CN105524073A - Method for extracting brucine N-oxide from nux vomica - Google Patents
Method for extracting brucine N-oxide from nux vomica Download PDFInfo
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- CN105524073A CN105524073A CN201510915496.XA CN201510915496A CN105524073A CN 105524073 A CN105524073 A CN 105524073A CN 201510915496 A CN201510915496 A CN 201510915496A CN 105524073 A CN105524073 A CN 105524073A
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- 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
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Abstract
The invention provides a method for extracting brucine N-oxide from nux vomica. The method comprises the following steps: grinding nux vomica into powder, immersing the nux vomica powder with ethanol or carrying out reflux extraction, drying a filtrate by distillation and dissolving with methanol-water solution; carrying out one-dimensional liquid phase separation, adopting a C18 chromatographic column, A phase water and B phase methanol binary liquid phase, carrying out 25% B-phase isocratic elution for 10 min, carrying out 35% B-phase isocratic elution for 20 min, carrying out 100% B-phase isocratic elution for 10 min, collecting 13.5-15.5min eluate, drying by distillation, and dissolving with methanol-water solution; carrying out two-dimensional liquid-phase separation, adopting a C18 chromatographic column, A phase water and B phase methanol binary liquid phase, carrying out 30% B-phase isocratic elution, collecting 17.5-20 min eluate, and drying by distillation to obtain brucine N-oxide with purity being 99%. The method can be directly applied to industrial production and provides source guarantee for potential role development of brucine N-oxide.
Description
Technical field
The present invention relates to technical field of extraction of Chinese traditional medicine, be specifically related to a kind of method extracting NSC 118079 from nux vomica.
Background technology
NSC 118079 is a kind of compound in Chinese medicine nux vomica, and structural formula is as follows:
Now the separation and Extraction of NSC 118079 is limited by the non-mechanized operating method of traditional Chinese medicine, complex steps, consuming time, effort, the compound amount obtained few and batch between difference large, be difficult to carry out clinical study.
In addition for NSC 118079, because in plant, content is few, its application also few people's research, therefore its latent effect has to be developed.
Summary of the invention
Technical problem to be solved by this invention is for above prior art problem, provides a kind of method extracting NSC 118079 from Chinese medicine nux vomica, and the method adopts high performance liquid chromatography to carry out separation and purification, separation method is stablized, repeatability is high, and preparation amount is large, simple to operately saves time.
The technical solution adopted in the present invention is:
From nux vomica, extract a method for NSC 118079, 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 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 13.5-15.5min elutriant 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 30% isocratic elution 20-40min; Collect 17.5-20min elutriant according to ultra-violet absorption spectrum, rotary evaporation is concentrated into dry, obtains NSC 118079 of the present invention.
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 nux vomica oxynitride 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, the nux vomica oxynitride purity obtained can reach 99%, and extract batch between consistence high, operability is good, degree of controllability is high, directly can apply to industrial production, for the latent effect exploitation of NSC 118079 provides source to ensure.
Accompanying drawing explanation
Shown in Fig. 1 is the one dimension chromatographic fractionation figure that the present invention extracts the method for NSC 118079 from nux vomica;
Shown in Fig. 2 is the Two way chromatograms separation graph that the present invention extracts the method for NSC 118079 from nux vomica;
Shown in Fig. 3 is that the HPLC of the object product that the present invention obtains tests pure figure;
Shown in Fig. 4 is the H of the object product that the present invention obtains
1nMR nuclear-magnetism figure;
Shown in Fig. 5 is the C of the object product that the present invention obtains
13nMR nuclear-magnetism figure.
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 13.5-15.5 minute (in Fig. 1 No. 3 peaks), carrying out rotary evaporation is concentrated into dry, for one dimension prepares crude product.Methanol-water (0.1% acetic acid) solubilize one dimension with 50% prepares crude product, and concentration is 50mg/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 30%B equality wash-out 30 minutes.Adopt UV-detector, select 210nm absorbing wavelength, preparation temperature is room temperature, sample size is 2000 μ L/ pins, and flow rate of mobile phase is 16mL/min, collects the cut at 17.5-20 minute (in Fig. 2 M-Et-3-3 peak), rotary evaporated to dryness, obtains object M-Et-3-3 component.
Carry out HPLC purity check to the M-Et-3-3 component obtained, detect purity through high performance liquid phase and reach 99%, be single compound, HPLC spectrogram is shown in Fig. 3.
Carry out the qualification such as NMR hydrogen spectrum, NMR carbon spectrum to the component obtained, as shown in Figure 4,5, show this M-Et-3-3 component is NSC 118079 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, 15g 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 150mg/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 180mL/ pin, and flow rate of mobile phase is 600mL/min, collects the cut of 13.5-15.5 minute, carries out rotary evaporation and be concentrated into dry, for one dimension prepares vauqueline crude product.Methanol-water (0.1% acetic acid) solubilize vauqueline crude product with 55%, 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 30%B equality 30 minutes wash-outs.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 16mL/min, and collect the cut of 17.5-20 minute, rotary evaporated to dryness, obtains object component.
Carry out HPLC purity check to the component obtained, detecting purity through high performance liquid phase and reach 99%, is single compound.
Carry out the qualification such as NMR hydrogen spectrum, NMR carbon spectrum to the component obtained, it is NSC 118079 that result shows this component.
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 100mL60%, 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 × 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 100mL/ pin, and flow rate of mobile phase is 600mL/min, collects the cut of 13.5-15.5 minute, carries out rotary evaporation and be concentrated into dry, for one dimension prepares vauqueline crude product.Methanol-water (0.1% acetic acid) solubilize vauqueline 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 30%B equality 30 minutes wash-outs.Adopt UV-detector 210nm Selective absorber wavelength, preparation temperature is room temperature, and sample size is 500 μ L/ pins, and flow rate of mobile phase is 16mL/min, and collect the cut of 17.5-20 minute, rotary evaporated to dryness, obtains object component.
Carry out HPLC purity check to the component obtained, detecting purity through high performance liquid phase and reach 99%, is single compound.
Carry out the qualification such as NMR hydrogen spectrum, NMR carbon spectrum to the component obtained, it is NSC 118079 that result shows this component.
The material that the embodiment of the present invention relates to, reagent and experimental installation, if no special instructions, be the common commercially available prod meeting traditional Chinese medicine extraction.
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. from nux vomica, extract a method for NSC 118079, it 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 13.5-15.5min elutriant 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 30% isocratic elution 20-40min; Collect 17.5-20min elutriant according to ultra-violet absorption spectrum, rotary evaporation is concentrated into dry, obtains NSC 118079 of the present invention.
2. the method extracting NSC 118079 from nux vomica 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 method extracting NSC 118079 from nux vomica 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 method extracting NSC 118079 from nux vomica 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 method extracting NSC 118079 from nux vomica 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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| CN201510915496.XA CN105524073A (en) | 2015-12-09 | 2015-12-09 | Method for extracting brucine N-oxide from nux vomica |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101804096A (en) * | 2010-02-08 | 2010-08-18 | 南京中医药大学 | Method for extracting purified total alkaloid of strychnos nux-vomica and application thereof in pharmacy |
| CN102432618A (en) * | 2011-12-08 | 2012-05-02 | 南京海昌中药集团有限公司 | Preparation process for separating and purifying strychnine from total alkali of nux vomica |
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2015
- 2015-12-09 CN CN201510915496.XA patent/CN105524073A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101804096A (en) * | 2010-02-08 | 2010-08-18 | 南京中医药大学 | Method for extracting purified total alkaloid of strychnos nux-vomica and application thereof in pharmacy |
| CN102432618A (en) * | 2011-12-08 | 2012-05-02 | 南京海昌中药集团有限公司 | Preparation process for separating and purifying strychnine from total alkali of nux vomica |
Non-Patent Citations (6)
| Title |
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| 孙丽荣等: "油炸马钱子的生物碱成分研究", 《红河学院学报》 * |
| 林学义、苏友新: "马钱子中生物碱成分及其分离纯化工艺研究进展", 《福建中医药》 * |
| 沈保家等: "二维色谱技术及其在中药领域中的应用", 《中国科学：化学》 * |
| 潘自皓等: "基于5种生物碱定量分析的马钱子总碱提取工艺优化", 《食品与生物技术学报》 * |
| 陈晓莉等: "HPLC法测定马钱子缓释片中士的宁与马钱子碱", 《哈尔滨商业大学学报（自然科学版）》 * |
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