CN105769761B - The method for preparing DNJ nano suspensions - Google Patents
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- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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Abstract
The invention discloses separation DNJ and the method for preparing DNJ nano suspensions, the method of DNJ is detached using mulberry tree as raw material, it is extracted by crushing, microwave coking, isolate I is obtained after being dissolved in water, which passes sequentially through fine sand activated carbon and silica gel, and chromatographic isolation formation isolate II, the isolate II obtains isolate III through ion exchange and ethanol precipitation twice, the isolated isolate IV of the oxidized aluminium chromatographic columns of isolate III crystallizes isolate IV with obtaining target product after recrystallizing.The method for preparing DNJ nano suspensions carries out esterification modification to DNJ components first, enhances that its is fat-soluble, is then combined by surfactant and DNJ is dispersed into high degree of dispersion population by high-pressure homogeneous processing.Present invention separation DNJ technologies are easy to get with raw material, simple for process, with obvious effects, and the characteristics of suitable for large-scale production, the present invention, which prepares DNJ nano suspensions, can improve the dissolubility of drug, raising bioavailability.
Description
The application is for original applying number：201410614225.6 original application day is on October 31st, 2014, entitled
The divisional application that the patent of invention of " separation DNJ and the method for preparing DNJ nano suspensions " proposes.
Technical field
The invention belongs to field of natural medicinal chemistry, are related to purification of pharmaceuticals technology, more particularly to from Sang Yuan scales
The method for isolating and purifying DNJ and preparing DNJ component nano suspensions.
Background technology
Diabetes have become one of the primary killers for endangering the health of the mankind, can cause eye, kidney, heart, blood vessel, nerve
Chronic lesion and dysfunction.It is estimated that the year two thousand fifty, the number for suffering from diabetes in the world is up to 300,000,000, wherein 90% is 2
Patients with type Ⅰ DM.Mainly there are sulfonylurea, biguanides, alpha-glucosidase to inhibit currently for the oral hypoglycemic agents of diabetes B
4 class of agent and thiazolidinediones.Wherein alpha-glucosidase restrainer blood sugar decreasing effect is notable, and the concern being subject to is the most extensive.
Acarbose, voglibose and the Miglitol listed at present belongs to such drug, but these types of drug all passing through
It learns synthetic method to obtain, easily causes the side reactions such as different degrees of flatulence, abdominal discomfort, Nausea and vomiting, borborygmus and diarrhea.
Therefore, it is the important of traditional Chinese medicine research field to explore exploitation high-efficiency low-toxicity plant source alpha-glucosidase inhibitor hypoglycemic drug
Developing goal.
DNJ (Chinese name 1-DNJ) is a kind of piperidine alkaloid, be widely present with the limb of mulberry tree, leaf and
In rhizome, structure is similar to glucose (structural formula is shown below), can be with the enteral phlorose of mammal
Glycosides enzyme combines, and affinity is significantly greater than the disaccharides such as maltose, sucrose, so as to which disaccharides blocked to be combined with alpha-glucosidase,
It is emulative that disaccharides is inhibited to be decomposed into glucose, it is finally reached the purpose of control blood-sugar content.
At present, it is limited by DNJ physicochemical properties itself, existing isolation technics or efficiency easy to operate is low or technology is answered
Miscellaneous small scale limits the functionization of natural DNJ and scale exploitation, and the market input amount of DNJ is caused to be far smaller than practical need
The amount of asking (related content can with reference to author related plant DNJ to recent domestic isolate and purify research in each method it is excellent
Bad made systematization summarizes and discusses that (plant source DNJ separating and purifying technologies are summarized, sericulture science, 2014,40 (3):0544-
0550)).In view of hypoglycemic performance excellent DNJ and its predicament encountered in large-scale promotion application, it is necessary to develop one
Kind is simple and efficient the method for purifying natural DNJ.
Drug is transported by blood or diffusion of body fluids, first has to have certain water solubility, but drug will be by lipid
Biomembrane reach each histocyte, it is also required to have appropriate fat-soluble.DNJ belongs to the substance of the fat-soluble difference of good water solubility,
Because being difficult to by being unsuitable for physiologic infusion medication during biomembrane arrival small intestine.The alpha-glucosaccharase of small intestine physiology epimere if oral
Enzyme easily be suppressed, in, the degree of susceptibility of hypomere carbohydrate digestion it is smaller, need to enhance itself and internal mucous membrane by certain method
The adhesiveness of tissue so as to extend the residence times of DNJ in vivo, improves its bioavilability.
Invention content
In view of this, present invention firstly provides the methods of separating-purifying DNJ in slave natural plants being simple and efficient a kind of.
On the basis of this, the present invention also provides a kind of methods for preparing DNJ nano suspensions.
A kind of method for detaching DNJ, includes the following steps：
1), drying and crushing mulberry material and extracting solution is obtained with dilute hydrochloric acid, ethanol solution or extracting in boiling water；
2), first by the extracting solution of step 1) concentrate after microwave coking, obtain powdered extract, then dissolved and from
The heart detaches supernatant liquor, and last concentrated supernatant obtains isolate I；
3), first chromatographic isolation, including：
A, isolate I is detached using chromatographic column i, this step stationary phase is fine sand and Mixture of Activated Carbon, and mobile phase is second
Alcoholic solution；
B, it concentrates eluent obtained by A and is further detached with chromatographic column ii, this step stationary phase is silica gel, and mobile phase is second
The mixed liquor of acetoacetic ester, ethyl alcohol；
C, eluent obtained by concentration B, obtains isolate II；
4), ion-exchange chromatography：Isolate II is made to pass through cation exchange resin first, and is carried out with calcium hydroxide solution
Then elution concentrates eluent obtained by ion exchange, ethyl alcohol is then added in into the eluent after concentration is precipitated, finally mistake
Filter is concentrated to give isolate III；
5) isolate III, is detached first with chromatographic column iii, this step stationary phase is aluminium oxide, and mobile phase is acetic acid second
Ester, alcohol mixeding liquid, then concentrate eluant obtain isolate IV；
6) isolate IV, is added in into ethanol solution crystallizing first, crystallized product is then added in into ethyl acetate, ethyl alcohol mixes
It is recrystallized in solution, is finally separating crystallized product and obtains separation target components.
Preferably, concentration of hydrochloric acid is 0.03~0.07mol/L when step 1) is extracted using dilute hydrochloric acid；It is carried using ethanol solution
Concentration of alcohol is 60~80% when taking, and liquid ratio is 20~45mL/g, and extraction time is 3~5 times, during extraction using 350~
20~40min of 490W Sonication assisted treatments；The use of boiled water temperature during extracting in boiling water is 95~100 DEG C, liquid ratio for 20~
45mL/g, 20~40min of extraction time.
Preferably, microwave power is 640~800W during the coking of step 2) microwave, and processing time is 2~6min.
Preferably, fine sand and activated carbon volume ratio are 2 in step 3)-A:1, ethanol solution concentration is 50~90%, elution
Flow velocity is 0.9~1.6BV/h, collects 1~30 column volume eluent；Ethyl acetate and ethyl alcohol volume ratio are 1 in step 3)-B:3
~2:1, elution flow rate is 0.5~1.3BV/h, collects 1~30 column volume eluent.
Preferably, step 4) calcium hydroxide solution pH is 10~12, and elution flow rate is 0.9~1.6BV/h, collects 1~30
Column volume eluent, ethyl alcohol account for the volume of the concentrated liquid a concentration of 70~85%.
Preferably, step 5) ethyl acetate and ethyl alcohol volume ratio are 1:3~2:1, elution flow rate is 0.3~0.8BV/h, is received
Collect 1~50 column volume eluent.
Preferably, step 6) crystallization temperature is 1~6 DEG C, and ethanol solution concentration is more than 90%, and the time is 6~48h, is tied again
Brilliant temperature is -18~-23 DEG C, and ethyl acetate is 2 with ethyl alcohol volume ratio:1~7:1, the time is 6~48h, according to crystallize-tying again
The sequence of crystalline substance-crystallization-recrystallization repeats 2~5 times.
The method that the present invention prepares DNJ nano suspensions, includes the following steps：
1) esterification target components, are prepared, including：
A, by the gauge of substance take 0.25~1.0 part of chlorination 1- butyl -3- methylimidazoles, DNJ0.03-0.07 parts, second,
Thirdth, fourth, penta, oneself or 0.3-0.5 parts of heptanoic anhydride and 0.003~0.012 part of eight water aluminum sulfate and reaction kettle hybrid reaction is added in；b、
The reaction product of ethyl acetate washing step a, upper liquid is taken after layering, and neutrality is adjusted to saturated sodium carbonate, must be esterified target group
Point；
2) solution A is prepared：The solution A contains mannitol or polyethylene glycol 400 by mass：0.2~3%, lactose：0.5
~2%, glucose：0.5~2%, sodium carboxymethylcellulose：0.3~5%, remaining is water；
3) surfactant formulatory solution B is added in into solution A, surfactant qualities percentage composition in the solution B
It is 0.5~5%, the surfactant is polyethylene glycol 1000 vitamin E succinic acid ester, PLURONICS F87, Tween 80, ovum
Any one in phosphatide, lauryl sodium sulfate or glyceryl triacetate；
4) by volume 3:2~2:3 mixing steps 1) gained esterification target components and solution B and ultrasonication is carried out,
Power is 140~280W, 15~45min of time during supersound process；
5) removal step 4) organic component in solution, high pressure homogenizer or the processing of ultra micro nano grinder are then moved to,
Obtain nano suspension.
Preferably, temperature is 80~160 DEG C during step 1) hybrid reaction, and the reaction time is 6~14h, and mixed liquor rotating speed is
300~500r/min, the conversion ratio of gained esterification target components is 63.2~87.3%, the quality of obtained esterification target components
A concentration of 0.7~1.25%.
Preferably, 5 are respectively recycled under 500bar, 1000bar, 1500bar pressure respectively during the high-pressure homogeneous processing of step 5)
~the l0 times or ultra micro nano grinder 2900rpm 45~60min that mills obtains the suspension that average grain diameter is 180~380nm
The beneficial effects of the present invention are：
The method of present invention separation DNJ is proposed using common mulberry as raw material on the basis of chromatographic separation technology principle
A set of simple, efficiently, practical DNJ separating and purifying technology routes.Microwave coking means are utilized in separation method of the present invention for the first time
Handle extract, make under the premise of DNJ stability is not damaged part biological macromolecular by coking into insoluble solid and compared with
It can be easily separated；The present invention is stationary phase using fine sand and activated carbon and coordinates using ethyl alcohol as mobile phase, can effectively remove separation
A large amount of impurity in object I, and the stationary phase may be reused, and contribute to cost savings；With hydrogen during ion exchange of the present invention
Calcium oxide solution elutes cation exchange resin instead of the ammonium hydroxide with strong and stimulating smell in traditional handicraft, in subsequent handling
It is not removed only with respect to alkaline matters such as sodium hydroxides by ethanol precipitation easily, and it is apparent to elute effect feasibility；The present invention into
Using aluminium oxide as stationary phase, ethyl acetate, alcohol mixeding liquid can improve separative efficiency and substantially drop one step as mobile phase
The reversible adsorption loss of low DNJ, improves purification efficiency；Each solvent can be recycled in purification process of the present invention, and each chromatographic column is filled out
Expect that cheap and technical process is easy to operate, repeated preferable, suitable for large-scale industrial production.
The method that the present invention prepares DNJ nano suspensions carries out esterification modification to DNJ components first, is allowed to become fat-soluble
It is easy to by force and in vivo active ingredient of the enzymolysis for DNJ, the present invention is further combined and high-pressure homogeneous place by surfactant
DNJ active ingredients are dispersed into high degree of dispersion population of the grain size less than 1000nm by reason, form stable nanometer colloidal dispersion,
The solubility and dissolution rate of insoluble drug can be improved；So as to solve the fat-soluble differences of DNJ, the problem of bioavailability is low.
Specific embodiment
The preferred embodiment of the present invention is described in detail below.
Disclosure is detached DNJ and the method for preparing DNJ nano suspensions by following embodiments.
The method for detaching DNJ, includes the following steps：
1), drying and crushing mulberry material and extracting solution is obtained with dilute hydrochloric acid, ethanol solution or extracting in boiling water；
Wherein：Concentration of hydrochloric acid is 0.03~0.07mol/L when being extracted using dilute hydrochloric acid；Ethyl alcohol when being extracted using ethanol solution
A concentration of 60~80%, liquid ratio is 20~45mL/g, and extraction time is 3~5 times, and 350~490W ultrasonic waves are utilized during extraction
20~40min of aid in treatment；The use of boiled water temperature during extracting in boiling water it is 95~100 DEG C, liquid ratio is 20~45mL/g, during extraction
Between 20~40min；
2), first by the extracting solution of step 1) concentrate after microwave coking, obtain powdered extract, then dissolved and from
The heart detaches supernatant liquor, and last concentrated supernatant obtains isolate I；
Wherein the coking of step 2) microwave when microwave power be 640~800W, processing time be 2~6min；
3), first chromatographic isolation, including：
A, isolate I is detached using chromatographic column i, this step stationary phase is fine sand and Mixture of Activated Carbon, and mobile phase is second
Alcoholic solution；Fine sand and activated carbon volume ratio are 2 in this step:1, ethanol solution concentration is 50~90%, elution flow rate for 0.9~
1.6BV/h collects 1~30 column volume eluent；
B, it concentrates eluent obtained by A and is further detached with chromatographic column ii, this step stationary phase is silica gel, and mobile phase is second
The mixed liquor of acetoacetic ester, ethyl alcohol；Ethyl acetate and ethyl alcohol volume ratio are 1 in this step:3~2:1, elution flow rate for 0.5~
1.3BV/h collects 1~30 column volume eluent；
C, eluent obtained by concentration B, obtains isolate II；
4), ion-exchange chromatography：Isolate II is made to pass through cation exchange resin first, and is carried out with calcium hydroxide solution
Then elution concentrates eluent obtained by ion exchange, ethyl alcohol is then added in into the eluent after concentration is precipitated, finally mistake
Filter is concentrated to give isolate III；Calcium hydroxide solution pH is 10~12 in this step, and elution flow rate is 0.9~1.6BV/h, is received
Collect 1~30 column volume eluent, ethyl alcohol accounts for the volume of the concentrated liquid a concentration of 70~85%；
5) isolate III, is detached first with chromatographic column iii, this step stationary phase is aluminium oxide, and mobile phase is acetic acid second
Ester, alcohol mixeding liquid, then concentrate eluant obtain isolate IV；Ethyl acetate and ethyl alcohol volume ratio are 1 in this step:3~
2:1, elution flow rate is 0.3~0.8BV/h, collects 1~50 column volume eluent；
6) isolate IV, is added in into ethanol solution crystallizing first, crystallized product is then added in into ethyl acetate, ethyl alcohol mixes
It is recrystallized in solution, is finally separating crystallized product and obtains separation target components；Crystallization temperature is 1~6 DEG C in this step, ethyl alcohol
Solution concentration is more than 90%, and the time is 6~48h, and recrystallization temperature is -18~-23 DEG C, and ethyl acetate is 2 with ethyl alcohol volume ratio:
1~7:1, the time is 6~48h, is repeated 2~5 times according to the sequence of crystallization-recrystallization-crystallization-recrystallization.
DNJ target components (the wherein matter of DNJ obtained by the method for preparing DNJ nano suspensions, preferably method described above
It is >=35% to measure percentage), specifically include following steps：
1) esterification target components, are prepared, including：
A, 0.25~1.0 part of chlorination 1- butyl -3- methylimidazoles, DNJ (DNJ standard items or containing DNJ are taken by the gauge of substance
Separation component) 0.03-0.07 parts, second, third, fourth, penta, 0.3-0.5 parts of oneself or heptanoic anhydride and eight water aluminum sulfate 0.003~
0.012 part and add in reaction kettle hybrid reaction；Temperature is 80~160 DEG C during this step hybrid reaction, and the reaction time is 6~14h,
Mixed liquor rotating speed is 300~500r/min；
B, the reaction product of ethyl acetate washing step a, upper liquid is taken after layering, is adjusted to neutrality with saturated sodium carbonate, is obtained
It is esterified target components；After testing, the conversion ratio of esterification target components is 63.2~87.3% obtained by this step, obtained esterification mesh
The mass concentration for marking component is 0.7~1.25%；
2) solution A is prepared：The solution A contains mannitol or polyethylene glycol 400 by mass：0.2~3%, lactose：0.5
~2%, glucose：0.5~2%, sodium carboxymethylcellulose：0.3~5%, remaining is water；
3) surfactant formulatory solution B is added in into solution A, surfactant qualities percentage composition in the solution B
It is 0.5~5%, the surfactant is polyethylene glycol 1000 vitamin E succinic acid ester, PLURONICS F87, Tween 80, ovum
Any one in phosphatide, lauryl sodium sulfate or glyceryl triacetate；
4) by volume 3:2~2:3 mixing steps 1) gained esterification target components and solution B and carry out ultrasonication；
Power is 140~280W when this step is ultrasonically treated, and the time is 15~45min；
5) removal step 4) organic component in solution, high pressure homogenizer or the processing of ultra micro nano grinder are then moved to,
Obtain nano suspension；Specific can be used respectively recycles 5~l0 times or surpasses under 500bar, 1000bar, 1500bar pressure respectively
Micro-nano pulverizer 2900rpm mills 45~60min, obtains the suspension that average grain diameter is 180~380nm.
Embodiment 1：
The method that this implementation prepares DNJ nano suspensions, includes the following steps：
(1), the extraction medicinal extract containing DNJ is prepared：It can be used one or more in following 3 kinds of methods：
Method 1：It is extracted after mulberry material drying and crushing with 70% ethanol solution ultrasonic wave auxiliary (420W, 30min), wherein
Liquid ratio is 40mL/g, and extraction time is 4 times, filters extracting solution, medicinal extract must be extracted after reduced pressure.5kg mulberry skin dry powder obtains
The purity of 0.50kg medicinal extract, wherein DNJ is 0.104%.
Method 2：With (100 DEG C, 30min) extractions of boiling pure water after mulberry material drying and crushing, wherein liquid ratio is 40mL/g,
Extraction time is 4 times, filters extracting solution, medicinal extract must be extracted after reduced pressure.5kg mulberry skin dry powder obtains 0.33kg medicinal extract, wherein DNJ
Purity be 0.141%.
Method 3：Mulberry material is after drying and crushing is handled with the dilute hydrochloric acid ultrasonic wave of 0.05mol/L auxiliary (420W, 30min)
Extraction, wherein liquid ratio are 40mL/g, and extraction time is 4 times, filters extracting solution, medicinal extract must be extracted after reduced pressure.5kg mulberry skins
Dry powder obtains 0.46kg medicinal extract, and the wherein purity of DNJ is 0.162%.
(2), above-mentioned medicinal extract scale separation Sang Yuan DNJ are utilized：It can be used one or more in following 7 kinds of methods：
Method 1：Medicinal extract handles (640W, 6min) with microwave coking, gained powder water dissolution, centrifugation (2000r/min,
20min), isolate I is obtained after supernatant is concentrated under reduced pressure；Isolate I is 2 by volume ratio:The double-deck color of 1 " fine sand-activated carbon "
Column is composed, 80% ethanol solution elution collects 1~27BV, flow velocity 1.1BV/h, eluent is concentrated under reduced pressure, then passes through silica gel color
Compose column, ethyl acetate:Ethyl alcohol=1:3 mixed solvent elution, collects 2~30BV, flow velocity 0.8BV/h, and eluent depressurizes dense
Contract to obtain isolate II；Isolate II is eluted by cation exchange resin column, the calcium hydroxide solution of PH 12, and collection 1~
28BV, flow velocity 1.1BV/h, eluent are precipitated after being concentrated under reduced pressure with the ethanol solution for accounting for the volume of the concentrated liquid a concentration of 75%, filter
Calcium hydroxide etc. is removed, isolate III is obtained after reduced pressure；Isolate III passes through chromatography on alumina column, ethyl acetate:Ethyl alcohol=
1:3 mixed solvent elution, collects 1~41BV, flow velocity 0.5BV/h, and eluent obtains isolate IV after being concentrated under reduced pressure；At 4 DEG C
Under the conditions of, 98% ethanol solution crystallizing 6h, under the conditions of -20 DEG C, ethyl acetate:Ethyl alcohol=2:1 mixed solution recrystallization
6h is continuously repeated 2 times, and it is 36.4% to obtain purity, and the DNJ that sample recovery rate is 39.3% detaches target components.
Method 2：Medicinal extract handles (800W, 2min) with microwave coking, gained powder water dissolution, centrifugation (5000r/min,
8min), isolate I is obtained after supernatant is concentrated under reduced pressure；Isolate I is 2 by volume ratio:The double-deck chromatography of 1 " fine sand-activated carbon "
Column, 50% ethanol solution elution, collects 2~25BV, flow velocity 0.9BV/h, eluent is concentrated under reduced pressure, then passes through silica gel chromatograph
Column, ethyl acetate:Ethyl alcohol=2:1 mixed solvent elution, collects 2~23BV, flow velocity 0.6BV/h, and eluent is concentrated under reduced pressure
Obtain isolate II；Isolate II is eluted by cation exchange resin column, the calcium hydroxide solution of PH 11, collects 2~27BV,
Flow velocity is 0.9BV/h, and eluent is precipitated after being concentrated under reduced pressure with the ethanol solution for accounting for the volume of the concentrated liquid a concentration of 78%, filters off hydrogen-oxygen
Change calcium etc., isolate III is obtained after reduced pressure；Isolate III passes through chromatography on alumina column, ethyl acetate:Ethyl alcohol=2:1 it is mixed
Bonding solvent elutes, and collects 1~41BV, flow velocity 0.3BV/h, and eluent obtains isolate IV after being concentrated under reduced pressure；Under the conditions of 4 DEG C,
98% ethanol solution crystallizing 48h, under the conditions of -20 DEG C, ethyl acetate:Ethyl alcohol=7:1 mixed solution recrystallization 48h, even
Continuous to repeat 4 times, it is 38.7% to obtain purity, and the DNJ that sample recovery rate is 32.6% detaches target components.
Method 3：Medicinal extract handles (720W, 3min) with microwave coking, gained powder water dissolution, centrifugation (3000r/min,
12min), isolate I is obtained after supernatant is concentrated under reduced pressure；Isolate I is 2 by volume ratio:The double-deck color of 1 " fine sand-activated carbon "
Column is composed, 70% ethanol solution elution collects 2~20BV, flow velocity 1.2BV/h, eluent is concentrated under reduced pressure, then passes through silica gel color
Compose column, ethyl acetate:Ethyl alcohol=1:1 mixed solvent elution, collects 2~16BV, flow velocity 0.8BV/h, and eluent depressurizes dense
Contract to obtain isolate II；Isolate II is eluted by cation exchange resin column, the calcium hydroxide solution of PH 11, and collection 3~
23BV, flow velocity 1.2BV/h, eluent are precipitated after being concentrated under reduced pressure with the ethanol solution for accounting for the volume of the concentrated liquid a concentration of 80%, filter
Calcium hydroxide etc. is removed, isolate III is obtained after reduced pressure；Isolate III passes through chromatography on alumina column, ethyl acetate:Ethyl alcohol=
1:1 mixed solvent elution, collects 2~38BV, flow velocity 0.6BV/h, and eluent obtains isolate IV after being concentrated under reduced pressure；At 4 DEG C
Under the conditions of, 98% ethanol solution crystallizing for 24 hours, under the conditions of -20 DEG C, ethyl acetate:Ethyl alcohol=5:1 mixed solution recrystallization
For 24 hours, it continuously repeats 3 times, it is 39.1% to obtain purity, and the DNJ that sample recovery rate is 37.9% detaches target components.
Method 4：Medicinal extract handles (640W, 5min) with microwave coking, gained powder water dissolution, centrifugation (4000r/min,
10min), isolate I is obtained after supernatant is concentrated under reduced pressure；Isolate I is 2 by volume ratio:The double-deck color of 1 " fine sand-activated carbon "
Column is composed, 90% ethanol solution elution collects 1~30BV, flow velocity 1.6BV/h, eluent is concentrated under reduced pressure, then passes through silica gel color
Compose column, ethyl acetate:Ethyl alcohol=2:3 mixed solvent elution, collects 1~30BV, flow velocity 1.3BV/h, and eluent depressurizes dense
Contract to obtain isolate II；Isolate II is eluted by cation exchange resin column, the calcium hydroxide solution of PH 11, and collection 1~
30BV, flow velocity 1.6BV/h, eluent are precipitated after being concentrated under reduced pressure with the ethanol solution for accounting for the volume of the concentrated liquid a concentration of 70%, filter
Calcium hydroxide etc. is removed, isolate III is obtained after reduced pressure；Isolate III passes through chromatography on alumina column, ethyl acetate:Ethyl alcohol=
2:3 mixed solvent elution, collects 1~50BV, flow velocity 0.8BV/h, and eluent obtains isolate IV after being concentrated under reduced pressure；At 4 DEG C
Under the conditions of, 98% ethanol solution crystallizing 12h, under the conditions of -20 DEG C, ethyl acetate:Ethyl alcohol=2:1 mixed solution recrystallization
12h is continuously repeated 5 times, and it is 35.0% to obtain purity, and the DNJ that sample recovery rate is 38.2% detaches target components.
Method 5：Medicinal extract handles (800W, 5min) with microwave coking, gained powder water dissolution, centrifugation (2000r/min,
18min), isolate I is obtained after supernatant is concentrated under reduced pressure；Isolate I is 2 by volume ratio:The double-deck color of 1 " fine sand-activated carbon "
Column is composed, 60% ethanol solution elution collects 2~26BV, flow velocity 1.0BV/h, eluent is concentrated under reduced pressure, then passes through silica gel color
Compose column, ethyl acetate:Ethyl alcohol=4:3 mixed solvent elution, collects 2~28BV, flow velocity 1.1BV/h, and eluent depressurizes dense
Contract to obtain isolate II；Isolate II is eluted by cation exchange resin column, the calcium hydroxide solution of PH 10, and collection 2~
28BV, flow velocity 1.2BV/h, eluent are precipitated after being concentrated under reduced pressure with the ethanol solution for accounting for the volume of the concentrated liquid a concentration of 75%, filter
Calcium hydroxide etc. is removed, isolate III is obtained after reduced pressure；Isolate III passes through chromatography on alumina column, ethyl acetate:Ethyl alcohol=
5:3 mixed solvent elution, collects 2~43BV, flow velocity 0.6BV/h, and eluent obtains isolate IV after being concentrated under reduced pressure；At 4 DEG C
Under the conditions of, 98% ethanol solution crystallizing 36h, under the conditions of -20 DEG C, ethyl acetate:Ethyl alcohol=4:1 mixed solution recrystallization
36h is continuously repeated 3 times, and it is 37.1% to obtain purity, and the DNJ that sample recovery rate is 35.5% detaches target components.
Method 6：Medicinal extract handles (800W, 4min) with microwave coking, gained powder water dissolution, centrifugation (3000r/min,
16min), isolate I is obtained after supernatant is concentrated under reduced pressure；Isolate I is 2 by volume ratio:The double-deck color of 1 " fine sand-activated carbon "
Column is composed, 60% ethanol solution elution collects 2~26BV, flow velocity 1.4BV/h, eluent is concentrated under reduced pressure, then passes through silica gel color
Compose column, ethyl acetate:Ethyl alcohol=1:1 mixed solvent elution, collects 2~28BV, flow velocity 0.5BV/h, and eluent depressurizes dense
Contract to obtain isolate II；Isolate II is eluted by cation exchange resin column, the calcium hydroxide solution of PH 10, and collection 2~
26BV, flow velocity 1.4BV/h, eluent are precipitated after being concentrated under reduced pressure with the ethanol solution for accounting for the volume of the concentrated liquid a concentration of 74%, filter
Calcium hydroxide etc. is removed, isolate III is obtained after reduced pressure；Isolate III passes through chromatography on alumina column, ethyl acetate:Ethyl alcohol=
4:3 mixed solvent elution, collects 2~44BV, flow velocity 0.5BV/h, and eluent obtains isolate IV after being concentrated under reduced pressure；At 4 DEG C
Under the conditions of, 98% ethanol solution crystallizing 40h, under the conditions of -20 DEG C, ethyl acetate:Ethyl alcohol=6:1 mixed solution recrystallization
40h is continuously repeated 4 times, and it is 38.0% to obtain purity, and the DNJ that sample recovery rate is 34.3% detaches target components.
Method 7：Medicinal extract handles (720W, 4min) with microwave coking, gained powder water dissolution, centrifugation (4000r/min,
12min), isolate I is obtained after supernatant is concentrated under reduced pressure；Isolate I is 2 by volume ratio:The double-deck color of 1 " fine sand-activated carbon "
Column is composed, 80% ethanol solution elution collects 2~30BV, flow velocity 1.3BV/h, eluent is concentrated under reduced pressure, then passes through silica gel color
Compose column, ethyl acetate:Ethyl alcohol=5:3 mixed solvent elution, collects 2~27BV, flow velocity 0.9BV/h, and eluent depressurizes dense
Contract to obtain isolate II；Isolate II is eluted by cation exchange resin column, the calcium hydroxide solution of PH 12, and collection 2~
30BV, flow velocity 1.3BV/h, eluent are precipitated after being concentrated under reduced pressure with the ethanol solution for accounting for the volume of the concentrated liquid a concentration of 85%, filter
Calcium hydroxide etc. is removed, isolate III is obtained after reduced pressure；Isolate III passes through chromatography on alumina column, ethyl acetate:Ethyl alcohol=
1:1 mixed solvent elution, collects 2~40BV, flow velocity 0.4BV/h, and eluent obtains isolate IV after being concentrated under reduced pressure；At 4 DEG C
Under the conditions of, 98% ethanol solution crystallizing 15h, under the conditions of -20 DEG C, ethyl acetate:Ethyl alcohol=3:1 mixed solution recrystallization
15h is continuously repeated 3 times, obtain purity be 37.2%, sample recovery rate be 36.7% DNJ detach target components.
(3), it is esterified modification using above-mentioned separation target components and prepares nano suspension：It can be used in following 6 kinds of methods
It is one or more：
Method 1：By ionic liquid chlorination 1- butyl -3- methylimidazoles (0.25mol), separation target components are (containing about DNJ
0.05mol), heptanoic anhydride (0.40mol) and aluminum sulfate octadecahydrate (2.0g) are sequentially added in reaction kettle, 80 DEG C, rotating speed 300r/
18h is reacted under the conditions of min.Ionic liquid is washed with ethyl acetate, upper liquid is taken after layering, neutrality is adjusted to saturated sodium carbonate,
Target components must be esterified, and (100%) conversion ratio 63.2%, selectivity, are reused after ionic liquid vacuum distillation drying.Stirring
It is lower to prepare containing the water that mass fraction is 0.2% mannitol, 0.5% lactose, 0.5% glucose and 0.3% sodium carboxymethylcellulose
Solution obtains solution A.Lauryl sodium sulfate in mass ratio 0.5% is dissolved in solution A, obtains solution B.Ester containing 1.25%DNJ
Change target components and solution B by volume 1:1 mixing, with power 140W ultrasonication 45min, decompression boils off organic solvent.
It is transferred to the broken machine 2900rpm of Ultramicro-powder nanometer to mill 45min, obtains average grain diameter as 345nm suspensions.It is dry that it is obtained after freeze-drying
Dry product.
Method 2：By ionic liquid chlorination 1- butyl -3- methylimidazoles (0.55mol), separation target components are (containing about DNJ
0.05mol), propionic andydride (0.40mol) aluminum sulfate octadecahydrate (4.4g) is sequentially added in reaction kettle, 160 DEG C, rotating speed 500r/min
Under the conditions of react 6h.Ionic liquid is washed with ethyl acetate, upper liquid is taken after layering, is adjusted to neutrality with saturated sodium carbonate, obtains ester
Changing target components, (100%) conversion ratio 75.3%, selectivity, are reused after ionic liquid vacuum distillation drying.Match under stirring
It makes containing the aqueous solution that mass fraction is 3% polyethylene glycol 400,2% lactose, 2% glucose and 4% sodium carboxymethylcellulose, obtains
Solution A.Tween 80 in mass ratio 1.2% is dissolved in solution A, obtains solution B.Esterification target components and solution containing 1.1%DNJ
B by volume 1:1 mixing, with power 280W ultrasonication 15min, decompression boils off organic solvent.It is transferred to Ultramicro-powder nanometer
Broken machine 2900rpm mills 52min, obtains average grain diameter as 270nm suspensions.Its dry product is obtained after freeze-drying.
Method 3：By ionic liquid chlorination 1- butyl -3- methylimidazoles (1.0mol), separation target components are (containing about DNJ
0.05mol), valeric anhydride (0.40mol) and aluminum sulfate octadecahydrate (8.0g) are sequentially added in reaction kettle, 100 DEG C, rotating speed 300r/
9h is reacted under the conditions of min.Ionic liquid is washed with ethyl acetate, upper liquid is taken after layering, is adjusted to neutrality with saturated sodium carbonate, obtains
Being esterified target components, (100%) conversion ratio 80.1%, selectivity, are reused after ionic liquid vacuum distillation drying.Under stirring
It is the water-soluble of 1.6% mannitol, 0.7% lactose, 0.7% glucose and 2.8% sodium carboxymethylcellulose to prepare containing mass fraction
Liquid obtains solution A.Glyceryl triacetate in mass ratio 2.4% is dissolved in solution A, obtains solution B.Esterification mesh containing 1.0%DNJ
Mark component and solution B by volume 1:1 mixing, with power 140W ultrasonication 45min, decompression boils off organic solvent.Transfer
It into high pressure homogenizer, is respectively recycled under 500bar, 1000bar, 1500bar pressure 5 times, obtains average grain diameter as 380nm suspensions.
Its dry product is obtained after freeze-drying.
Method 4：By ionic liquid chlorination 1- butyl -3- methylimidazoles (0.7mol), separation target components are (containing about DNJ
0.05mol), butyric anhydride (0.40mol) and aluminum sulfate octadecahydrate (5.6g) are sequentially added in reaction kettle, 120 DEG C, rotating speed 400r/
12h is reacted under the conditions of min.Ionic liquid is washed with ethyl acetate, upper liquid is taken after layering, neutrality is adjusted to saturated sodium carbonate,
Target components must be esterified, and (100%) conversion ratio 87.3%, selectivity, are reused after ionic liquid vacuum distillation drying.Stirring
The lower water for preparing mass fraction as 1.2% polyethylene glycol 400,1.5% lactose, 1.5% glucose and 3% sodium carboxymethylcellulose
Solution obtains solution A.Polyethylene glycol 1000 vitamin E succinic acid ester in mass ratio 2% is dissolved in solution A, obtains solution B.Contain
The esterification target components of 0.83%DNJ and solution B by volume 1:1 mixing, with power 210W ultrasonication 30min, decompression
Boil off organic solvent.It is transferred in high pressure homogenizer, is respectively recycled l0 times under 500bar, 1000bar, 1500bar pressure, must be averaged
Grain size is 180nm suspensions.Its dry product is obtained after freeze-drying.
Method 5：By ionic liquid chlorination 1- butyl -3- methylimidazoles (0.85mol), separation target components are (containing about DNJ
0.05mol), acetic anhydride (0.40mol) and aluminum sulfate octadecahydrate (6.8g) are sequentially added in reaction kettle, 160 DEG C, rotating speed 500r/
15h is reacted under the conditions of min.Ionic liquid is washed with ethyl acetate, upper liquid is taken after layering, neutrality is adjusted to saturated sodium carbonate,
Target components must be esterified, and (100%) conversion ratio 81.8%, selectivity, are reused after ionic liquid vacuum distillation drying.Stirring
Lower prepare containing mass fraction is 2% polyethylene glycol 400,1.2% lactose, 1.2% glucose and 1.6% sodium carboxymethylcellulose
Aqueous solution obtains solution A.Lecithin in mass ratio 5% is dissolved in solution A, obtains solution B.Esterification target group containing 0.91%DNJ
Point with solution B by volume 1:1 mixing, with power 210W ultrasonication 30min, decompression boils off organic solvent.It is transferred to super
The broken machine 2900rpm of micro mist nanometer mills 60min, obtains average grain diameter as 300nm suspensions.Its dry product is obtained after freeze-drying.
Method 6：By ionic liquid chlorination 1- butyl -3- methylimidazoles (0.4mol), DNJ standard items (0.05mol), caproic acid
Acid anhydride (0.40mol) and aluminum sulfate octadecahydrate (3.2g) are sequentially added in reaction kettle, 130 DEG C, react under the conditions of rotating speed 400r/min
9h.Ionic liquid is washed with ethyl acetate, upper liquid is taken after layering, neutrality is adjusted to saturated sodium carbonate, obtain esterification target components
(100%) conversion ratio 70.4%, selectivity, are reused after ionic liquid vacuum distillation drying.Stirring is lower to prepare containing quality point
Number is the aqueous solution of 2% mannitol, 1% lactose, 1% glucose and 5% sodium carboxymethylcellulose, obtains solution A.By poloxamer
188 in mass ratio 2% are dissolved in solution A, obtain solution B.Esterification target components containing 0.70%DNJ and solution B by volume 1:1
Mixing, with power 280W ultrasonication 15min, decompression boils off organic solvent.It is transferred in high pressure homogenizer, 500bar,
It is respectively recycled under 1000bar, 1500bar pressure 7 times, obtains average grain diameter as 210nm suspensions.Its dry product is obtained after freeze-drying.
Remarks：In separation DNJ and during preparing DNJ nano suspensions, almost each step will detect the content of DNJ with
It determines the operation sequence of subsequent step and obtains the results such as the rate of output, concentration；The present invention detects the content of DNJ using following methods：
Step 1：DNJ derivatization steps are：
30 μ L DNJ standard solutions or extracting solution are mixed with the potassium borate buffer (pH 8.5) of 30 μ L 0.4mol/L
In 0.5mL centrifuge tubes, the FMOC-Cl of 60 μ L 5mmol/L is added in, water-bath 20min under the conditions of 25 DEG C adds 30 μ L 1mol/L's
Glycine terminates reaction.Stablize newly-generated DNJ-FMOC with 30 μ L 0.1% (v/v) acetic acid solutions, determined with 120 μ L distilled water
Hold.0.22 μm of nylon leaching film is finally crossed, it is to be measured.
Step 2：HPLC is detected：
Utilize the sample to be tested of HPLC detecting steps 1, detection 5 μm of C of reverse phase18(4.6 × 150mm) chromatographic column
(Waters, Atlantis, Ireland), mobile phase are acetonitrile：The volume ratio of 0.1% acetic acid is 55:45, flow velocity 1mL/min,
30 DEG C, Detection wavelength 254nm of column temperature, 10 μ L of sample size, each 3 repetitions of sample.
It should be noted that step (1), (two) form the present invention separation DNJ methods technical solution, step (1),
(2), (three) form the technical solution that the present invention prepares the method for DNJ nano suspensions, but the DNJ sources of step (3) are unlimited
In the DNJ target components obtained by step (1), (two) method or the DNJ obtained by other methods.It additionally needs
Bright, in above-described embodiment, step (1) includes 3 kinds of specific processing procedures, specific processing procedure during step (2) includes 7, step
(3) including 6 kinds of specific processing procedures, the method that DNJ nano suspensions are prepared in embodiment 1 can be appointing for above three step
Meaning combination.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (3)
1. prepare the method for DNJ nano suspensions, which is characterized in that include the following steps：
1）Esterification target components are prepared, including：
A, by the gauge of substance take 0.25 ~ 1.0 part of chlorination 1- butyl -3- methylimidazoles, DNJ0.03-0.07 parts, second, third, fourth,
Pentath, oneself or 0.3-0.5 parts of heptanoic anhydride and 0.003 ~ 0.012 part of aluminum sulfate octadecahydrate and reaction kettle hybrid reaction is added in；B, acetic acid
The reaction product of ethyl ester washing step a, upper liquid is taken after layering, and neutrality is adjusted to saturated sodium carbonate, obtains esterification target components；
2）Prepare solution A：The solution A contains mannitol or polyethylene glycol 400 by mass：0.2 ~ 3%, lactose：0.5 ~ 2%, Portugal
Grape sugar：0.5 ~ 2 %, sodium carboxymethylcellulose：0.3 ~ 5 %, remaining is water；
3）Surfactant formulatory solution B is added in into solution A, surfactant qualities percentage composition is 0.5 in the solution B
~ 5%, the surfactant is polyethylene glycol 1000 vitamin E succinic acid ester, PLURONICS F87, Tween 80, lecithin, ten
Any one in sodium dialkyl sulfate or glyceryl triacetate；
4）By volume 3:2~2:3 mixing steps 1）Gained is esterified target components and solution B and carries out ultrasonication, at ultrasound
Power is 140 ~ 280W, 15 ~ 45min of time during reason；
5）Removal step 4）Then organic component in solution moves to high pressure homogenizer or the processing of ultra micro nano grinder, obtains
Nano suspension.
2. the method for DNJ nano suspensions is prepared according to claim 1, it is characterised in that：Step 1）Temperature during hybrid reaction
It is 80 ~ 160 DEG C to spend, and the reaction time is 6 ~ 14h, and mixed liquor rotating speed is 300 ~ 500r/min, and gained is esterified the conversion of target components
Rate is 63.2 ~ 87.3%, and the mass concentration of obtained esterification target components is 0.7 ~ 1.25%.
3. the method for DNJ nano suspensions is prepared according to claim 1, it is characterised in that：Step 5）High-pressure homogeneous processing
When respectively recycled 5 ~ l0 times under 500bar, 1000bar, 1500bar pressure respectively or ultra micro nano grinder 2900rpm mills
45 ~ 60min obtains the suspension that average grain diameter is 180 ~ 380nm.
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| CN101355971A (en) * | 2005-05-17 | 2009-01-28 | 阿米库斯治疗学公司 | Method for the treatment of pompe disease using 1-deoxynojirimycin and derivatives |
| CN102232937A (en) * | 2010-04-30 | 2011-11-09 | 天津药物研究院 | Nanometer preparation and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101355971A (en) * | 2005-05-17 | 2009-01-28 | 阿米库斯治疗学公司 | Method for the treatment of pompe disease using 1-deoxynojirimycin and derivatives |
| CN102232937A (en) * | 2010-04-30 | 2011-11-09 | 天津药物研究院 | Nanometer preparation and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 桑树中1-脱氧野尻霉素(DNJ)的研究进展;姚瑜 等;《蚕学通讯》;20070630;第27卷(第2期);第35-38页 * |
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