CN104402801B - Separate DNJ and the method preparing DNJ nano suspension - Google Patents
Separate DNJ and the method preparing DNJ nano suspension Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
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- 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
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Abstract
The invention discloses the method separating DNJ and preparing DNJ nano suspension, separate the method for DNJ with mulberry for raw material, extract through pulverizing, microwave coking, obtaining separator I after being dissolved in water, this separator passes sequentially through fine sand-activated carbon and twice chromatographic isolation of silica gel forms separator II, described separator II and obtain separator III through ion exchange with ethanol precipitation, the oxidized aluminum chromatographic column of this separator III separates and obtains separator IV, obtains target product after separator IV crystallization and recrystallization. Prepare the method for DNJ nano suspension first DNJ component carries out esterification to modify, strengthen that it is fat-soluble, then pass through surfactant and combine and high pressure homogenize processes and DNJ is dispersed into high degree of dispersion population. The present invention separates DNJ technology to be had raw material and is easy to get, and technique is simple, and effect is obvious, is suitable to the feature of large-scale production, and the present invention prepares DNJ nano suspension can improve the dissolubility of medicine, improves bioavailability.
Description
Technical field
The invention belongs to field of natural medicinal chemistry, relate to purification of pharmaceuticals technology, particularly to from Sang Yuan scale separation purification DNJ 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 type 2 diabetes mellitus. Oral antidiabetic drug currently for type 2 diabetes mellitus mainly has sulfonylurea, biguanides, alpha-glucosidase inhibitor and thiazolidinediones 4 class. Wherein alpha-glucosidase inhibitor blood sugar decreasing effect is notable, and attention is the most extensive. 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, it is easy to cause the side reactions such as flatulence in various degree, abdominal discomfort, Nausea and vomiting, borborygmus and diarrhoea. Therefore, the important development target that exploitation high-efficiency low-toxicity plant source alpha-glucosidase inhibitor hypoglycemic drug is Chinese medicine research field is explored.
DNJ (Chinese name 1-DNJ) is a kind of piperidine alkaloid, it is widely present in the branch with mulberry, leaves and rhizome, its structure similar to glucose (structural formula is as shown below), can be combined with mammiferous enteral alpha-glucosidase, and affinity is significantly greater than the disaccharide such as maltose, sucrose, be combined thus having blocked disaccharide with alpha-glucosidase, emulative inhibit disaccharide to be decomposed into glucose, be finally reached the purpose controlling blood sugar content.
At present; restriction 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 exploitation; 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 separation purification research of plant DNJ; silkworm industry science; 2014,40 (3): 0544-0550)). The blood sugar lowering 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, by blood or diffusion of body fluids transhipment, first has to have certain water solublity, but medicine to pass through lipid biomembrane and arrive each histiocyte, 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 intestinal by biomembrane. If oral, the alpha-glucosidase of small intestinal physiology epimere is easily suppressed, in, the degree of susceptibility of hypomere carbohydrate digestion less, requiring over certain method and strengthen the adhesiveness of itself and internal mucous membrane tissue, thus extending the DNJ holdup time in vivo, improving its bioavailability.
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 extracting solution with dilute hydrochloric acid, alcoholic solution or boiling water extraction;
2), first by step 1) extracting solution concentration after microwave coking, obtain powdered extract, then dissolved the also centrifugation supernatant, last concentrated supernatant, obtain separator I;
3), first chromatographic isolation, including:
A, utilizing chromatographic column i to separate separator I, this step is fixing is fine sand and Mixture of Activated Carbon mutually, and mobile phase is alcoholic solution;
B, concentration A gained eluent also separate further with chromatographic column ii, and this step is fixing is silica gel mutually, and mobile phase is the mixed liquor of ethyl acetate, ethanol;
C, concentration B gained eluent, obtain separator II;
4), ion-exchange chromatography: first make separator II pass through cation exchange resin, and carry out eluting with aqua calcis, then concentration ion clearing house obtains eluent, then adds ethanol in the eluent after concentration and precipitates, and last filtering and concentrating obtains separator III;
5), separating separator III first with chromatographic column iii, this step is fixing is aluminium oxide mutually, and mobile phase is ethyl acetate, alcohol mixeding liquid, and then concentrate eluant obtains separator IV;
6), first by separator IV add alcoholic solution crystallization, then crystallized product is added recrystallization in ethyl acetate, alcohol mixed solution, is finally separating crystallized product and namely obtains separate targets component.
Preferably, step 1) use dilute hydrochloric acid extraction time concentration of hydrochloric acid be 0.03～0.07mol/L;When using alcoholic 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 treatment 20～40min during extraction; During use boiling water extraction, 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 eluent; 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 eluent.
Preferably, step 4) aqua calcis pH is 10～12, elution flow rate is 0.9～1.6BV/h, collects 1～30 column volume eluent, 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 eluent.
Preferably, step 6) crystallization temperature is 1～6 DEG C, ethanol solution concentration is more than 90%, 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.
The present invention prepares the method for DNJ nano suspension, comprises the following steps:
1), esterification target components is prepared, including:
A, take by amount of substance chlorination 1-butyl-3-Methylimidazole. 0.25～1.0 part, DNJ0.03-0.07 part, second, third, fourth, penta, oneself or heptanoic anhydride 0.3-0.5 part and 0.003～0.012 part of eight water aluminum sulfate add reactor hybrid reaction; B, ethyl acetate washing step a product, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, obtain esterification target components;
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 carboxymethyl cellulose: 0.3～5%, and all the other are 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, sodium lauryl sulphate or glyceryl triacetate;
4) 3:2～2:3 blend step 1 by volume) gained esterification target components and solution B carry out ultrasonic Treatment, during supersound process, power is 140～280W, 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 response 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 homogenize 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 mean diameter is 180～380nm
The beneficial effects of the present invention is:
The present invention separates the method for DNJ with common Mulberry for 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 part biological macromole be become insoluble solid to be easier to separate by coking under the premise not damaging DNJ stability; The present invention with fine sand and activated carbon for fixing mutually and coordinate with ethanol for mobile phase, it is possible to effectively remove a large amount of impurity in separator I, and this fixed and can reuse mutually, contributes to cost savings; The ammonia in traditional handicraft with strong and stimulating abnormal smells from the patient is replaced to carry out eluting cation 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 sodium hydroxide, and eluting effect feasibility is obvious; The present invention is further using aluminium oxide as fixing phase, and ethyl acetate, alcohol mixeding liquid are as mobile phase, it is possible to improve separation 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 better, 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, so as to become fat-soluble being prone to by force and in vivo the effective ingredient that enzymolysis is DNJ, the present invention is combined by surfactant further and high pressure homogenize processes and DNJ effective ingredient is dispersed into the particle diameter high degree of dispersion population less than 1000nm, form stable nanometer colloidal dispersion, it is possible to improve dissolubility and the dissolution rate of insoluble drug; Thus solving 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 extracting solution with dilute hydrochloric acid, alcoholic solution or boiling water extraction;
Wherein: during use dilute hydrochloric acid extraction, concentration of hydrochloric acid is 0.03～0.07mol/L; When using alcoholic 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 treatment 20～40min during extraction; During use boiling water extraction, boiled water temperature is 95～100 DEG C, and liquid ratio is 20～45mL/g, extraction time 20～40min;
2), first by step 1) extracting solution concentration after microwave coking, obtain powdered extract, then dissolved the also centrifugation supernatant, 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 is fine sand and Mixture of Activated Carbon mutually, and mobile phase is alcoholic solution; 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 eluent;
B, concentration A gained eluent also separate further with chromatographic column ii, and this step is fixing is silica gel mutually, and mobile phase is the mixed liquor of ethyl acetate, ethanol; 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 eluent;
C, concentration B gained eluent, obtain separator II;
4), ion-exchange chromatography: first make separator II pass through cation exchange resin, and carry out eluting with aqua calcis, then concentration ion clearing house obtains eluent, then adds ethanol in the eluent after concentration and precipitates, and 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 eluent, 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 aluminium oxide mutually, and mobile phase is ethyl acetate, alcohol mixeding liquid, 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 eluent;
6), first by separator IV add alcoholic solution crystallization, then crystallized product is added recrystallization in ethyl acetate, alcohol mixed solution, is finally separating crystallized product and namely obtains separate targets component; 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.
The method preparing DNJ nano suspension, it is preferable that the DNJ target components (wherein the mass percent of DNJ is >=35%) obtained by method described above, specifically includes following steps:
1), esterification target components is prepared, including:
A, take by amount of substance chlorination 1-butyl-3-Methylimidazole. 0.25～1.0 part, DNJ (DNJ standard substance 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 0.003～0.012 part of eight water aluminum sulfate add reactor hybrid reaction; During this step hybrid reaction, temperature is 80～160 DEG C, and the response time is 6～14h, and mixed liquor rotating speed is 300～500r/min;
B, ethyl acetate washing step a product, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, obtain esterification target components; 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 carboxymethyl cellulose: 0.3～5%, and all the other are 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, sodium lauryl sulphate or glyceryl triacetate;
4) 3:2～2:3 blend step 1 by volume) gained esterification target components and solution B carry out ultrasonic Treatment; During this step supersound process, 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 adopt each circulation 5～l0 time under 500bar, 1000bar, 1500bar pressure respectively, or ultra micro nano grinder 2900rpm mills 45～60min, obtains the suspension that mean diameter is 180～380nm.
Embodiment 1:
The method of DNJ nano suspension is prepared in this enforcement, comprises the following steps:
(1), the preparation extraction extractum containing DNJ: can adopt in following 3 kinds of methods one or more:
Method 1: extracting with the alcoholic 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 extracting solution, must extract extractum after concentrating under reduced pressure. 5kg Cortex Mori dry powder obtains 0.50kg extractum, 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 extracting solution, must extract extractum after concentrating under reduced pressure. 5kg Cortex Mori dry powder obtains 0.33kg extractum, and wherein the purity of DNJ is 0.141%.
Method 3: extracting with the dilute 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 extracting solution, must extract extractum after concentrating under reduced pressure. 5kg Cortex Mori dry powder obtains 0.46kg extractum, and wherein the purity of DNJ is 0.162%.
(2), utilize above-mentioned extractum scale to separate Sang Yuan DNJ: can adopt in following 7 kinds of methods one or more:
Method 1: extractum microwave coking processes (640W, 6min), gained powder water dissolution, centrifugal (2000r/min, 20min), obtains separator I after supernatant concentrating under reduced pressure; Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the alcoholic solution eluting of 80%, collect 1～27BV, flow velocity is 1.1BV/h, and eluent concentrating under reduced pressure, again through silica gel chromatographic column, ethyl acetate: the mixed solvent eluting of ethanol=1:3, collecting 2～30BV, flow velocity is 0.8BV/h, and eluent concentrating under reduced pressure obtains separator II; Separator II passes through cation exchange resin column, the aqua calcis eluting of PH12, collects 1～28BV, flow velocity is 1.1BV/h, with accounting for the alcoholic solution precipitation that concentrated liquid volume concentrations is 75%, elimination calcium hydroxide etc. after eluent concentrating under reduced pressure, after concentrating under reduced pressure, obtain separator III; Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent eluting of ethanol=1:3, collects 1～41BV, and flow velocity is 0.5BV/h, obtains separator IV after eluent concentrating under reduced pressure; Under 4 DEG C of conditions, the alcoholic solution crystallization 6h of 98%, under-20 DEG C of conditions, ethyl acetate: the mixed solution recrystallization 6h of ethanol=2:1, continuously repeat 2 times, obtaining purity is 36.4%, and sample recovery rate is the DNJ separate targets component of 39.3%.
Method 2: extractum microwave coking processes (800W, 2min), gained powder water dissolution, centrifugal (5000r/min, 8min), obtains separator I after supernatant concentrating under reduced pressure; Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the alcoholic solution eluting of 50%, collect 2～25BV, flow velocity is 0.9BV/h, and eluent concentrating under reduced pressure, again through silica gel chromatographic column, ethyl acetate: the mixed solvent eluting of ethanol=2:1, collecting 2～23BV, flow velocity is 0.6BV/h, and eluent concentrating under reduced pressure obtains separator II; Separator II passes through cation exchange resin column, the aqua calcis eluting of PH11, collects 2～27BV, flow velocity is 0.9BV/h, with accounting for the alcoholic solution precipitation that concentrated liquid volume concentrations is 78%, elimination calcium hydroxide etc. after eluent concentrating under reduced pressure, after concentrating under reduced pressure, obtain separator III;Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent eluting of ethanol=2:1, collects 1～41BV, and flow velocity is 0.3BV/h, obtains separator IV after eluent concentrating under reduced pressure; Under 4 DEG C of conditions, the alcoholic solution crystallization 48h of 98%, under-20 DEG C of conditions, ethyl acetate: the mixed solution recrystallization 48h of ethanol=7:1, continuously repeat 4 times, obtaining purity is 38.7%, and sample recovery rate is the DNJ separate targets component of 32.6%.
Method 3: extractum microwave coking processes (720W, 3min), gained powder water dissolution, centrifugal (3000r/min, 12min), obtains separator I after supernatant concentrating under reduced pressure; Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the alcoholic solution eluting of 70%, collect 2～20BV, flow velocity is 1.2BV/h, and eluent concentrating under reduced pressure, again through silica gel chromatographic column, ethyl acetate: the mixed solvent eluting of ethanol=1:1, collecting 2～16BV, flow velocity is 0.8BV/h, and eluent concentrating under reduced pressure obtains separator II; Separator II passes through cation exchange resin column, the aqua calcis eluting of PH11, collects 3～23BV, flow velocity is 1.2BV/h, with accounting for the alcoholic solution precipitation that concentrated liquid volume concentrations is 80%, elimination calcium hydroxide etc. after eluent concentrating under reduced pressure, after concentrating under reduced pressure, obtain separator III; Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent eluting of ethanol=1:1, collects 2～38BV, and flow velocity is 0.6BV/h, obtains separator IV after eluent concentrating under reduced pressure; Under 4 DEG C of conditions, the alcoholic solution crystallization 24h of 98%, under-20 DEG C of conditions, ethyl acetate: the mixed solution recrystallization 24h of ethanol=5:1, continuously repeat 3 times, obtaining purity is 39.1%, and sample recovery rate is the DNJ separate targets component of 37.9%.
Method 4: extractum microwave coking processes (640W, 5min), gained powder water dissolution, centrifugal (4000r/min, 10min), obtains separator I after supernatant concentrating under reduced pressure; Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the alcoholic solution eluting of 90%, collect 1～30BV, flow velocity is 1.6BV/h, and eluent concentrating under reduced pressure, again through silica gel chromatographic column, ethyl acetate: the mixed solvent eluting of ethanol=2:3, collecting 1～30BV, flow velocity is 1.3BV/h, and eluent concentrating under reduced pressure obtains separator II; Separator II passes through cation exchange resin column, the aqua calcis eluting of PH11, collects 1～30BV, flow velocity is 1.6BV/h, with accounting for the alcoholic solution precipitation that concentrated liquid volume concentrations is 70%, elimination calcium hydroxide etc. after eluent concentrating under reduced pressure, after concentrating under reduced pressure, obtain separator III; Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent eluting of ethanol=2:3, collects 1～50BV, and flow velocity is 0.8BV/h, obtains separator IV after eluent concentrating under reduced pressure; Under 4 DEG C of conditions, the alcoholic solution crystallization 12h of 98%, under-20 DEG C of conditions, ethyl acetate: the mixed solution recrystallization 12h of ethanol=2:1, continuously repeat 5 times, obtaining purity is 35.0%, and sample recovery rate is the DNJ separate targets component of 38.2%.
Method 5: extractum microwave coking processes (800W, 5min), gained powder water dissolution, centrifugal (2000r/min, 18min), obtains separator I after supernatant concentrating under reduced pressure; Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the alcoholic solution eluting of 60%, collect 2～26BV, flow velocity is 1.0BV/h, and eluent concentrating under reduced pressure, again through silica gel chromatographic column, ethyl acetate: the mixed solvent eluting of ethanol=4:3, collecting 2～28BV, flow velocity is 1.1BV/h, and eluent concentrating under reduced pressure obtains separator II;Separator II passes through cation exchange resin column, the aqua calcis eluting of PH10, collects 2～28BV, flow velocity is 1.2BV/h, with accounting for the alcoholic solution precipitation that concentrated liquid volume concentrations is 75%, elimination calcium hydroxide etc. after eluent concentrating under reduced pressure, after concentrating under reduced pressure, obtain separator III; Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent eluting of ethanol=5:3, collects 2～43BV, and flow velocity is 0.6BV/h, obtains separator IV after eluent concentrating under reduced pressure; Under 4 DEG C of conditions, the alcoholic solution crystallization 36h of 98%, under-20 DEG C of conditions, ethyl acetate: the mixed solution recrystallization 36h of ethanol=4:1, continuously repeat 3 times, obtaining purity is 37.1%, and sample recovery rate is the DNJ separate targets component of 35.5%.
Method 6: extractum microwave coking processes (800W, 4min), gained powder water dissolution, centrifugal (3000r/min, 16min), obtains separator I after supernatant concentrating under reduced pressure; Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the alcoholic solution eluting of 60%, collect 2～26BV, flow velocity is 1.4BV/h, and eluent concentrating under reduced pressure, again through silica gel chromatographic column, ethyl acetate: the mixed solvent eluting of ethanol=1:1, collecting 2～28BV, flow velocity is 0.5BV/h, and eluent concentrating under reduced pressure obtains separator II; Separator II passes through cation exchange resin column, the aqua calcis eluting of PH10, collects 2～26BV, flow velocity is 1.4BV/h, with accounting for the alcoholic solution precipitation that concentrated liquid volume concentrations is 74%, elimination calcium hydroxide etc. after eluent concentrating under reduced pressure, after concentrating under reduced pressure, obtain separator III; Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent eluting of ethanol=4:3, collects 2～44BV, and flow velocity is 0.5BV/h, obtains separator IV after eluent concentrating under reduced pressure; Under 4 DEG C of conditions, the alcoholic solution crystallization 40h of 98%, under-20 DEG C of conditions, ethyl acetate: the mixed solution recrystallization 40h of ethanol=6:1, continuously repeat 4 times, obtaining purity is 38.0%, and sample recovery rate is the DNJ separate targets component of 34.3%.
Method 7: extractum microwave coking processes (720W, 4min), gained powder water dissolution, centrifugal (4000r/min, 12min), obtains separator I after supernatant concentrating under reduced pressure; Separator I is by " fine sand-activated carbon " that volume ratio is 2:1 double-deck chromatographic column, the alcoholic solution eluting of 80%, collect 2～30BV, flow velocity is 1.3BV/h, and eluent concentrating under reduced pressure, again through silica gel chromatographic column, ethyl acetate: the mixed solvent eluting of ethanol=5:3, collecting 2～27BV, flow velocity is 0.9BV/h, and eluent concentrating under reduced pressure obtains separator II; Separator II passes through cation exchange resin column, the aqua calcis eluting of PH12, collects 2～30BV, flow velocity is 1.3BV/h, with accounting for the alcoholic solution precipitation that concentrated liquid volume concentrations is 85%, elimination calcium hydroxide etc. after eluent concentrating under reduced pressure, after concentrating under reduced pressure, obtain separator III; Separator III passes through chromatography on alumina post, ethyl acetate: the mixed solvent eluting of ethanol=1:1, collects 2～40BV, and flow velocity is 0.4BV/h, obtains separator IV after eluent concentrating under reduced pressure; Under 4 DEG C of conditions, the alcoholic solution crystallization 15h of 98%, under-20 DEG C of conditions, ethyl acetate: the mixed solution recrystallization 15h of ethanol=3:1, continuously repeat 3 times, must obtain purity is 37.2%, and sample recovery rate is the DNJ separate targets component of 36.7%.
(3), utilize the esterification of above-mentioned separate targets component modify and prepare nano suspension: can adopt in following 6 kinds of methods one or more:
Method 1: by ionic liquid chlorination 1-butyl-3-Methylimidazole. (0.25mol), separate targets component (containing about DNJ0.05mol), heptanoic anhydride (0.40mol) and aluminum sulfate octadecahydrate (2.0g) are sequentially added in reactor, 80 DEG C, rotating speed 300r/min when reaction 18h. Wash ionic liquid by ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 63.2%, selectivity 100%) must be esterified, reuse after ionic liquid vacuum distillation drying. The lower preparation of stirring, containing the aqueous solution that mass fraction is 0.2% mannitol, 0.5% lactose, 0.5% glucose and 0.3% sodium carboxymethyl cellulose, obtains solution A. Sodium lauryl sulphate 0.5% is dissolved in solution A in mass ratio, obtains solution B. Esterification target components containing 1.25%DNJ mixes with solution B 1:1 by volume, and with power 140W ultrasonic Treatment 45min, decompression boils off organic solvent. Transferring to the broken machine 2900rpm of superfine powder nanometer to mill 45min, obtaining mean diameter is 345nm suspension. Its dry product is obtained after lyophilization.
Method 2: by ionic liquid chlorination 1-butyl-3-Methylimidazole. (0.55mol), separate targets component (containing about DNJ0.05mol), propionic andydride (0.40mol) aluminum sulfate octadecahydrate (4.4g) is sequentially added in reactor, 160 DEG C, rotating speed 500r/min when reaction 6h. Wash ionic liquid by ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 75.3%, selectivity 100%) must be esterified, reuse after ionic liquid vacuum distillation drying. The lower preparation of stirring, containing the aqueous solution that mass fraction is 3% PEG400,2% lactose, 2% glucose and 4% sodium carboxymethyl cellulose, obtains solution A. Tween 80 1.2% is dissolved in solution A in mass ratio, obtains solution B. Esterification target components containing 1.1%DNJ mixes with solution B 1:1 by volume, and with power 280W ultrasonic Treatment 15min, decompression boils off organic solvent. Transferring to the broken machine 2900rpm of superfine powder nanometer to mill 52min, obtaining mean diameter is 270nm suspension. Its dry product is obtained after lyophilization.
Method 3: by ionic liquid chlorination 1-butyl-3-Methylimidazole. (1.0mol), separate targets component (containing about DNJ0.05mol), valeric anhydride (0.40mol) and aluminum sulfate octadecahydrate (8.0g) are sequentially added in reactor, 100 DEG C, rotating speed 300r/min when reaction 9h. Wash ionic liquid by ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 80.1%, selectivity 100%) must be esterified, reuse after ionic liquid vacuum distillation drying. The lower preparation of stirring, containing the aqueous solution that mass fraction is 1.6% mannitol, 0.7% lactose, 0.7% glucose and 2.8% sodium carboxymethyl cellulose, obtains solution A. Glyceryl triacetate 2.4% is dissolved in solution A in mass ratio, obtains solution B. Esterification target components containing 1.0%DNJ mixes with solution B 1:1 by volume, and with power 140W ultrasonic Treatment 45min, decompression boils off organic solvent. Transferring in high pressure homogenizer, each circulation 5 times under 500bar, 1000bar, 1500bar pressure, obtaining mean diameter is 380nm suspension. Its dry product is obtained after lyophilization.
Method 4: by ionic liquid chlorination 1-butyl-3-Methylimidazole. (0.7mol), separate targets component (containing about DNJ0.05mol), butyryl oxide. (0.40mol) and aluminum sulfate octadecahydrate (5.6g) are sequentially added in reactor, 120 DEG C, rotating speed 400r/min when reaction 12h.Wash ionic liquid by ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 87.3%, selectivity 100%) must be esterified, reuse after ionic liquid vacuum distillation drying. The lower preparation mass fraction of stirring is the aqueous solution of 1.2% PEG400,1.5% lactose, 1.5% glucose and 3% sodium carboxymethyl cellulose, obtains solution A. Polyethylene glycol 1000 vitamin E succinic acid ester 2% is dissolved in solution A in mass ratio, obtains solution B. Esterification target components containing 0.83%DNJ mixes with solution B 1:1 by volume, and with power 210W ultrasonic Treatment 30min, decompression boils off organic solvent. Transferring in high pressure homogenizer, each circulation l0 time under 500bar, 1000bar, 1500bar pressure, obtaining mean diameter is 180nm suspension. Its dry product is obtained after lyophilization.
Method 5: by ionic liquid chlorination 1-butyl-3-Methylimidazole. (0.85mol), separate targets component (containing about DNJ0.05mol), acetic anhydride (0.40mol) and aluminum sulfate octadecahydrate (6.8g) are sequentially added in reactor, 160 DEG C, rotating speed 500r/min when reaction 15h. Wash ionic liquid by ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 81.8%, selectivity 100%) must be esterified, reuse after ionic liquid vacuum distillation drying. The lower preparation of stirring, containing the aqueous solution that mass fraction is 2% PEG400,1.2% lactose, 1.2% glucose and 1.6% sodium carboxymethyl cellulose, obtains solution A. Lecithin 5% is dissolved in solution A in mass ratio, obtains solution B. Esterification target components containing 0.91%DNJ mixes with solution B 1:1 by volume, and with power 210W ultrasonic Treatment 30min, decompression boils off organic solvent. Transferring to the broken machine 2900rpm of superfine powder nanometer to mill 60min, obtaining mean diameter is 300nm suspension. Its dry product is obtained after lyophilization.
Method 6: by ionic liquid chlorination 1-butyl-3-Methylimidazole. (0.4mol), DNJ standard substance (0.05mol), caproic anhydride (0.40mol) and aluminum sulfate octadecahydrate (3.2g) are sequentially added in reactor, 130 DEG C, rotating speed 400r/min when reaction 9h. Wash ionic liquid by ethyl acetate, take upper liquid after layering, be adjusted to neutrality with saturated sodium carbonate, target components (conversion ratio 70.4%, selectivity 100%) must be esterified, reuse after ionic liquid vacuum distillation drying. The lower preparation of stirring, containing the aqueous solution that mass fraction is 2% mannitol, 1% lactose, 1% glucose and 5% sodium carboxymethyl cellulose, obtains solution A. PLURONICS F87 2% is dissolved in solution A in mass ratio, obtains solution B. Esterification target components containing 0.70%DNJ mixes with solution B 1:1 by volume, and with power 280W ultrasonic Treatment 15min, decompression boils off organic solvent. Transferring in high pressure homogenizer, each circulation 7 times under 500bar, 1000bar, 1500bar pressure, obtaining mean diameter is 210nm suspension. Its dry product is obtained after lyophilization.
Remarks: in separating DNJ and prepare the process of 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 adopts the content of following method detection DNJ:
Step 1:DNJ derivatization step is:
The potassium borate buffer (pH8.5) of 30 μ LDNJ standard solutions or extracting solution and 30 μ L0.4mol/L is mixed in 0.5mL centrifuge tube, add the FMOC-Cl of 60 μ L5mmol/L, 25 DEG C of Water Under bath 20min, the glycine adding 30 μ L1mol/L terminates reaction.Newly-generated DNJ-FMOC is stablized, with 120 μ L distilled water constant volumes with 30 μ L0.1% (v/v) acetic acid solutions. Finally cross 0.22 μm of nylon leaching film, to be measured.
Step 2:HPLC detects:
Utilize the testing sample of HPLC detecting step 1, detect with anti-phase 5 μm of C18(4.6 × 150mm) chromatographic column (Waters, Atlantis, Ireland), mobile phase is 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) namely 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 processing procedure 3 kinds concrete, step (two) includes concrete processing procedure in 7, step (three) includes processing procedure 6 kinds concrete, and the method preparing DNJ nano suspension in embodiment 1 can be the combination in any of above three step.
What 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 (7)
1. the method separating DNJ, it is characterised in that comprise the following steps:
1), drying and crushing Mulberry material obtain extracting solution with dilute hydrochloric acid, alcoholic solution or boiling water extraction;
2), first by microwave coking after the extracting solution concentration of step 1), obtain powdered extract, then dissolved and the centrifugation supernatant, last concentrated supernatant, obtain separator I;
3), first chromatographic isolation, including:
A, utilize chromatographic columnSeparating separator I, this step is fixing is fine sand and Mixture of Activated Carbon mutually, and mobile phase is alcoholic solution;
B, concentration A gained eluent also use chromatographic columnSeparating further, this step is fixing is silica gel mutually, and mobile phase is the mixed liquor of ethyl acetate, ethanol;
C, concentration B gained eluent, obtain separator II;
4), ion-exchange chromatography: first make separator II pass through cation exchange resin, and carry out eluting with aqua calcis, then concentration ion clearing house obtains eluent, then adds ethanol in the eluent after concentration and precipitates, and last filtering and concentrating obtains separator III;
5), first with chromatographic columnSeparating separator III, this step is fixing is aluminium oxide mutually, and mobile phase is ethyl acetate, alcohol mixeding liquid, and then concentrate eluant obtains separator IV;
6), first by separator IV add alcoholic solution crystallization, then crystallized product is added recrystallization in ethyl acetate, alcohol mixed solution, is finally separating crystallized product and namely obtains separate targets component.
2. the method separating DNJ according to claim 1, it is characterized in that: during step 1) use dilute hydrochloric acid extraction, concentration of hydrochloric acid is 0.03 ~ 0.07mol/L, when using alcoholic solution to extract, concentration of alcohol is 60 ~ 80%, liquid ratio is 20 ~ 45mL/g, extraction time is 3 ~ 5 times, utilizes 350 ~ 490W Sonication assisted treatment 20 ~ 40min during extraction;During use boiling water extraction, boiled water temperature is 95 ~ 100 DEG C, and liquid ratio is 20 ~ 45mL/g, extraction time 20 ~ 40min.
3. the method separating DNJ according to claim 1, it is characterised in that: step 2) microwave coking time microwave power be 640 ~ 800W, the process time is 2 ~ 6min.
4. the method separating DNJ according to claim 1, it is characterised in that: in step 3)-A, 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 eluent; In step 3)-B, 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 eluent.
5. the method separating DNJ according to claim 1, it is characterised in that: step 4) aqua calcis pH is 10 ~ 12, and elution flow rate is 0.9 ~ 1.6BV/h, collects 1 ~ 30 column volume eluent, and it is 70 ~ 85% that ethanol accounts for concentrated liquid volume concentrations.
6. the method separating DNJ according to claim 1, it is characterised in that: step 5) 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 eluent.
7. the method separating DNJ according to claim 1, it is characterized in that: step 6) crystallization temperature is 1 ~ 6 DEG C, ethanol solution concentration is more than 90%, 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.
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