CN104447778B - A kind of preparation method of 4-hydroxyl icajine - Google Patents
A kind of preparation method of 4-hydroxyl icajine Download PDFInfo
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- CN104447778B CN104447778B CN201410707741.3A CN201410707741A CN104447778B CN 104447778 B CN104447778 B CN 104447778B CN 201410707741 A CN201410707741 A CN 201410707741A CN 104447778 B CN104447778 B CN 104447778B
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- RNZRHJNFQWMXHB-UHFFFAOYSA-N N-methyl-sec-pseudostrychnine Natural products O1CC=C(C2CC3=O)CN(C)CCC43C3=CC=CC=C3N3C(=O)CC1C2C34 RNZRHJNFQWMXHB-UHFFFAOYSA-N 0.000 title claims abstract description 56
- ZMTRTSSBHBKGMR-UHFFFAOYSA-N Pseudostrychnin Natural products O=C1CC2OCC=C3CNCCC45C(C2C3CC4=O)N1c6ccccc56 ZMTRTSSBHBKGMR-UHFFFAOYSA-N 0.000 title claims abstract description 56
- PKPARRIYUZPZFV-LBPSXOOBSA-N icajine Natural products CN1CC[C@]23[C@@H]4[C@H]5[C@@H](CC2=O)C(=CCOC5=CC(=O)N4c6ccccc36)C1 PKPARRIYUZPZFV-LBPSXOOBSA-N 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- 210000000582 semen Anatomy 0.000 claims abstract description 27
- 238000010828 elution Methods 0.000 claims abstract description 11
- 238000004780 2D liquid chromatography Methods 0.000 claims abstract description 10
- -1 hydroxyl icajine Chemical compound 0.000 claims abstract description 6
- 238000000746 purification Methods 0.000 claims abstract description 4
- 239000012071 phase Substances 0.000 claims description 77
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 60
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 31
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 24
- 208000035126 Facies Diseases 0.000 claims description 23
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 17
- 239000007791 liquid phase Substances 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 14
- 238000002390 rotary evaporation Methods 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- 238000000862 absorption spectrum Methods 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- 239000012046 mixed solvent Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 2
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 2
- 238000010992 reflux Methods 0.000 claims 1
- 238000010898 silica gel chromatography Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 238000002955 isolation Methods 0.000 abstract description 2
- 239000006166 lysate Substances 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 229930014626 natural product Natural products 0.000 abstract description 2
- 238000005457 optimization Methods 0.000 abstract description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract 2
- 239000012043 crude product Substances 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- 239000000377 silicon dioxide Substances 0.000 description 14
- GCFHZZWXZLABBL-UHFFFAOYSA-N ethanol;hexane Chemical compound CCO.CCCCCC GCFHZZWXZLABBL-UHFFFAOYSA-N 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 239000012982 microporous membrane Substances 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 229930013930 alkaloid Natural products 0.000 description 3
- 230000000202 analgesic effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- RNZRHJNFQWMXHB-ZXXLSYNSSA-N icajine Chemical compound O1CC=C([C@@H]2CC3=O)CN(C)CC[C@]43C3=CC=CC=C3N3C(=O)C[C@H]1[C@H]2[C@H]34 RNZRHJNFQWMXHB-ZXXLSYNSSA-N 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000050051 Chelone glabra Species 0.000 description 1
- 102000009660 Cholinergic Receptors Human genes 0.000 description 1
- 108010009685 Cholinergic Receptors Proteins 0.000 description 1
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 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
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000004262 preparative liquid chromatography Methods 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Cosmetics (AREA)
Abstract
The present invention relates to the preparation method of a kind of 4 hydroxyl icajines, use two-dimensional liquid chromatography technology, 4 hydroxyl icajines are extracted from natural product Semen Strychni sporophore, pass through chromatographic column, lysate, flow equal reasonable selection, flow velocity, type of elution, collect the optimization of the process conditions such as section, make whole isolation and purification method practical, easy and simple to handle, automation equipment degree is high, just can carry out under normal temperature and pressure, repeatability is high, between the material composition batch obtained, concordance is high, operability is good, and the 4 hydroxyl icajine purity height obtained, more than 99% can be reached, can directly apply to commercial production.
Description
Technical field
The present invention relates to production of chemicals field, the preparation method of a kind of 4-hydroxyl icajine.
Background technology
Alkaloid compound is widely distributed in Chinese medicine animal drugs, and its chemical constitution complexity is various, biological alive
Property is notable.The alkaloid of some novel structures has noticeable physiology and pharmacologically active, it may be possible to control
Treat the important sources of series of human major disease medicine.4-hydroxyl icajine is exactly such a kind of alkaloid
Compounds, is a kind of important active skull cap components being widely present in plant, and its structure is as follows:
Having now been found that 4-hydroxyl icajine has a following effect: first, it is to the cell of proliferation and differentiation and tumor
Cell has its proliferation and differentiation of suppression and Cytotoxic effect, inducing cell apoptosis, and therefore, 4-hydroxyl depends on
The activity of card essence suppression tumor.Second, whole central nervous system has excitation, and improves corticocerebral
Sensory centre function.3rd, analgesic activity: experiment shows the writhing method with mouse peritoneal injection acetic acid, mice
Intramuscular injection, the ED50 of analgesic activity is that 12.68mg/Kg. hot plate method in mice proves there is analgesic activity.Its analgesia
Effect has certain contacting with M cholinoceptor.
Up to the present, the method carrying disubstituted-4-hydroxy icajine in natural extracts has been reported less,
And comparatively laborious, not to mention extract high-purity or monomer 4-hydroxyl icajine, this is in certain journey
Also counteracts that on degree that 4-hydroxyl icajine is as the progress in terms of tcm development.
Summary of the invention
The technical problem to be solved is to provide the preparation method of a kind of 4-hydroxyl icajine.
For solving above-mentioned technical problem, the technical scheme is that
The preparation method of a kind of 4-hydroxyl icajine, specifically comprises the following steps that
(1), with Semen Strychni sporophore as raw material, after grinds powder, soak by ethyl acetate or backflow carries
Taking 1-2h, extract 1-3 time, filter, gained filtrate carries out rotary evaporated to dryness, with volume fraction of ethanol is
The ethanol of 20-50%-normal hexane binary mixed solvent dissolves, and obtaining Semen Strychni concentration is 50-200mg/mL's
Extracting solution；
(2) extracting solution obtained is carried out liquid chromatograph separation: the flowing of employing is two end number mixing organic facies mutually,
Wherein A phase is normal hexane, and B phase is ethanol；Sample size is 1000-5000 μ L mL/ pin；Flow rate of mobile phase is
50-200mL/min；Detector is UV-detector, detects wavelength 200-260nm；Type of elution: B phase is dense
Spend 0% isocratic 5min, B phase concentration 0%-64% gradient 5-35min, B phase concentration 64%-100% gradient 35-45min；
Collect peak 7 (retention time 28-33min) therein according to ultra-violet absorption spectrum to steam as purpose component, rotation
Send to do, obtain one-dimensional liquid phase component；
(3) one-dimensional liquid phase is dissolved with the ethanol that volume fraction of ethanol is 25-50%-normal hexane binary mixed solvent
Component, being dissolved to concentration is 50-100mg/mL；
(4) two-dimensional HPLC separation is carried out: the flowing of employing is mutually for two end number mixing organic facies, wherein A phase
Being normal hexane, B phase is ethanol；Sample size is 500-2000 μ L/ pin；Flow rate of mobile phase is 10-30mL/min；；
Detector is UV-detector, detects wavelength 200-260nm；Isocratic elution mode: 40%B equality 45min
Eluting；According to ultra-violet absorption spectrum collection 5 peaks (retention time 31-32min) therein as purpose component,
Rotary evaporation is concentrated to dryness, and obtains target product 4-hydroxyl icajine.
Preferably, the preparation method of above-mentioned 4-hydroxyl icajine, in described step (2), liquid chromatograph uses
Chromatographic column be purification on normal-phase silica gel axial pressure chromatographic column (Innovel silica 150 × 250mm, 10 μm, ), during use, column temperature is room temperature or 25-40 DEG C.
Preferably, the preparation method of above-mentioned 4-hydroxyl icajine, in described step (4), two-dimensional liquid chromatography is adopted
Chromatographic column be DAISO silica gel chromatographic column (DAISO Silica 20 × 250mm, 5 μm,),
During use, column temperature is room temperature or 25-40 DEG C.
The invention has the beneficial effects as follows:
The preparation method of above-mentioned 4-hydroxyl icajine, uses two-dimensional liquid chromatography technology, from natural product Semen Strychni
Sub-sporophore carries disubstituted-4-hydroxy icajine, by chromatographic column, lysate, equal rationally the selecting of flowing,
The optimization of the process conditions such as flow velocity, type of elution, collection section so that whole isolation and purification method conscientiously may be used
OK, easy and simple to handle, automation equipment degree is high, just can carry out under normal temperature and pressure, and repeatability is high, obtains
Between material composition batch, concordance is high, and operability is good, and the 4-hydroxyl icajine purity obtained is high, can
To reach more than 99%, can directly apply to commercial production.
Accompanying drawing explanation
Fig. 1 is to carry the one-dimensional of disubstituted-4-hydroxy icajine in embodiment 2 from Semen Strychni sporophore efficiently to prepare liquid
Phase chromatogram；
Fig. 2 is to carry the one-dimensional of disubstituted-4-hydroxy icajine in embodiment 2 from Semen Strychni sporophore efficiently to prepare liquid
The two-dimensional highly effective preparative liquid chromatography figure of phase chromatographic isolation gained fraction 7；
Fig. 3 is the analytical type high-efficient liquid phase chromatogram of the purpose component obtained in embodiment 2；
Fig. 4 is 4-hydroxyl icajine1HNMR spectrogram；
Fig. 5 is 4-hydroxyl icajine13CNMR spectrogram.
Detailed description of the invention
In order to make those skilled in the art be better understood from technical scheme, below in conjunction with the accompanying drawings and
Technical scheme of the present invention is described in further detail by detailed description of the invention.Except as otherwise noted, originally
All scientific and technical terms that invention uses have the phase being generally understood that with the technical field of the invention personnel
Same implication.
Embodiment 1:
With Semen Strychni sporophore as raw material, extract rotation by ethyl acetate after grinds powder and steam the horse obtaining being dried
Weigh 5g after Semen Strychni coarse powder, be dissolved in the ethanol-hexane solution of 100mL 20%, prepare Semen Strychni extract molten
Liquid, concentration is 50mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph and prepare.One-dimensional liquid phase
Chromatograph employing Innovel silica 150 × 250m, 10 μm,Flowing use mutually for organic facies
Mixing, wherein organic facies A phase is normal hexane, and B phase is ethanol, type of elution: the isocratic 5min of B phase concentration 0%,
B phase concentration 0%-64% gradient 5-35min, B phase concentration 64%-100% gradient 35-45min.Use UV-detector
210nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 5000 μ L/ pins, and flow rate of mobile phase is
80mL/min, collects the fraction of 28-33 minute, carries out rotary evaporation and is concentrated to dryness, for one-dimensional preparation 4-hydroxyl
Icajine crude product.Ethanol with 40%-hexane solution dissolves 4-hydroxyl icajine crude product, and concentration is 80mg/mL,
Through filtering with microporous membrane, carrying out two-dimensional liquid chromatography and prepare, chromatographic column is DAISO Silica 20 × 250mm,
5 μm,The mixing for organic facies that flowing uses mutually, wherein organic facies A phase is normal hexane, and B phase is
Ethanol, uses 45 minutes eluting of 40%B equality.UV-detector 210nm is used to select absorbing wavelength, preparation
Temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 16mL/min, collects 31-32 minute
Fraction, rotary evaporated to dryness, obtain 4-hydroxyl icajine compound, through liquid-phase chromatographic analysis, purity is
98.9%, in the one-dimensional 4-of preparation hydroxyl icajine crude product, the content of 4-hydroxyl icajine is 23%.
Embodiment 2:
With Semen Strychni sporophore as raw material, extract rotation by ethyl acetate after grinds powder and steam the horse obtaining being dried
Weigh 10g after Semen Strychni coarse powder, be dissolved in the ethanol-hexane solution of 100mL 30%, prepare Semen Strychni extract molten
Liquid, concentration is 100mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph and prepare.One-dimensional liquid
Phase chromatograph employing Innovel silica 150 × 250mm, 10 μm,Flowing use mutually for having
The mixing of machine phase, wherein organic facies A phase is normal hexane, and B phase is ethanol, type of elution: B phase concentration 0% is isocratic
5min, B phase concentration 0%-64% gradient 5-35min, B phase concentration 64%-100% gradient 35-45min.Use ultraviolet
Detector 210nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 5000 μ L/ pins, and flowing is flowed mutually
Speed is 80mL/min, collects the fraction of 28-33 minute, carries out rotary evaporation and is concentrated to dryness, for one-dimensional preparation
4-hydroxyl icajine crude product, result is as shown in Figure 1.Ethanol with 50%-hexane solution dissolves 4-hydroxyl according to card
Essence crude product, concentration is 100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, chromatographic column
For DAISO Silica 20 × 250mm, 5 μm,The mixing for organic facies that flowing uses mutually,
Wherein organic facies A phase is normal hexane, and B phase is ethanol, uses 45 minutes eluting of 40%B equality.Use ultraviolet
Detector 210nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 1000 μ L/ pins, and flow phase
Flow velocity is 16mL/min, collects the fraction of 31-32 minute, rotary evaporated to dryness, obtains 4-hydroxyl icajine
Compound, result is as shown in Figure 2.The component obtained is carried out efficient liquid phase chromatographic analysis, result such as Fig. 3 institute
Showing, purity is 99.4%, and in the one-dimensional 4-of preparation hydroxyl icajine crude product, the content of 4-hydroxyl icajine is 35%.
After measured, as illustrated in figures 4-5, product is target product 4-hydroxyl icajine.
Embodiment 3:
With Semen Strychni sporophore as raw material, extract rotation by ethyl acetate after grinds powder and steam the horse obtaining being dried
Weigh 5g after Semen Strychni coarse powder, be dissolved in the ethanol-hexane solution of 200mL 30%, prepare Semen Strychni extract molten
Liquid, concentration is 25mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph and prepare.One-dimensional liquid phase
Chromatograph employing Innovel silica 150 × 250mm, 10 μm,Flowing use mutually for organic
The mixing of phase, wherein organic facies A phase is normal hexane, and B phase is ethanol, type of elution: the isocratic 5min of B phase concentration 0%,
B phase concentration 0%-64% gradient 5-35min, B phase concentration 64%-100% gradient 35-45min.Use UV-detector
210nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 5000 μ L/ pins, and flow rate of mobile phase is 80
ML/min, collects the fraction of 28-33 minute, carries out rotary evaporation and is concentrated to dryness, and depends on for one-dimensional preparation 4-hydroxyl
Card essence crude product.Ethanol with 30%-hexane solution dissolves 4-hydroxyl icajine crude product, and concentration is 100mg/mL,
Through filtering with microporous membrane, carrying out two-dimensional liquid chromatography and prepare, chromatographic column is DAISO Silica 20 × 250mm,
5 μm,The mixing for organic facies that flowing uses mutually, wherein organic facies A phase is normal hexane, and B phase is
Ethanol, uses 45 minutes eluting of 40%B equality.UV-detector 210nm is used to select absorbing wavelength, preparation
Temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 16mL/min, collects 31-32 minute
Fraction, rotary evaporated to dryness, obtain 4-hydroxyl icajine compound, the component obtained carried out high-efficient liquid
Analysis of hplc, purity is 98.2%, one-dimensional prepares the content of 4-hydroxyl icajine in 4-hydroxyl icajine crude product
It is 18%.
Embodiment 4:
With Semen Strychni sporophore as raw material, extract rotation by ethyl acetate after grinds powder and steam the horse obtaining being dried
Weigh 20g after Semen Strychni coarse powder, be dissolved in the ethanol-hexane solution of 100mL 50%, prepare Semen Strychni extract molten
Liquid, concentration is 200mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph and prepare.One-dimensional liquid
Phase chromatograph employing Innovel silica 150 × 250mm, 10 μm,Flowing use mutually for having
The mixing of machine phase, wherein organic facies A phase is normal hexane, and B phase is ethanol, type of elution: B phase concentration 0% is isocratic
5min, B phase concentration 0%-64% gradient 5-35min, B phase concentration 64%-100% gradient 35-45min.Use ultraviolet
Detector 210nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 5000 μ L/ pins, and flowing is flowed mutually
Speed is 80mL/min, collects the fraction of 28-33 minute, carries out rotary evaporation and is concentrated to dryness, for one-dimensional preparation
4-hydroxyl icajine crude product.Ethanol with 50%-hexane solution dissolves 4-hydroxyl icajine crude product, and concentration is
100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, and chromatographic column is DAISO Silica
20 × 250mm, 5 μm,The mixing for organic facies that flowing uses mutually, wherein organic facies A phase is
Normal hexane, B phase is ethanol, uses 45 minutes eluting of 40%B equality.UV-detector 210nm is used to select
Absorbing wavelength, preparation temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 16mL/min,
Collect the fraction of 31-32 minute, rotary evaporated to dryness, obtain 4-hydroxyl icajine compound, to the group obtained
Dividing and carry out efficient liquid phase chromatographic analysis, purity is 99.1%, 4-hydroxyl in one-dimensional preparation 4-hydroxyl icajine crude product
The content of icajine is 27%.
Embodiment 5:
With Semen Strychni sporophore as raw material, extract rotation by ethyl acetate after grinds powder and steam the horse obtaining being dried
Weigh 16g after Semen Strychni coarse powder, be dissolved in the ethanol-hexane solution of 200mL 40%, prepare Semen Strychni extract molten
Liquid, concentration is 80mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph and prepare.One-dimensional liquid phase
Chromatograph employing Innovel silica 150 × 250mm, 10 μm,Flowing use mutually for organic
The mixing of phase, wherein organic facies A phase is normal hexane, and B phase is ethanol, type of elution: the isocratic 5min of B phase concentration 0%,
B phase concentration 0%-64% gradient 5-35min, B phase concentration 64%-100% gradient 35-45min.Use UV-detector
210nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 5000 μ L/ pins, and flow rate of mobile phase is 80
ML/min, collects the fraction of 28-33 minute, carries out rotary evaporation and is concentrated to dryness, and depends on for one-dimensional preparation 4-hydroxyl
Card essence crude product.Ethanol with 40%-hexane solution dissolves 4-hydroxyl icajine crude product, and concentration is 100mg/mL,
Through filtering with microporous membrane, carrying out two-dimensional liquid chromatography and prepare, chromatographic column is DAISO Silica 20 × 250mm,
5 μm,The mixing for organic facies that flowing uses mutually, wherein organic facies A phase is normal hexane, and B phase is
Ethanol, uses 45 minutes eluting of 40%B equality.UV-detector 210nm is used to select absorbing wavelength, preparation
Temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 16mL/min, collects 31-32 minute
Fraction, rotary evaporated to dryness, obtain 4-hydroxyl icajine compound, the component obtained carried out high-efficient liquid
Analysis of hplc, purity is 98.2%, one-dimensional prepares the content of 4-hydroxyl icajine in 4-hydroxyl icajine crude product
It is 29%.
Embodiment 6:
With Semen Strychni sporophore as raw material, extract rotation by ethyl acetate after grinds powder and steam the horse obtaining being dried
Weigh 40g after Semen Strychni coarse powder, be dissolved in the ethanol-hexane solution of 1000mL 25%, prepare Semen Strychni extract
Solution, concentration is 40mg/mL, crosses 0.45 μm microporous filter membrane, carries out one-dimensional liquid chromatograph and prepare.One-dimensional liquid
Phase chromatograph employing Innovel silica 150 × 250mm, 10 μm,Flowing use mutually for having
The mixing of machine phase, wherein organic facies A phase is normal hexane, and B phase is ethanol, type of elution: B phase concentration 0% is isocratic
5min, B phase concentration 0%-64% gradient 5-35min, B phase concentration 64%-100% gradient 35-45min.Use ultraviolet
Detector 210nm selects absorbing wavelength, and preparation temperature is room temperature, and sample size is 5000 μ L/ pins, and flowing is flowed mutually
Speed is 80mL/min, collects the fraction of 28-33 minute, carries out rotary evaporation and is concentrated to dryness, for one-dimensional preparation
4-hydroxyl icajine crude product.Ethanol with 30%-hexane solution dissolves 4-hydroxyl icajine crude product, and concentration is
100mg/mL, through filtering with microporous membrane, carries out two-dimensional liquid chromatography and prepares, and chromatographic column is DAISO Silica
20 × 250mm, 5 μm,The mixing for organic facies that flowing uses mutually, wherein organic facies A phase is
Normal hexane, B phase is ethanol, uses 45 minutes eluting of 40%B equality.UV-detector 210nm is used to select
Absorbing wavelength, preparation temperature is room temperature, and sample size is 1000 μ L/ pins, and flow rate of mobile phase is 16mL/min,
Collect the fraction of 31-32 minute, rotary evaporated to dryness, obtain 4-hydroxyl icajine compound, to the group obtained
Dividing and carry out efficient liquid phase chromatographic analysis, purity is 98.9%, 4-hydroxyl in one-dimensional preparation 4-hydroxyl icajine crude product
The content of icajine is 21%.
Person of ordinary skill in the field should note, and the technical method of the present invention is not limited only to from Semen Strychni
Son carries disubstituted-4-hydroxy icajine, it is also possible to be applied to the extraction of 4-hydroxyl icajine in other medical material.
Material, reagent and the experimental facilities that the present embodiments relate to, if no special instructions, is and meets
The common commercially available prod of field of medicine preparation.
Above-mentioned retouch in detail what the preparation method of this kind of 4-hydroxyl icajine was carried out with reference to detailed description of the invention
State, be illustrative rather than determinate, can according to restriction scope list several embodiments, because of
This changing and modifications under without departing from present general inventive concept, within should belonging to protection scope of the present invention.
Claims (3)
1. the preparation method of a 4-hydroxyl icajine, it is characterised in that: specifically comprise the following steps that
(1) with Semen Strychni sporophore as raw material, after grinds powder, soak by ethyl acetate
Or reflux, extract, 1-2h, extracts 1-3 time, filtering, gained filtrate carries out rotary evaporation extremely
Dry, dissolve with the ethanol that volume fraction of ethanol is 20-50%-normal hexane binary mixed solvent,
It is the extracting solution of 50-200mg/mL to Semen Strychni concentration；
(2) extracting solution obtained is carried out liquid chromatograph separation: the flowing of employing is mutually for binary
Mixing organic facies, wherein A phase is normal hexane, and B phase is ethanol；Sample size is 1000-5000 μ L
/ pin；Flow rate of mobile phase is 50-200mL/min；Detector is UV-detector, detects wavelength
200-260nm；Type of elution: B phase concentration 0% isocratic 5min, B phase concentration 0%-64% gradient
5-35min, B phase concentration 64%-100% gradient 35-45min；It is collected according to ultra-violet absorption spectrum
In peak 7 as purpose component, its retention time is 28-33min, and rotary evaporated to dryness obtains
One-dimensional liquid phase component；
(3) dissolve with the ethanol that volume fraction of ethanol is 25-50%-normal hexane binary mixed solvent
One-dimensional liquid phase component, being dissolved to concentration is 50-100mg/mL；
(4) two-dimensional HPLC separation is carried out: the flowing of employing is two end number mixing organic facies mutually,
Wherein A phase is normal hexane, and B phase is ethanol；Sample size is 500-2000 μ L/ pin；Flowing is flowed mutually
Speed is 10-30mL/min；Detector is UV-detector, detects wavelength 200-260nm；Deng
Degree type of elution: 40%B equality 45min eluting；Collect therein according to ultra-violet absorption spectrum
Peak 5 is as purpose component, and its retention time is 31-32min, and rotary evaporation is concentrated to dryness,
To target product 4-hydroxyl icajine.
The preparation method of 4-hydroxyl icajine the most according to claim 1, it is characterised in that:
The chromatographic column that in described step (2), liquid chromatograph uses is purification on normal-phase silica gel axial pressure chromatographic column,
During use, column temperature is 25-40 DEG C.
The preparation method of 4-hydroxyl icajine the most according to claim 1, its feature exists
In: the chromatographic column that in described step (4), two-dimensional liquid chromatography uses is DAISO silica gel chromatography
Post, during use, column temperature is 25-40 DEG C.
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