medicinal material Cortex Mori is through after the cutting, with water as solvent extraction 3 times, extraction time was respectively 2 hours, 1 hour, 1 hour, extracting temperature is 50 ℃, quantity of solvent is respectively 7 times of Cortex Mori raw medicinal material weight, 5 times, 5 times, extracting solution merges, centrifugal behind 60 ℃ of following concentrating under reduced pressure, get supernatant, last 001*7 gel type cation exchange resin column, the pH value of upper prop liquid is 4～5, the effluent pH value drops to 1～2, illustrate that effective ingredient has exchanged on the resin, being eluted to pH value with deionized water is 5～6, the 0.5M ammonia eluting of 4～6 times of column volumes of reuse, ammonia eluent concentrating under reduced pressure is waved most ammonia, pH value is 8～10,201*4 gel strong basic type anion-exchange resin post on the concentrated solution, and be washed to neutrality, pH value is 13～14 upper prop effluent and the last D151 macropore weak-type cation exchange resin column of water lotion series connection, washing also is collected into 2 column volume water lotions, and the pH value that collection is got off from the weak-type cation exchange column is 8～9 effluent and water lotion, and drying under reduced pressure obtains Cortex Mori extract.
4, the preparation method of Cortex Mori extract as claimed in claim 1 is characterized in that this preparation method of extract is as follows:
The raw medicinal material Cortex Mori is through after the cutting, alcoholic solution with 15% is as solvent extraction 4 times, extraction time was respectively 3 hours, 2 hours, 1 hour, 1 hour, and extracting temperature is 80 ℃, and quantity of solvent is respectively 8 times, 6 times, 5 times, 5 times of Cortex Mori raw medicinal material weight; Extracting solution merges, centrifugal behind 55 ℃ of following concentrating under reduced pressure, get supernatant, last 001*7 gel type cation exchange resin column, the pH value of upper prop liquid is 4～5, the effluent pH value drops to 1～2, being eluted to pH value with deionized water is 5～6, the 1.0M ammonia eluting of 2～4 times of column volumes of reuse, ammonia eluent concentrating under reduced pressure is waved most ammonia, pH value is 9, D201 macropore strong basic type anion-exchange resin post on the concentrated solution, and be washed to neutrality, pH value is 13～14 upper prop effluent and the last 110 gel weak-type cation exchange resin columns of water lotion series connection, washing also is collected into 2 column volume water lotions, and the pH value that collection is got off from the weak-type cation exchange resin column is 8～9 effluent and water lotion, and spray drying obtains Cortex Mori extract.
5, the preparation method of Cortex Mori extract as claimed in claim 1 is characterized in that this preparation method of extract is as follows:
The raw medicinal material Cortex Mori is through after the cutting, and as solvent extraction 2 times, extraction time was respectively 3 hours, 2 hours with water, and extracting temperature is 30 ℃, and quantity of solvent is respectively 10 times, 8 times of Cortex Mori raw medicinal material weight; Extracting solution merges, centrifugal behind 70 ℃ of following concentrating under reduced pressure, get supernatant, pass through carbon column, remove pigment, ethanol elution with 15%, 001*4 type cation exchange resin column on 15% the ethanol elution, the pH value of upper prop liquid is 6, the effluent pH value drops to 1～2, being eluted to pH value with deionized water is 5～6, the ammonia eluting of the 0.2M of 6～8 times of column volumes of reuse is waved most ammonia with ammonia eluent concentrating under reduced pressure, and pH value is 9,201*4 gel strong basic type anion-exchange resin post on the concentrated solution, and being washed to neutrality, pH value is 13～14 upper prop effluent and the last WK40 macropore weak-type cation exchange resin column of water lotion series connection, washes and be collected into 2 column volume water lotions, the pH value that collection is got off from the weak-type cation exchange column is 8～9 effluent and water lotion, and lyophilization obtains Cortex Mori extract.
6, the method for quality control of the Cortex Mori extract of being made by the described preparation method of claim 1-2 is characterized in that this method is as follows:
According to high effective liquid chromatography for measuring:
The a chromatographic condition: closing silica gel with octadecane base silane Key is filler; With acetonitrile-0.1% acetic acid solution=40-60: 60-40 is mobile phase, flow velocity 1.0ml/min, 25 ℃ of column temperatures; Fluorescence detector excitation wavelength 254nm, emission wavelength 322nm, sample size 10 μ l;
The preparation of b reference substance solution: precision takes by weighing 1-deoxynojirimycin 2-4mg, puts in the 10ml volumetric flask, adds redistilled water to dissolving and being diluted to scale, shakes up; Precision is measured 0.30-0.50ml, puts in the 5ml volumetric flask to add redistilled water to scale, shakes up; With redistilled water mother solution is diluted to the standard solution series of variable concentrations, precision is measured the 10ul standard solution, carries out derivatization and liquid-phase chromatographic analysis by the following step, with concentration of standard solution chromatographic peak area is carried out linear regression, the drawing standard curve;
The preparation of c sample solution: precision takes by weighing extract 0.03-0.06g, puts in the 10ml volumetric flask, adds the hydrochloric acid solution of 0.05-0.15mol/L, ultrasonicly make its dissolving and be diluted to scale, shake up, precision is measured 0.30-0.60ml, put in the 100ml volumetric flask and add deionized water, shake up to scale, standby;
D derivatization process: the standard solution 10 μ l that precision is measured said extracted liquid or variable concentrations put in the 1.5ml tool plug centrifuge tube, and accurate adding 10 μ l pH values are the sodium borate buffer liquid of 8-9, shake up; The accurate 20 μ l chloro-carbonic acids-9-fluorenes methyl ester that adds shakes up; Put into 20-30 ℃ of circulator bath constant temperature 10-20 minute, and took out; Add the 10ul0.1M glycine, shake up; Add 0.95ml 0.1% acetic acid, shake up, filter, get 10 μ l sample introduction analyses with the disposable aspiration needle filter; By external standard method calculation sample concentration, the content of 1-deoxynojirimycin is between 500mg/g～900mg/g in the extract.
7, the method for quality control of Cortex Mori extract as claimed in claim 6 is characterized in that this method is as follows:
According to high effective liquid chromatography for measuring:
The a chromatographic condition: closing silica gel with octadecane base silane Key is filler; With acetonitrile-0.1% acetic acid solution=55: 45 was mobile phase, flow velocity 1.0ml/min, 25 ℃ of column temperatures; Fluorescence detector excitation wavelength 254nm, emission wavelength 322nm, sample size 10 μ l;
The preparation of b reference substance solution: precision takes by weighing 1-deoxynojirimycin 2.29mg, puts in the 10ml volumetric flask, adds redistilled water to dissolving and being diluted to scale, shakes up; Precision is measured 0.40ml, puts in the 5ml volumetric flask to add redistilled water to scale, shakes up; With redistilled water mother solution is diluted to the standard solution series of variable concentrations, precision is measured the 10ul standard solution, carries out derivatization and liquid-phase chromatographic analysis by the following step;
The preparation of c sample solution: precision takes by weighing extract 0.05g, puts in the 10ml volumetric flask, adds the hydrochloric acid solution of 0.05mol/L, ultrasonicly make its dissolving and be diluted to scale, shake up, precision is measured 0.50ml, put in the 100ml volumetric flask and add deionized water, shake up to scale, standby;
D derivatization process: precision measures said extracted liquid or different standard solution 10 μ l with concentration put in the 1.5ml tool plug centrifuge tube, and the accurate 10 μ l pH values that add are 8.5 sodium borate buffer liquid, shake up; The accurate 20 μ l chloro-carbonic acids-9-fluorenes methyl ester that adds shakes up; Put into 30 ℃ of circulator bath constant temperature 10 minutes, and took out; Add the 0.1M glycine of 10ul, shake up; Add 0.95ml 0.1% acetic acid, shake up, filter, get 10 μ l sample introduction analyses with the disposable aspiration needle filter, the record chromatogram, with the calculated by peak area sample concentration, the content of 1-deoxynojirimycin is between 500mg/g～900mg/g in the extract by external standard method.
8, a kind of content of 1-deoxynojirimycin is the Cortex Mori extract of 500mg/g～900mg/g.
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