CN105384747A - Brucine extraction method - Google Patents
Brucine extraction method Download PDFInfo
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- CN105384747A CN105384747A CN201510828006.2A CN201510828006A CN105384747A CN 105384747 A CN105384747 A CN 105384747A CN 201510828006 A CN201510828006 A CN 201510828006A CN 105384747 A CN105384747 A CN 105384747A
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- RRKTZKIUPZVBMF-UHFFFAOYSA-N brucine Natural products C1=2C=C(OC)C(OC)=CC=2N(C(C2)=O)C3C(C4C5)C2OCC=C4CN2C5C31CC2 RRKTZKIUPZVBMF-UHFFFAOYSA-N 0.000 title claims abstract description 55
- RRKTZKIUPZVBMF-IBTVXLQLSA-N brucine Chemical compound O([C@@H]1[C@H]([C@H]2C3)[C@@H]4N(C(C1)=O)C=1C=C(C(=CC=11)OC)OC)CC=C2CN2[C@@H]3[C@]41CC2 RRKTZKIUPZVBMF-IBTVXLQLSA-N 0.000 title claims abstract description 54
- 238000000605 extraction Methods 0.000 title claims abstract description 14
- QMGVPVSNSZLJIA-UHFFFAOYSA-N Nux Vomica Natural products C1C2C3C4N(C=5C6=CC=CC=5)C(=O)CC3OCC=C2CN2C1C46CC2 QMGVPVSNSZLJIA-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 24
- 244000107975 Strychnos nux-vomica Species 0.000 claims abstract description 23
- 238000010828 elution Methods 0.000 claims abstract description 12
- 238000000926 separation method Methods 0.000 claims abstract description 8
- 239000012071 phase Substances 0.000 claims description 95
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 87
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- 239000007791 liquid phase Substances 0.000 claims description 44
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000004811 liquid chromatography Methods 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 10
- 238000010992 reflux Methods 0.000 claims description 9
- 238000001228 spectrum Methods 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 8
- 238000010829 isocratic elution Methods 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 238000002390 rotary evaporation Methods 0.000 claims description 8
- 239000012046 mixed solvent Substances 0.000 claims description 6
- 238000000862 absorption spectrum Methods 0.000 claims description 4
- 238000007654 immersion Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 5
- 229930014626 natural product Natural products 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 235000013399 edible fruits Nutrition 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 description 33
- 239000012043 crude product Substances 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- QMGVPVSNSZLJIA-FVWCLLPLSA-N strychnine Chemical compound O([C@H]1CC(N([C@H]2[C@H]1[C@H]1C3)C=4C5=CC=CC=4)=O)CC=C1CN1[C@@H]3[C@]25CC1 QMGVPVSNSZLJIA-FVWCLLPLSA-N 0.000 description 14
- 239000000284 extract Substances 0.000 description 12
- 238000001914 filtration Methods 0.000 description 12
- 239000006096 absorbing agent Substances 0.000 description 10
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 229960005453 strychnine Drugs 0.000 description 8
- 238000001556 precipitation Methods 0.000 description 7
- 238000004780 2D liquid chromatography Methods 0.000 description 6
- 241001279009 Strychnos toxifera Species 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 235000012054 meals Nutrition 0.000 description 6
- 239000012982 microporous membrane Substances 0.000 description 6
- 210000000582 semen Anatomy 0.000 description 6
- 238000010025 steaming Methods 0.000 description 6
- 229930013930 alkaloid Natural products 0.000 description 4
- 238000002425 crystallisation Methods 0.000 description 4
- 238000004262 preparative liquid chromatography Methods 0.000 description 4
- -1 Alkaloid compound Chemical class 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 150000003797 alkaloid derivatives Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000050051 Chelone glabra Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 241001113787 Strychnos Species 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000000954 anitussive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000001670 myorelaxant effect Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
- 230000001457 vasomotor Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a brucine extraction method. By using a two-dimensional liquid chromatogram technology, brucine is extracted from the fruit body of nux vomica as a natural product; by reasonably selecting chromatographic columns, dissolved solutions, mobile phases and the like, and optimizing process conditions such as flow speed, elution modes and collection sections, a whole separation and purification method is practicable, the operation is simple and convenient, the automation degree of instruments is high, the method can be performed under normal temperature and normal pressure, the repeatability is high, obtained material components in different batches are high in consistency, the operability is good, and the obtained brucine has high purity capable of reaching more than 99 percent, and can be directly applied to industrial production.
Description
Technical field
The present invention relates to natural compounds extractive technique field, be specifically related to a kind of extracting method of brucine.
Background technology
Alkaloid compound is widely distributed in Chinese medicine animal drugs, and its chemical structure complexity is various, and biological activity is remarkable.The alkaloid of some novel structures has noticeable physiology and pharmacologically active, is likely the important sources for the treatment of series of human major disease medicine.Brucine (also known as Strychnine) is exactly so a kind of alkaloid compound, and be a kind of important active skull cap components be extensively present in plant materials, its structure is as follows:
Current discovery brucine has following effect: the first, and it has its proliferation and differentiation of suppression and Cytotoxic effect to the cell of proliferation and differentiation and tumour cell, cell death inducing, therefore, and the activity of brucine Tumor suppression; The second, to the effect of Digestive tract, directly test proof with patient's intestines impotence pipe, brucine does not have excitation to human intestines and stomach's unstriated muscle, to gastral unique effect, is the bitter taste due to it, increases the secretion of stomach reflectingly; 3rd, impact on respiratory system: vauqueline act as 1/8 of brucine, it is reported the mouse that it causes ammoniacal liquor and SO2 and cough have stronger antitussive effect, the effect of oral comparatively abdominal injection is remarkable, separately has Eradicates phlegm effect (the phenol red method of small white mouse); 4th, brucine has curariform action mask, to some bacterium, has restraining effect in vitro, and multiple Strychnos is produced in Africa, and wherein some also has myorelaxant effects; 5th, alkaloid brucine in seed, absorbs very soon after oral and works, the reflection function of first excited spinal cord, secondly the respiratory centre maincenter in excited oblongata and vasomotor center, and improve the function of pallium sensorium (cortex analyzer).
The classical way that bibliographical information extracts Strychnine from nux vomica is as follows: soak nux vomica with liming, then does solvent extraction with benzene and with hcl as extraction agent after reclaiming most of benzene, extraction liquid separates out Strychnine coarse crystallization, and recrystallization obtains highly purified strychnine hydrochloride.Shortcoming is that solvent price is high, and environmental pollution is large, poor stability (toxicity of benzene is large), and strict to equipment requirements, need be strictly on guard against that solvent is revealed.
Notification number is that the Chinese invention patent of CN101838273B discloses a kind of method extracting Strychnine from nux vomica, successively by water refluxing extraction, add polymeric flocculant adsorption clarification, pH is regulated to separate out precipitate A, sulfuric acid or phosphoric acid dissolution precipitation A, then regulate pH to separate out precipitate B, diluted hydrochloric acid dissolution precipitate B, crystallization, recrystallization obtains the finished product that purity is up to 95.7.The method complex steps, and owing to adopting refluxing extraction and crystallization method, poor repeatability, success ratio is not high, result by experiment condition and environmental influence large.
Notification number is that the Chinese invention patent of CN104072505A discloses a kind of method simultaneously extracting vauqueline and Strychnine, main employing supersound extraction, macroporous resin adsorption, the method of sulfuric acid ethanol periodic crystallisation obtains strychnine hydrochloride, the Strychnine that the Strychnine that the method obtains is not pure, and also there is poor repeatability in method itself, the problems such as system stability difference.
So, up to the present, the method extracting brucine in natural product extract is generally speaking more loaded down with trivial details, poor repeatability, operability is not high, say nothing of and extract high purity or monomer brucine, this also counteracts that the progress of brucine as tcm development aspect to a certain extent.
Summary of the invention
Technical problem to be solved by this invention is for above prior art problem, a kind of method extracting brucine from nux vomica is provided, the method adopts two-dimensional highly effective liquid phase chromatographic to carry out separation and purification, separation method is stablized, repeatability is high, preparation amount is large, simple to operately saves time, and the brucine monomeric compound purity obtained can reach more than 99%.
The technical solution adopted in the present invention is:
An extracting method for brucine, the method comprises the following steps:
(1) with nux vomica sporophore for raw material, after grinds powder, by alcohol immersion or refluxing extraction, filter, gained filtrate carries out rotary evaporated to dryness, dissolves, obtain the extracting solution that solid content is 50-200mg/mL with the methanol-water binary mixed solvent that methyl alcohol volume fraction is 50-70%;
(2) carry out liquid chromatography separation to the extracting solution obtained: the chromatographic column of employing is C18 chromatographic column, and moving phase is two end number mixing liquid phase, wherein A phase is water, B phase is methyl alcohol; Sample size is 50-200mL/ pin; Flow rate of mobile phase is 100-1000mL/min; Detector is UV-detector, determined wavelength 200-260nm; Type of elution is B phase concentration 25% isocratic elution 10min, B phase concentration 35% isocratic elution 20min, B phase concentration 100% isocratic elution 10min; According to ultra-violet absorption spectrum collection, wherein No. 7 peaks (retention time 23-25min) are as object component, and rotary evaporated to dryness, obtains one dimension liquid phase component;
(3) dissolve one dimension liquid phase component with the methanol-water binary mixed solvent that methyl alcohol volume fraction is 50-70%, being dissolved to solid content is 50-100mg/mL;
(4) carry out two-dimensional HPLC separation: the chromatographic column of employing is C18 chromatographic column, the moving phase of employing is two end number mixing liquid phase, and wherein A phase is water, B phase is methyl alcohol; Sample size is 500-2000 μ L/ pin; Flow rate of mobile phase is 10-30mL/min; Detector is UV-detector, determined wavelength 200-260nm; Type of elution is B phase concentration 33% isocratic elution 40-50min; According to ultra-violet absorption spectrum collection, wherein No. 1 peak (retention time 18-20min) is as object component, and rotary evaporation is concentrated into dry, obtains target product brucine.
In described step (1), refluxing extraction can repeat 1-3 time, each 1-2h.
In described step (1), (3), methanol-water binary mixed solvent is containing 0.1% (mass percent) acetic acid.
In described step (2), (4), the A phase of two end number mixing liquid phase is containing 0.1% (mass percent) acetic acid.
In described step (2) C18 chromatographic column be specially anti-phase C18 axial pressure chromatographic column (150 × 250mm, 10 μm,
), during use, column temperature is room temperature or 25-40 DEG C.
In described step (4) C18 chromatographic column be specially China spectrum C18 chromatographic column (20 × 250mm, 5 μm,
), during use, column temperature is room temperature or 25-40 DEG C.
The present invention selects two-dimensional highly effective liquid phase chromatographic technology to extract brucine from natural product nux vomica sporophore, by choose reasonable chromatographic column, lysate, flow equal, in conjunction with to flow velocity, type of elution, collect groping of the processing condition such as section, make whole separation purification method practical, easy and simple to handle, preparation cycle is short, automation equipment degree is high, reagent consumption is little, just can carry out under normal temperature and pressure, preparation amount is large, repeatability is high, between the material composition that obtains batch, consistence is high, operability is good, degree of controllability is high, and the brucine purity obtained is high, reach more than 99%, directly can apply to industrial production.
Accompanying drawing explanation
Shown in Fig. 1 is the one dimension high performance preparative liquid chromatography figure that the embodiment of the present invention 1 extracts brucine from nux vomica sporophore;
Shown in Fig. 2 is the two-dimensional highly effective preparative liquid chromatography figure that the embodiment of the present invention 1 extracts separating obtained No. 7 cuts of one dimension high performance preparative liquid chromatography of brucine from nux vomica sporophore;
Shown in Fig. 3 is the analysis mode high-efficient liquid phase chromatogram of the object component obtained in the embodiment of the present invention 1;
Shown in Fig. 4 is the object of the invention component
1hNMR spectrogram;
Shown in Fig. 5 is the object of the invention component
13cNMR spectrogram.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail.Iting is noted that following illustrating is all exemplary, being intended to the invention provides further instruction.Except as otherwise noted, all Science and Technology terms that the present invention uses have the identical meanings usually understood with the technical field of the invention personnel.
1, raw material, material:
Raw material nux vomica sporophore is plucked from Hebei and is passed through qualification.
2, reagent:
Hplc grade methanol, acetic acid, ethanol are purchased from Tianjin Concord Technology Co., Ltd..
3, plant and instrument:
Supper micron mill is purchased from Sanqing Stainless Steel Apparatus Co., Ltd., Shandong; Tabletop refrigerated centrifuge is purchased from Thermo company; Rotary Evaporators is purchased from Shanghai Ya Rong biochemical instrument factory; Anti-phase C18 axial pressure chromatographic column (150 × 250mm, 10 μm,
) purchased from Hanbon Sci. & Tech. Co., Ltd.; China spectrum C18 chromatographic column (20 × 250mm, 5 μm,
) purchased from Shanghai spectral technology service centre of China; Preparative high performance liquid chromatography instrument is purchased from Hanbon Sci. & Tech. Co., Ltd..
Embodiment 1:
With nux vomica sporophore for raw material, alcohol reflux 1.5h is used after grinds powder, filter, get precipitation and repeat extraction 2 times, merge gained filtrate, 10g is taken after revolving the nux vomica meal steaming and obtain drying, be dissolved in methanol-water (0.1% acetic acid) solution of 100mL55%, obtained Semen Strychni extract solution, concentration is 100mg/mL, cross 0.45 μm of millipore filtration, carry out the preparation of one dimension liquid chromatography.One dimension liquid chromatography adopts InnovelC18150 × 250mm, 10 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (containing 0.1% acetic acid), B phase is methyl alcohol, type of elution: the degree such as degree such as B phase concentration 25% grade 10min, B phase concentration 35% 20min, B phase concentration 100% etc. spends 10min.Adopt UV-detector, select 210nm absorbing wavelength, preparation temperature is room temperature, sample size is 150mL/ pin, and flow rate of mobile phase is 600mL/min, collects the cut at 23-25 minute (in Fig. 1 No. 7 peaks), carrying out rotary evaporation is concentrated into dry, for one dimension prepares brucine crude product.Methanol-water (0.1% acetic acid) solubilize brucine crude product with 55%, concentration is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, and chromatographic column is China spectrum C1820 × 250mm, 5 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, employing 33%B equality 45 minutes wash-outs.Adopt UV-detector, select 210nm absorbing wavelength, preparation temperature is room temperature, sample size is 1000 μ L/ pins, and flow rate of mobile phase is 16mL/min, collects the cut at 18-20 minute (in Fig. 2 No. 1 peak), rotary evaporated to dryness, obtains brucine compound.Carry out efficient liquid phase chromatographic analysis to the component obtained, as shown in Figure 3, purity is 99.6% to result; In one dimension preparation, the content of brucine crude product is 19%.Carry out nuclear magnetic resonance spectroscopy to the object component obtained, as illustrated in figures 4-5, display object product is brucine to result.
Embodiment 2:
With nux vomica sporophore for raw material, alcohol reflux 2h is used after grinds powder, filter, get precipitation and repeat extraction 2 times, merge gained filtrate, 5g is taken after revolving the nux vomica meal steaming and obtain drying, be dissolved in methanol-water (0.1% acetic acid) solution of 100mL50%, obtained Semen Strychni extract solution, concentration is 50mg/mL, cross 0.45 μm of millipore filtration, carry out the preparation of one dimension liquid chromatography.One dimension liquid chromatography adopts InnovelC18150 × 250m, 10 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, type of elution: the degree such as degree such as B phase concentration 25% grade 10min, B phase concentration 35% 20min, B phase concentration 100% etc. spends 10min.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, and sample size is 150mL/ pin, and flow rate of mobile phase is 600mL/min, collects the cut of 23-25 minute, carries out rotary evaporation and be concentrated into dry, for one dimension prepares brucine crude product.Methanol-water (0.1% acetic acid) solubilize brucine crude product with 50%, concentration is 80mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, and chromatographic column is China spectrum C1820 × 250mm, 5 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, employing 33%B equality 45 minutes wash-outs.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, and sample size is 1000 μ L/ pins, flow rate of mobile phase is 16mL/min, collects the cut of 18-20 minute, rotary evaporated to dryness, obtain object component, through liquid-phase chromatographic analysis, purity is 98.8%; In one dimension preparation, the content of brucine crude product is 13%.After measured, this object product is brucine.
Embodiment 3:
With nux vomica sporophore for raw material, alcohol immersion 2h is used after grinds powder, filter, get precipitation repetition 2 times, merge gained filtrate, 5g is taken after revolving the nux vomica meal steaming and obtain drying, be dissolved in methanol-water (0.1% acetic acid) solution of 200mL60%, obtained Semen Strychni extract solution, concentration is 25mg/mL, cross 0.45 μm of millipore filtration, carry out the preparation of one dimension liquid chromatography.One dimension liquid chromatography adopts InnovelC18150 × 250mm, 10 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, type of elution: the degree such as degree such as B phase concentration 25% grade 10min, B phase concentration 35% 20min, B phase concentration 100% etc. spends 10min.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, and sample size is 150mL/ pin, and flow rate of mobile phase is 600mL/min, collects the cut of 23-25 minute, carries out rotary evaporation and be concentrated into dry, for one dimension prepares brucine crude product.Methanol-water (0.1% acetic acid) solubilize brucine crude product with 60%, concentration is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, and chromatographic column is China spectrum C1820 × 250mm, 5 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, employing 33%B equality 40 minutes wash-outs.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, sample size is 1000 μ L/ pins, flow rate of mobile phase is 16mL/min, collect the cut of 18-20 minute, rotary evaporated to dryness, obtains object component, carry out efficient liquid phase chromatographic analysis to the component obtained, purity is 98.5%; In one dimension preparation, the content of brucine crude product is 18%.After measured, this object product is brucine.
Embodiment 4:
With nux vomica sporophore for raw material, alcohol immersion 2h is used after grinds powder, filter, get precipitation repetition 3 times, merge gained filtrate, 20g is taken after revolving the nux vomica meal steaming and obtain drying, be dissolved in methanol-water (0.1% acetic acid) solution of 100mL60%, obtained Semen Strychni extract solution, concentration is 200mg/mL, cross 0.45 μm of millipore filtration, carry out the preparation of one dimension liquid chromatography.One dimension liquid chromatography adopts InnovelC18150 × 250mm, 10 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, type of elution: the degree such as degree such as B phase concentration 25% grade 10min, B phase concentration 35% 20min, B phase concentration 100% etc. spends 10min.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, and sample size is 150mL/ pin, and flow rate of mobile phase is 600mL/min, collects the cut of 23-25 minute, carries out rotary evaporation and be concentrated into dry, for one dimension prepares brucine crude product.Methanol-water (0.1% acetic acid) solubilize brucine crude product with 60%, concentration is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, and chromatographic column is China spectrum C1820 × 250mm, 5 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, employing 33%B equality 40 minutes wash-outs.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, sample size is 1000 μ L/ pins, flow rate of mobile phase is 16mL/min, collect the cut of 18-20 minute, rotary evaporated to dryness, obtains object component, carry out efficient liquid phase chromatographic analysis to the component obtained, purity is 99.1%; In one dimension preparation, the content of brucine crude product is 17%.After measured, this object product is brucine.
Embodiment 5:
With nux vomica sporophore for raw material, alcohol reflux 1.5h is used after grinds powder, filter, get precipitation and repeat extraction 2 times, merge gained filtrate, 16g is taken after revolving the nux vomica meal steaming and obtain drying, be dissolved in methanol-water (0.1% acetic acid) solution of 200mL65%, obtained Semen Strychni extract solution, concentration is 80mg/mL, cross 0.45 μm of millipore filtration, carry out the preparation of one dimension liquid chromatography.One dimension liquid chromatography adopts InnovelC18150 × 250mm, 10 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, type of elution: the degree such as degree such as B phase concentration 25% grade 10min, B phase concentration 35% 20min, B phase concentration 100% etc. spends 10min.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, and sample size is 150mL/ pin, and flow rate of mobile phase is 600mL/min, collects the cut of 23-25 minute, carries out rotary evaporation and be concentrated into dry, for one dimension prepares brucine crude product.Methanol-water (0.1% acetic acid) solubilize brucine crude product with 65%, concentration is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, and chromatographic column is China spectrum C1820 × 250mm, 5 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, employing 33%B equality 40 minutes wash-outs.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, sample size is 1000 μ L/ pins, flow rate of mobile phase is 16mL/min, collect the cut of 18-20 minute, rotary evaporated to dryness, obtains object component, carry out efficient liquid phase chromatographic analysis to the component obtained, purity is 98.7%; In one dimension preparation, the content of brucine crude product is 19%.After measured, product is target product brucine.
Embodiment 6:
With nux vomica sporophore for raw material, alcohol reflux 1.5h is used after grinds powder, filter, get precipitation and repeat extraction 2 times, merge gained filtrate, 40g is taken after revolving the nux vomica meal steaming and obtain drying, be dissolved in methanol-water (0.1% acetic acid) solution of 1000mL70%, obtained Semen Strychni extract solution, concentration is 40mg/mL, cross 0.45 μm of millipore filtration, carry out the preparation of one dimension liquid chromatography.One dimension liquid chromatography adopts InnovelC18150 × 250mm, 10 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), B phase is methyl alcohol, type of elution: the degree such as degree such as B phase concentration 25% grade 10min, B phase concentration 35% 20min, B phase concentration 100% etc. spends 10min.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, and sample size is 150mL/ pin, and flow rate of mobile phase is 600mL/min, collects the cut of 23-25 minute, carries out rotary evaporation and be concentrated into dry, for one dimension prepares brucine crude product.Methanol-water (0.1% acetic acid) solubilize brucine crude product with 70%, concentration is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography preparation, and chromatographic column is China spectrum C1820 × 250mm, 5 μm,
what moving phase adopted be the mixing of binary liquid phase, and wherein binary liquid phase A phase is water (0.1% acetic acid), and B phase is methyl alcohol, employing 33%B equality 40 minutes wash-outs.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, sample size is 1000 μ L/ pins, flow rate of mobile phase is 16mL/min, collect the cut of 18-20 minute, rotary evaporated to dryness, obtains object component, carry out efficient liquid phase chromatographic analysis to the component obtained, purity is 98.9%; In one dimension preparation, the content of brucine crude product is 21%.After measured, this object product is brucine.
The high performance preparative liquid chromatography collection of illustrative plates of embodiment 2-6 is similar to Example 1, the analysis mode high-efficient liquid phase chromatogram of the object component obtained,
1hNMR spectrogram and
13cNMR spectrogram and embodiment 1 together, are limit by length and are not just enumerated display one by one here.
The material that the embodiment of the present invention relates to, reagent and experimental installation, if no special instructions, be the commercially available prod meeting effective ingredients in plant and extract.
The above, be only the preferred embodiments of the present invention, should be understood that; for those skilled in the art; under the prerequisite not departing from core technology of the present invention, can also make improvements and modifications, these improvements and modifications also should belong to scope of patent protection of the present invention.Any change in the implication suitable with claims of the present invention and scope, all should think to be included in the scope of claims.
Claims (5)
1. an extracting method for brucine, is characterized in that comprising the following steps:
(1) with nux vomica sporophore for raw material, after grinds powder, by alcohol immersion or refluxing extraction, filter, gained filtrate carries out rotary evaporated to dryness, dissolves, obtain the extracting solution of solid content 50-200mg/mL with the methanol-water binary mixed solvent that methyl alcohol volume fraction is 50-70%;
(2) carry out the separation of one dimension liquid chromatography to the extracting solution obtained: the chromatographic column of employing is C18 chromatographic column, and moving phase is two end number mixing liquid phase, wherein A phase is water, B phase is methyl alcohol; Sample size is 50-200mL/ pin; Flow rate of mobile phase is 100-1000mL/min; Detector is UV-detector, determined wavelength 200-260nm; Type of elution is B phase concentration 25% isocratic elution 10min, B phase concentration 35% isocratic elution 20min, B phase concentration 100% isocratic elution 10min; Collect object component according to ultra-violet absorption spectrum, rotary evaporated to dryness, obtains one dimension liquid phase component;
(3) dissolve one dimension liquid phase component with the methanol-water binary mixed solvent that methyl alcohol volume fraction is 50-70%, being dissolved to solid content is 50-100mg/mL;
(4) carry out two-dimensional HPLC separation: the chromatographic column of employing is C18 chromatographic column, and moving phase is two end number mixing liquid phase, wherein A phase is water, B phase is methyl alcohol; Sample size is 500-2000 μ L/ pin; Flow rate of mobile phase is 10-30mL/min; Detector is UV-detector, determined wavelength 200-260nm; Type of elution is B phase concentration 33% isocratic elution 40-50min; Collect object component according to ultra-violet absorption spectrum, rotary evaporation is concentrated into dry, obtains target product brucine.
2. the extracting method of a kind of brucine according to claim 1, is characterized in that: in described step (1), (3), methanol-water binary mixed solvent is containing 0.1% acetic acid.
3. the extracting method of a kind of brucine according to claim 1, is characterized in that: in described step (2), (4), the A phase of two end number mixing liquid phase is containing 0.1% acetic acid.
4. the extracting method of a kind of brucine according to claim 1, is characterized in that: in described step (2), C18 chromatographic column is specially anti-phase C18 axial pressure chromatographic column, and during use, column temperature is room temperature or 25-40 DEG C.
5. the extracting method of a kind of brucine according to claim 1, is characterized in that: in described step (4), C18 chromatographic column is specially China spectrum C18 chromatographic column, and during use, column temperature is room temperature or 25-40 DEG C.
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