CN105769761A - Method for preparing DNJ nanosuspension - Google Patents

Method for preparing DNJ nanosuspension Download PDF

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CN105769761A
CN105769761A CN201610117593.9A CN201610117593A CN105769761A CN 105769761 A CN105769761 A CN 105769761A CN 201610117593 A CN201610117593 A CN 201610117593A CN 105769761 A CN105769761 A CN 105769761A
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dnj
solution
outlier
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CN105769761B (en
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徐立
刘超
喻艳
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Southwest University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • 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
    • A61K47/38—Cellulose; Derivatives thereof

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  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
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Abstract

The invention discloses a method for separating DNJ and a method for preparing a DNJ nanosuspension.According to the method for separating DNJ, mulberry leaves are used as a raw material and subjected to smashing extraction, microwave scorching and water dissolution, and then an outlier I is obtained; the outlier I is subjected to fine sand-activated carbon chromatographic separation and silica gel chromatographic separation in sequence to form an outlier II; the outlier II is subjected to ion exchange and ethanol precipitation to obtain an outlier III; the outlier III is subjected to aluminum oxide chromatographic column separation to obtain an outlier IV; the outlier IV is crystallized and recrystallized to obtain the target product.According to the method for preparing the DNJ nanosuspension, firstly, DNJ components are subjected to esterification modification so that the lipid solubility of the DNJ components can be enhanced, and then DNJ is dispersed into high-dispersity particle swarms by means of surfactant combination and high-pressure homogenization.The DNG separation technology has the advantages that raw materials are easy to obtain, the process is simple, the effect is obvious, and the technology is suitable for large-scale production.The prepared DNJ nanosuspension can improve the solubility of medicine and bioavailability.

Description

The method preparing DNJ nano suspension
The application is for original applying number: 201410614225.6, and original application day is on October 31st, 2014, the divisional application that the patent of invention of entitled " separating DNJ and the method preparing DNJ nano suspension " proposes.
Technical field
The invention belongs to field of natural medicinal chemistry, relate to purification of pharmaceuticals technology, particularly to from the isolated and purified DNJ of Sang Yuan scale and the method for preparing DNJ component nano suspension.
Background technology
One of diabetes healthy primary killers having become the harm mankind, can cause eye, kidney, heart, blood vessel, neural chronic lesion and dysfunction.According to estimates, to the year two thousand fifty, the number suffering from diabetes in the world is up to 300,000,000, and wherein 90% is diabetes B.OHA currently for diabetes B mainly has sulfonylurea, biguanides, alpha-glucosidase restrainer and thiazolidinediones 4 class.Wherein alpha-glucosidase restrainer blood sugar decreasing effect is notable, and attention is the most extensive.The side reactions such as the acarbose, voglibose and the Miglitol that have listed at present belong to this type of medicine, but this several drugs is all obtained by chemical synthesis process, easy initiation flatulence, abdominal discomfort, Nausea and vomiting, borborygmus and diarrhoea in various degree.Therefore, the important development target that exploitation high-efficiency low-toxicity plant source alpha-glucosidase inhibitor hypoglycemic drug is traditional Chinese medicine research field is explored.
DNJ (Chinese name 1-DNJ) is a kind of piperidine alkaloid, it is widely present in the limb with mulberry tree, leaf and rhizome, its structure similar to glucose (structural formula is shown below), can be combined with the enteral alpha-glucosidase of mammal, and affinity is significantly greater than the disaccharides such as maltose, sucrose, thus blocked disaccharides and be combined with alpha-glucosidase, emulative inhibit disaccharides to be decomposed into glucose, be finally reached the purpose controlling blood-sugar content.
At present; limited by the physicochemical property of DNJ own; existing isolation technics or efficiency simple to operate is low or technical sophistication small scale; limit the practical of natural DNJ and scale is developed; the market input amount causing DNJ is far smaller than actual demand amount, and (related content is referred to author and sums up and discuss (plant source DNJ separating and purifying technology summary to recent domestic about good and bad the made systematization of each method in the isolated and purified research of plant DNJ; silkworm industry science; 2014,40 (3): 0544-0550)).The hypoglycemic performance excellent in view of DNJ and the predicament run at large-scale promotion application thereof, it is necessary to develop a kind of simple method efficiently purifying natural DNJ.
Medicine is transported by blood or diffusion of body fluids, first has to have certain water solubility, but medicine to arrive each histocyte by lipid biomembrane, also requires that it has suitable fat-soluble.DNJ belongs to the material of the fat-soluble difference of good water solubility, is unsuitable for physiologic infusion medication because being difficult to arrive small intestine by biomembrane.If oral, the alpha-glucosidase of small intestine physiology epimere is easily suppressed, in, the degree of susceptibility of hypomere carbohydrate digestion less, need to be strengthened by certain method the adhesiveness of itself and internal mucous membrane tissue, thus extend the DNJ holdup time in vivo, improve its bioavilability.
Summary of the invention
In view of this, present invention firstly provides a kind of simple efficient method of separating-purifying DNJ from natural plants.On this basis, the present invention also provides for a kind of method preparing DNJ nano suspension.
A kind of method separating DNJ, comprises the following steps:
1), drying and crushing mulberry material obtain extract with watery hydrochloric acid, ethanol solution or extracting in boiling water；
2), first by step 1) extract concentrate after microwave coking, obtain powdered extract, then dissolved also centrifugation supernatant liquor, last concentrated supernatant, obtain separator I；
3), first chromatographic isolation, including:
A, utilizing chromatographic column i to separate separator I, this step is fixing be fine sand and Mixture of Activated Carbon mutually, and flowing is ethanol solution mutually；
B, concentration A gained eluent also separate further by chromatographic column ii, and this step is fixing is silica gel mutually, and flowing is the mixed liquor of ethyl acetate, ethanol mutually；
C, concentration B gained eluent, obtain separator II；
4), ion-exchange chromatography: first make separator II pass through cationic ion-exchange resin, and elute with aqua calcis, then concentrating ion clearing house and obtain eluent, then add ethanol in the eluent after concentrating and precipitate, last filtering and concentrating obtains separator III；
5), separating separator III first with chromatographic column iii, this step is fixing is aluminum oxide mutually, and flowing is ethyl acetate, alcohol mixeding liquid mutually, and then concentrate eluant obtains separator IV；
6), first by separator IV add ethanol solution crystallization, then crystallized product is added in ethyl acetate, alcohol mixed solution and recrystallize, be finally separating crystallized product and i.e. obtain separating target components.
Preferably, step 1) use watery hydrochloric acid extract time concentration of hydrochloric acid be 0.03～0.07mol/L；When using ethanol solution to extract, concentration of alcohol is 60～80%, and liquid ratio is 20～45mL/g, and extraction time is 3～5 times, utilizes 350～490W Sonication assisted treatments 20～40min during extraction；When using extracting in boiling water, boiled water temperature is 95～100 DEG C, and liquid ratio is 20～45mL/g, extraction time 20～40min.
Preferably, step 2) microwave coking time microwave power be 640～800W, the process time is 2～6min.
Preferably, step 3) fine sand is 2:1 with activated carbon volume ratio in-A, ethanol solution concentration is 50～90%, and elution flow rate is 0.9～1.6BV/h, collects 1～30 column volume eluents；Step 3) ethyl acetate and ethanol volume ratio are 1:3～2:1 in-B, elution flow rate is 0.5～1.3BV/h, collects 1～30 column volume eluents.
Preferably, step 4) aqua calcis pH is 10～12, elution flow rate is 0.9～1.6BV/h, collects 1～30 column volume eluents, and it is 70～85% that ethanol accounts for concentrated liquid volume concentrations.
Preferably, step 5) ethyl acetate is 1:3～2:1 with ethanol volume ratio, elution flow rate is 0.3～0.8BV/h, collects 1～50 column volume eluents.
Preferably, step 6) crystallization temperature is 1～6 DEG C, ethanol solution concentration is more than 90%, time is 6～48h, and recrystallization temperature is-18～-23 DEG C, and ethyl acetate and ethanol volume ratio are 2:1～7:1, time is 6～48h, repeats 2～5 times according to the order of crystallization-recrystallization-crystallization-recrystallization.
The present invention prepares the method for DNJ nano suspension, comprises the following steps:
1), esterification target components is prepared, including:
A, take chlorination 1-butyl-3-methylimidazole 0.25～1.0 parts, DNJ0.03-0.07 part, second, third, fourth, penta, oneself or heptanoic anhydride 0.3-0.5 part and eight water aluminum sulfate 0.003～0.012 part by the gauge of material and add reactor hybrid reaction；B, the product of ethyl acetate washing step a, take upper liquid, be adjusted to neutrality with saturated sodium carbonate, obtain esterification target components after layering；
2) preparation solution A: described solution A is by mass containing mannitol or PEG400: 0.2～3%, lactose: 0.5～2%, glucose: 0.5～2%, sodium carboxymethylcellulose: 0.3～5%, remaining is water；
3) in solution A, surfactant formulatory solution B is added, in described solution B, surfactant qualities percentage composition is 0.5～5%, and described surfactant is any one in polyethylene glycol 1000 vitamin E succinic acid ester, PLURONICS F87, Tween 80, lecithin, lauryl sodium sulfate or glyceryl triacetate；
4) 3:2～2:3 blend step 1 by volume) gained esterification target components and solution B carry out ultrasonic wave process, time ultrasonically treated, power is 140～280W, the time 15～45min；
5) removal step 4) organic component in solution, then move to high pressure homogenizer or ultra micro nano grinder processes, obtain nano suspension.
Preferably, step 1) hybrid reaction time temperature be 80～160 DEG C, 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%, and the mass concentration of obtained esterification target components is 0.7～1.25%.
Preferably, step 5) high-pressure homogeneous each circulation 5～l0 time under 500bar, 1000bar, 1500bar pressure respectively when processing, or ultra micro nano grinder 2900rpm mills 45～60min, obtains the suspension that average grain diameter is 180～380nm
The beneficial effects of the present invention is:
The present invention separates the method for DNJ with common mulberry as raw material, proposes a set of simple, efficient, practical DNJ separating and purifying technology route on the basis of chromatographic separation technology principle.Separation method of the present invention utilizes first microwave coking means process extract, make the big molecule of part biological be become insoluble solid to be easier to separate by coking on the premise of not damaging DNJ stability；The present invention and coordinates with ethanol for flowing phase with fine sand and activated carbon for fixing phase, can effectively remove a large amount of impurity in separator I, and this is fixed and can reuse mutually, contributes to cost savings；Replace that traditional handicraft has the ammoniacal liquor of strong and stimulating smell to elute cationic ion-exchange resin with aqua calcis during ion of the present invention exchange, subsequent handling is not easily removed by ethanol precipitation only with respect to alkaline matters such as NaOH, and it is obvious to elute effect feasibility；The present invention is further using aluminum oxide as fixing phase, and ethyl acetate, alcohol mixeding liquid, as flowing phase, can improve separative efficiency and the reversible adsorption loss of DNJ is greatly reduced, improving purification efficiency；Each solvent reusable edible in purification process of the present invention, each chromatographic column filler is cheap and technical process is simple to operate, repeated preferably, it is adaptable to large-scale industrial production.
The present invention prepares the method for DNJ nano suspension and first DNJ component carries out esterification modification, it is allowed to become fat-soluble and is prone to by force and in vivo the active ingredient that enzymolysis is DNJ, the present invention is combined by surfactant further and DNJ active ingredient is dispersed into the particle diameter high degree of dispersion population less than 1000nm by high-pressure homogeneous process, form stable nanometer colloidal dispersion, solubility and the dissolution rate of insoluble drug can be improved；Thus solve the fat-soluble difference of DNJ, the problem that bioavailability is low.
Detailed description of the invention
Below the preferred embodiments of the present invention are described in detail.
Following embodiment separates DNJ and the method preparing DNJ nano suspension by open.
The method separating DNJ, comprises the following steps:
1), drying and crushing mulberry material obtain extract with watery hydrochloric acid, ethanol solution or extracting in boiling water；
Wherein: when using watery hydrochloric acid to extract, concentration of hydrochloric acid is 0.03～0.07mol/L；When using ethanol solution to extract, concentration of alcohol is 60～80%, and liquid ratio is 20～45mL/g, and extraction time is 3～5 times, utilizes 350～490W Sonication assisted treatments 20～40min during extraction；When using extracting in boiling water, boiled water temperature is 95～100 DEG C, and liquid ratio is 20～45mL/g, extraction time 20～40min；
2), first by step 1) extract concentrate after microwave coking, obtain powdered extract, then dissolved also centrifugation supernatant liquor, last concentrated supernatant, obtain separator I；
Wherein step 2) microwave coking time microwave power be 640～800W, the process time is 2～6min；
3), first chromatographic isolation, including:
A, utilizing chromatographic column i to separate separator I, this step is fixing be fine sand and Mixture of Activated Carbon mutually, and flowing is ethanol solution mutually；In this step, fine sand and activated carbon volume ratio are 2:1, and ethanol solution concentration is 50～90%, and elution flow rate is 0.9～1.6BV/h, collect 1～30 column volume eluents；
B, concentration A gained eluent also separate further by chromatographic column ii, and this step is fixing is silica gel mutually, and flowing is the mixed liquor of ethyl acetate, ethanol mutually；In this step, ethyl acetate and ethanol volume ratio are 1:3～2:1, and elution flow rate is 0.5～1.3BV/h, collect 1～30 column volume eluents；
C, concentration B gained eluent, obtain separator II；
4), ion-exchange chromatography: first make separator II pass through cationic ion-exchange resin, and elute with aqua calcis, then concentrating ion clearing house and obtain eluent, then add ethanol in the eluent after concentrating and precipitate, last filtering and concentrating obtains separator III；In this step, aqua calcis pH is 10～12, and elution flow rate is 0.9～1.6BV/h, collects 1～30 column volume eluents, and it is 70～85% that ethanol accounts for concentrated liquid volume concentrations；
5), separating separator III first with chromatographic column iii, this step is fixing is aluminum oxide mutually, and flowing is ethyl acetate, alcohol mixeding liquid mutually, and then concentrate eluant obtains separator IV；In this step, ethyl acetate and ethanol volume ratio are 1:3～2:1, and elution flow rate is 0.3～0.8BV/h, collect 1～50 column volume eluents；
6), first by separator IV add ethanol solution crystallization, then crystallized product is added in ethyl acetate, alcohol mixed solution and recrystallize, be finally separating crystallized product and i.e. obtain separating target components；In this step, crystallization temperature is 1～6 DEG C, and ethanol solution concentration is more than 90%, and the time is 6～48h, recrystallization temperature is-18～-23 DEG C, ethyl acetate and ethanol volume ratio are 2:1～7:1, and the time is 6～48h, repeat 2～5 times according to the order of crystallization-recrystallization-crystallization-recrystallization.
DNJ target components (wherein the mass percent of DNJ is >=35%) obtained by the method preparing DNJ nano suspension, preferably method described above, specifically includes following steps:
1), esterification target components is prepared, including:
A, take chlorination 1-butyl-3-methylimidazole 0.25～1.0 parts, DNJ (DNJ standard items or the separation component containing DNJ) 0.03-0.07 part, second, third, fourth, penta, oneself or heptanoic anhydride 0.3-0.5 part and eight water aluminum sulfate 0.003～0.012 part by the gauge of material and add reactor hybrid reaction；During this step hybrid reaction, temperature is 80～160 DEG C, and the reaction time is 6～14h, and mixed liquor rotating speed is 300～500r/min；
B, the product of ethyl acetate washing step a, take upper liquid, be adjusted to neutrality with saturated sodium carbonate, obtain esterification target components after layering；After testing, the conversion ratio of this step gained esterification target components is 63.2～87.3%, and the mass concentration of obtained esterification target components is 0.7～1.25%；
2) preparation solution A: described solution A is by mass containing mannitol or PEG400: 0.2～3%, lactose: 0.5～2%, glucose: 0.5～2%, sodium carboxymethylcellulose: 0.3～5%, remaining is water；
3) in solution A, surfactant formulatory solution B is added, in described solution B, surfactant qualities percentage composition is 0.5～5%, and described surfactant is any one in polyethylene glycol 1000 vitamin E succinic acid ester, PLURONICS F87, Tween 80, lecithin, lauryl sodium sulfate or glyceryl triacetate；
4) 3:2～2:3 blend step 1 by volume) gained esterification target components and solution B carry out ultrasonic wave process；When this step is ultrasonically treated, power is 140～280W, and the time is 15～45min；
5) removal step 4) organic component in solution, then move to high pressure homogenizer or ultra micro nano grinder processes, obtain nano suspension；Specifically can use each circulation 5～l0 time 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.
Embodiment 1:
The method of DNJ nano suspension is prepared in this enforcement, comprises the following steps:
(1), preparation is containing the extraction medicinal extract of DNJ: can use one or more in following 3 kinds of methods:
Method 1: extracting with the ethanol solution ultrasonic assistant (420W, 30min) of 70% after mulberry material drying and crushing, wherein liquid ratio is 40mL/g, and extraction time is 4 times, filters extract, must extract medicinal extract after reduced pressure concentration.5kg mulberry skin dry powder obtains 0.50kg medicinal extract, and wherein the purity of DNJ is 0.104%.
Method 2: extracting by boiling pure water (100 DEG C, 30min) after mulberry material drying and crushing, wherein liquid ratio is 40mL/g, and extraction time is 4 times, filters extract, must extract medicinal extract after reduced pressure concentration.5kg mulberry skin dry powder obtains 0.33kg medicinal extract, and wherein the purity of DNJ is 0.141%.
Method 3: extracting with the watery hydrochloric acid ultrasonic assistant (420W, 30min) of 0.05mol/L after mulberry material drying pulverization process, wherein liquid ratio is 40mL/g, and extraction time is 4 times, filters extract, must extract medicinal extract after reduced pressure concentration.5kg mulberry skin dry powder obtains 0.46kg medicinal extract, and wherein the purity of DNJ is 0.162%.
(2) above-mentioned medicinal extract scale, is utilized to separate Sang Yuan DNJ: can use one or more in following 7 kinds of methods:
Method 1: medicinal extract microwave coking processes (640W, 6min), and gained powder use water dissolves, centrifugal (2000r/min, 20min), obtains separator I after supernatant reduced pressure concentration；Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the ethanol solution wash-out of 80%, collect 1～27BV, flow velocity is 1.1BV/h, eluent reduced pressure concentration, then passes through silica gel chromatographic column, ethyl acetate: the mixed solvent wash-out of ethanol=1:3, collecting 2～30BV, flow velocity is 0.8BV/h, and eluent reduced pressure concentration obtains separator II；Separator II passes through cation exchange resin column, the aqua calcis wash-out of PH 12, collects 1～28BV, flow velocity is 1.1BV/h, with accounting for the ethanol solution precipitation that concentrated liquid volume concentrations is 75% after eluent reduced pressure concentration, filter off calcium hydroxide etc., after reduced pressure concentration, obtain separator III；Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent wash-out of ethanol=1:3, collects 1～41BV, and flow velocity is 0.5BV/h, obtains separator IV after eluent reduced pressure concentration；Under the conditions of 4 DEG C, the ethanol solution of 98% crystallization 6h, under the conditions of-20 DEG C, ethyl acetate: the mixed solution recrystallization 6h of ethanol=2:1, continuously repeat 2 times, obtaining purity is 36.4%, sample recovery rate be 39.3% DNJ separate target components.
Method 2: medicinal extract microwave coking processes (800W, 2min), and gained powder use water dissolves, centrifugal (5000r/min, 8min), obtains separator I after supernatant reduced pressure concentration；Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the ethanol solution wash-out of 50%, collect 2～25BV, flow velocity is 0.9BV/h, eluent reduced pressure concentration, then passes through silica gel chromatographic column, ethyl acetate: the mixed solvent wash-out of ethanol=2:1, collecting 2～23BV, flow velocity is 0.6BV/h, and eluent reduced pressure concentration obtains separator II；Separator II passes through cation exchange resin column, the aqua calcis wash-out of PH 11, collects 2～27BV, flow velocity is 0.9BV/h, with accounting for the ethanol solution precipitation that concentrated liquid volume concentrations is 78% after eluent reduced pressure concentration, filter off calcium hydroxide etc., after reduced pressure concentration, obtain separator III；Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent wash-out of ethanol=2:1, collects 1～41BV, and flow velocity is 0.3BV/h, obtains separator IV after eluent reduced pressure concentration；Under the conditions of 4 DEG C, the ethanol solution of 98% crystallization 48h, under the conditions of-20 DEG C, ethyl acetate: the mixed solution recrystallization 48h of ethanol=7:1, continuously repeat 4 times, obtaining purity is 38.7%, sample recovery rate be 32.6% DNJ separate target components.
Method 3: medicinal extract microwave coking processes (720W, 3min), and gained powder use water dissolves, centrifugal (3000r/min, 12min), obtains separator I after supernatant reduced pressure concentration；Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the ethanol solution wash-out of 70%, collect 2～20BV, flow velocity is 1.2BV/h, eluent reduced pressure concentration, then passes through silica gel chromatographic column, ethyl acetate: the mixed solvent wash-out of ethanol=1:1, collecting 2～16BV, flow velocity is 0.8BV/h, and eluent reduced pressure concentration obtains separator II；Separator II passes through cation exchange resin column, the aqua calcis wash-out of PH 11, collects 3～23BV, flow velocity is 1.2BV/h, with accounting for the ethanol solution precipitation that concentrated liquid volume concentrations is 80% after eluent reduced pressure concentration, filter off calcium hydroxide etc., after reduced pressure concentration, obtain separator III；Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent wash-out of ethanol=1:1, collects 2～38BV, and flow velocity is 0.6BV/h, obtains separator IV after eluent reduced pressure concentration；Under the conditions of 4 DEG C, the ethanol solution of 98% crystallization 24h, under the conditions of-20 DEG C, ethyl acetate: the mixed solution recrystallization 24h of ethanol=5:1, continuously repeat 3 times, obtaining purity is 39.1%, sample recovery rate be 37.9% DNJ separate target components.
Method 4: medicinal extract microwave coking processes (640W, 5min), and gained powder use water dissolves, centrifugal (4000r/min, 10min), obtains separator I after supernatant reduced pressure concentration；Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the ethanol solution wash-out of 90%, collect 1～30BV, flow velocity is 1.6BV/h, eluent reduced pressure concentration, then passes through silica gel chromatographic column, ethyl acetate: the mixed solvent wash-out of ethanol=2:3, collecting 1～30BV, flow velocity is 1.3BV/h, and eluent reduced pressure concentration obtains separator II；Separator II passes through cation exchange resin column, the aqua calcis wash-out of PH 11, collects 1～30BV, flow velocity is 1.6BV/h, with accounting for the ethanol solution precipitation that concentrated liquid volume concentrations is 70% after eluent reduced pressure concentration, filter off calcium hydroxide etc., after reduced pressure concentration, obtain separator III；Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent wash-out of ethanol=2:3, collects 1～50BV, and flow velocity is 0.8BV/h, obtains separator IV after eluent reduced pressure concentration；Under the conditions of 4 DEG C, the ethanol solution of 98% crystallization 12h, under the conditions of-20 DEG C, ethyl acetate: the mixed solution recrystallization 12h of ethanol=2:1, continuously repeat 5 times, obtaining purity is 35.0%, sample recovery rate be 38.2% DNJ separate target components.
Method 5: medicinal extract microwave coking processes (800W, 5min), and gained powder use water dissolves, centrifugal (2000r/min, 18min), obtains separator I after supernatant reduced pressure concentration；Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the ethanol solution wash-out of 60%, collect 2～26BV, flow velocity is 1.0BV/h, eluent reduced pressure concentration, then passes through silica gel chromatographic column, ethyl acetate: the mixed solvent wash-out of ethanol=4:3, collecting 2～28BV, flow velocity is 1.1BV/h, and eluent reduced pressure concentration obtains separator II；Separator II passes through cation exchange resin column, the aqua calcis wash-out of PH 10, collects 2～28BV, flow velocity is 1.2BV/h, with accounting for the ethanol solution precipitation that concentrated liquid volume concentrations is 75% after eluent reduced pressure concentration, filter off calcium hydroxide etc., after reduced pressure concentration, obtain separator III；Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent wash-out of ethanol=5:3, collects 2～43BV, and flow velocity is 0.6BV/h, obtains separator IV after eluent reduced pressure concentration；Under the conditions of 4 DEG C, the ethanol solution of 98% crystallization 36h, under the conditions of-20 DEG C, ethyl acetate: the mixed solution recrystallization 36h of ethanol=4:1, continuously repeat 3 times, obtaining purity is 37.1%, sample recovery rate be 35.5% DNJ separate target components.
Method 6: medicinal extract microwave coking processes (800W, 4min), and gained powder use water dissolves, centrifugal (3000r/min, 16min), obtains separator I after supernatant reduced pressure concentration；Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the ethanol solution wash-out of 60%, collect 2～26BV, flow velocity is 1.4BV/h, eluent reduced pressure concentration, then passes through silica gel chromatographic column, ethyl acetate: the mixed solvent wash-out of ethanol=1:1, collecting 2～28BV, flow velocity is 0.5BV/h, and eluent reduced pressure concentration obtains separator II；Separator II passes through cation exchange resin column, the aqua calcis wash-out of PH 10, collects 2～26BV, flow velocity is 1.4BV/h, with accounting for the ethanol solution precipitation that concentrated liquid volume concentrations is 74% after eluent reduced pressure concentration, filter off calcium hydroxide etc., after reduced pressure concentration, obtain separator III；Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent wash-out of ethanol=4:3, collects 2～44BV, and flow velocity is 0.5BV/h, obtains separator IV after eluent reduced pressure concentration；Under the conditions of 4 DEG C, the ethanol solution of 98% crystallization 40h, under the conditions of-20 DEG C, ethyl acetate: the mixed solution recrystallization 40h of ethanol=6:1, continuously repeat 4 times, obtaining purity is 38.0%, sample recovery rate be 34.3% DNJ separate target components.
Method 7: medicinal extract microwave coking processes (720W, 4min), and gained powder use water dissolves, centrifugal (4000r/min, 12min), obtains separator I after supernatant reduced pressure concentration；Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the ethanol solution wash-out of 80%, collect 2～30BV, flow velocity is 1.3BV/h, eluent reduced pressure concentration, then passes through silica gel chromatographic column, ethyl acetate: the mixed solvent wash-out of ethanol=5:3, collecting 2～27BV, flow velocity is 0.9BV/h, and eluent reduced pressure concentration obtains separator II；Separator II passes through cation exchange resin column, the aqua calcis wash-out of PH 12, collects 2～30BV, flow velocity is 1.3BV/h, with accounting for the ethanol solution precipitation that concentrated liquid volume concentrations is 85% after eluent reduced pressure concentration, filter off calcium hydroxide etc., after reduced pressure concentration, obtain separator III；Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent wash-out of ethanol=1:1, collects 2～40BV, and flow velocity is 0.4BV/h, obtains separator IV after eluent reduced pressure concentration；Under the conditions of 4 DEG C, the ethanol solution of 98% crystallization 15h, under the conditions of-20 DEG C, ethyl acetate: the mixed solution recrystallization 15h of ethanol=3:1, continuously repeat 3 times, must obtain purity is 37.2%, sample recovery rate be 36.7% DNJ separate target components.
(3) esterification of above-mentioned separation target components, is utilized to modify and prepare nano suspension: can use one or more in following 6 kinds of methods:
Method 1: by ionic liquid chlorination 1-butyl-3-methylimidazole (0.25mol), separate target components (containing about DNJ 0.05mol), heptanoic anhydride (0.40mol) and aluminum sulfate octadecahydrate (2.0g) are sequentially added in reactor, 80 DEG C, react 18h under the conditions of rotating speed 300r/min.Wash ionic liquid with ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 63.2%, selective 100%) must be esterified, reuse after ionic liquid vacuum distillation drying.The lower preparation of stirring is 0.2% mannitol, 0.5% lactose, 0.5% glucose and the aqueous solution of 0.3% sodium carboxymethylcellulose containing mass fraction, obtains solution A.Lauryl sodium sulfate in mass ratio 0.5% is dissolved in solution A, obtains solution B.Esterification target components containing 1.25%DNJ mixes with solution B 1:1 by volume, processes 45min with power 140W ultrasonic wave, and decompression boils off organic solvent.Transferring to Ultramicro-powder nanometer broken machine 2900rpm mill 45min, obtaining average grain diameter is 345nm suspension.Its dry product is obtained after freeze-drying.
Method 2: by ionic liquid chlorination 1-butyl-3-methylimidazole (0.55mol), separate target components (containing about DNJ 0.05mol), propionic andydride (0.40mol) aluminum sulfate octadecahydrate (4.4g) is sequentially added in reactor, 160 DEG C, react 6h under the conditions of rotating speed 500r/min.Wash ionic liquid with ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 75.3%, selective 100%) must be esterified, reuse after ionic liquid vacuum distillation drying.The lower preparation of stirring is 3% PEG400,2% lactose, 2% glucose and the aqueous solution of 4% sodium carboxymethylcellulose containing mass fraction, obtains solution A.Tween 80 in mass ratio 1.2% is dissolved in solution A, obtains solution B.Esterification target components containing 1.1%DNJ mixes with solution B 1:1 by volume, processes 15min with power 280W ultrasonic wave, and decompression boils off organic solvent.Transferring to Ultramicro-powder nanometer broken machine 2900rpm mill 52min, obtaining average grain diameter is 270nm suspension.Its dry product is obtained after freeze-drying.
Method 3: by ionic liquid chlorination 1-butyl-3-methylimidazole (1.0mol), separate target components (containing about DNJ 0.05mol), valeric anhydride (0.40mol) and aluminum sulfate octadecahydrate (8.0g) are sequentially added in reactor, 100 DEG C, react 9h under the conditions of rotating speed 300r/min.Wash ionic liquid with ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 80.1%, selective 100%) must be esterified, reuse after ionic liquid vacuum distillation drying.The lower preparation of stirring is 1.6% mannitol, 0.7% lactose, 0.7% glucose and the aqueous solution of 2.8% sodium carboxymethylcellulose containing mass fraction, obtains solution A.Glyceryl triacetate in mass ratio 2.4% is dissolved in solution A, obtains solution B.Esterification target components containing 1.0%DNJ mixes with solution B 1:1 by volume, processes 45min with power 140W ultrasonic wave, and decompression boils off organic solvent.Transferring in high pressure homogenizer, each circulation 5 times under 500bar, 1000bar, 1500bar pressure, obtaining average grain diameter is 380nm suspension.Its dry product is obtained after freeze-drying.
Method 4: by ionic liquid chlorination 1-butyl-3-methylimidazole (0.7mol), separate target components (containing about DNJ 0.05mol), butyric anhydride (0.40mol) and aluminum sulfate octadecahydrate (5.6g) are sequentially added in reactor, 120 DEG C, react 12h under the conditions of rotating speed 400r/min.Wash ionic liquid with ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 87.3%, selective 100%) must be esterified, reuse after ionic liquid vacuum distillation drying.The lower preparation mass fraction of stirring is 1.2% PEG400,1.5% lactose, 1.5% glucose and the aqueous solution of 3% sodium carboxymethylcellulose, obtains solution A.Polyethylene glycol 1000 vitamin E succinic acid ester in mass ratio 2% is dissolved in solution A, obtains solution B.Esterification target components containing 0.83%DNJ mixes with solution B 1:1 by volume, processes 30min with power 210W ultrasonic wave, and decompression boils off organic solvent.Transferring in high pressure homogenizer, each circulation l0 time under 500bar, 1000bar, 1500bar pressure, obtaining average grain diameter is 180nm suspension.Its dry product is obtained after freeze-drying.
Method 5: by ionic liquid chlorination 1-butyl-3-methylimidazole (0.85mol), separate target components (containing about DNJ 0.05mol), acetic anhydride (0.40mol) and aluminum sulfate octadecahydrate (6.8g) are sequentially added in reactor, 160 DEG C, react 15h under the conditions of rotating speed 500r/min.Wash ionic liquid with ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 81.8%, selective 100%) must be esterified, reuse after ionic liquid vacuum distillation drying.The lower preparation of stirring is 2% PEG400,1.2% lactose, 1.2% glucose and the aqueous solution of 1.6% sodium carboxymethylcellulose containing mass fraction, obtains solution A.Lecithin in mass ratio 5% is dissolved in solution A, obtains solution B.Esterification target components containing 0.91%DNJ mixes with solution B 1:1 by volume, processes 30min with power 210W ultrasonic wave, and decompression boils off organic solvent.Transferring to Ultramicro-powder nanometer broken machine 2900rpm mill 60min, obtaining average grain diameter is 300nm suspension.Its dry product is obtained after freeze-drying.
Method 6: by ionic liquid chlorination 1-butyl-3-methylimidazole (0.4mol), DNJ standard items (0.05mol), caproic anhydride (0.40mol) and aluminum sulfate octadecahydrate (3.2g) are sequentially added in reactor, 130 DEG C, react 9h under the conditions of rotating speed 400r/min.Wash ionic liquid with ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 70.4%, selective 100%) must be esterified, reuse after ionic liquid vacuum distillation drying.The lower preparation of stirring is 2% mannitol, 1% lactose, 1% glucose and the aqueous solution of 5% sodium carboxymethylcellulose containing mass fraction, obtains solution A.PLURONICS F87 in mass ratio 2% is dissolved in solution A, obtains solution B.Esterification target components containing 0.70%DNJ mixes with solution B 1:1 by volume, processes 15min with power 280W ultrasonic wave, and decompression boils off organic solvent.Transferring in high pressure homogenizer, each circulation 7 times under 500bar, 1000bar, 1500bar pressure, obtaining average grain diameter is 210nm suspension.Its dry product is obtained after freeze-drying.
Remarks: during separation DNJ and preparation DNJ nano suspension, almost each step will detect the content of DNJ to determine the operation sequence of subsequent step and to obtain the result such as the rate of output, concentration；The present invention use following method detection DNJ content:
Step 1:DNJ derivatization step is:
The potassium borate buffer (pH 8.5) of 30 μ L DNJ standard solutions or extract and 30 μ L 0.4mol/L is mixed in 0.5mL centrifuge tube, add the FMOC-Cl of 60 μ L 5mmol/L, water-bath 20min under the conditions of 25 DEG C, the glycine adding 30 μ L 1mol/L terminates reaction.Newly-generated DNJ-FMOC is stablized, with 120 μ L distilled water constant volumes with 30 μ L 0.1% (v/v) acetic acid solutions.Finally cross 0.22 μm nylon leaching film, to be measured.
Step 2:HPLC detects:
Utilize the testing sample of HPLC detecting step 1, detect by anti-phase 5 μm C18(4.6 × 150mm) chromatographic column (Waters, Atlantis, Ireland), flowing is mutually for acetonitrile: the volume ratio of 0.1% acetic acid is 55:45, flow velocity 1mL/min, column temperature 30 DEG C, detection wavelength 254nm, sample size 10 μ L, the repetition of 3, each sample.
It should be noted that, step (one), (two) i.e. constitute the present invention and separate the technical scheme of DNJ method, step (one), (two), (three) constitute the technical scheme that the present invention prepares the method for DNJ nano suspension, but the DNJ of step (3) source is not limited to the DNJ target components obtained by step (), (two) method, it is also possible to be the DNJ obtained by additive method.It is further to note that, in above-described embodiment, step (one) includes 3 kinds of concrete processing procedures, step (two) includes concrete processing procedure in 7, step (three) includes 6 kinds of concrete processing procedures, and the method preparing DNJ nano suspension in embodiment 1 can be any combination of above three step.
Finally illustrate is, preferred embodiment above is only in order to illustrate technical scheme and unrestricted, although the present invention being described in detail by above preferred embodiment, but skilled artisan would appreciate that, in the form and details it can be made various change, without departing from claims of the present invention limited range.

Claims (3)

1. the method preparing DNJ nano suspension, it is characterised in that comprise the following steps:
1), esterification target components is prepared, including:
A, take by the gauge of material chlorination 1-butyl-3-methylimidazole 0.25～1.0 parts, DNJ0.03-0.07 part, second, third, fourth, Penta, oneself or heptanoic anhydride 0.3-0.5 part and eight water aluminum sulfate 0.003～0.012 part add reactor hybrid reaction；B, acetic acid The product of ethyl ester washing step a, takes upper liquid, is adjusted to neutrality with saturated sodium carbonate after layering, must be esterified target group Point；
2) preparation solution A: described solution A is by mass containing mannitol or PEG400: 0.2～3%, lactose: 0.5～2%, Glucose: 0.5～2%, sodium carboxymethylcellulose: 0.3～5%, remaining is water；
3) adding surfactant formulatory solution B in solution A, in described solution B, surfactant qualities percentage composition is 0.5～5%, described surfactant be polyethylene glycol 1000 vitamin E succinic acid ester, PLURONICS F87, Tween 80, Any one in lecithin, lauryl sodium sulfate or glyceryl triacetate；
4) 3:2～2:3 blend step 1 by volume) gained esterification target components and solution B carry out ultrasonic wave process, ultrasonic During process, power is 140～280W, the time 15～45min；
5) removal step 4) organic component in solution, then move to high pressure homogenizer or ultra micro nano grinder processes, obtain Nano suspension.
The method preparing DNJ nano suspension the most according to claim 1, it is characterised in that: step 1) hybrid reaction time temperature Degree is 80～160 DEG C, and the reaction time is 6～14h, and mixed liquor rotating speed is 300～500r/min, gained esterification target components Conversion ratio is 63.2～87.3%, and the mass concentration of obtained esterification target components is 0.7～1.25%.
The method preparing DNJ nano suspension the most according to claim 1, it is characterised in that: step 5) high-pressure homogeneous process Time each circulation 5～l0 time under 500bar, 1000bar, 1500bar pressure respectively, or ultra micro nano grinder 2900rpm stone roller Mill 45～60min, obtains the suspension that average grain diameter is 180～380nm.
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