CN101318981B - Method for separating benzoxazole oxazinone glycoside compounds from acanthus - Google Patents
Method for separating benzoxazole oxazinone glycoside compounds from acanthus Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 14
- -1 benzoxazole oxazinone glycoside compounds Chemical class 0.000 title abstract description 5
- 229930182470 glycoside Natural products 0.000 title description 3
- 241000207965 Acanthaceae Species 0.000 title 1
- 150000001875 compounds Chemical class 0.000 claims abstract description 50
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical group CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 49
- 239000002904 solvent Substances 0.000 claims abstract description 45
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000000203 mixture Substances 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000012488 sample solution Substances 0.000 claims abstract description 10
- 244000153294 Acanthus ilicifolius Species 0.000 claims abstract description 7
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical group CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 claims abstract description 4
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical group CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229940093499 ethyl acetate Drugs 0.000 claims abstract description 3
- JMMWKPVZQRWMSS-UHFFFAOYSA-N isopropanol acetate Natural products CC(C)OC(C)=O JMMWKPVZQRWMSS-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229940011051 isopropyl acetate Drugs 0.000 claims abstract description 3
- GWYFCOCPABKNJV-UHFFFAOYSA-N isovaleric acid Chemical compound CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 claims abstract description 3
- YKYONYBAUNKHLG-UHFFFAOYSA-N n-Propyl acetate Natural products CCCOC(C)=O YKYONYBAUNKHLG-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229940090181 propyl acetate Drugs 0.000 claims abstract description 3
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000284 extract Substances 0.000 claims description 16
- 230000014759 maintenance of location Effects 0.000 claims description 14
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 9
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 5
- 238000005070 sampling Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 abstract description 6
- 238000010262 high-speed countercurrent chromatography Methods 0.000 abstract description 4
- 239000003960 organic solvent Substances 0.000 abstract description 2
- 230000000144 pharmacologic effect Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract 1
- 229940035429 isobutyl alcohol Drugs 0.000 abstract 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- OUSLYTBGQGKTME-UHFFFAOYSA-N DIBOA-Glc Chemical compound OC1C(O)C(O)C(CO)OC1OC1C(=O)N(O)C2=CC=CC=C2O1 OUSLYTBGQGKTME-UHFFFAOYSA-N 0.000 description 15
- 239000012071 phase Substances 0.000 description 14
- PYQSUTLVBSTCSK-UHFFFAOYSA-N Blepharin Chemical compound OC1C(O)C(O)C(CO)OC1OC1C(=O)NC2=CC=CC=C2O1 PYQSUTLVBSTCSK-UHFFFAOYSA-N 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 239000012074 organic phase Substances 0.000 description 8
- BCMCBBGGLRIHSE-UHFFFAOYSA-N 1,3-benzoxazole Chemical compound C1=CC=C2OC=NC2=C1 BCMCBBGGLRIHSE-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
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- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a method for separating a benzoxazine ketone compound (R is H or OH) as shown in a formula (1) from Acanthus ilicifolius L. The method is characterized in that: an upper phase of a solvent system consisting of three compositions a, b and c according to a volume ratio of 2-5 to 3-6 to 5-10; a lower phase of the solvent system is used as a mobile phase, wherein, the composition a is ethyl acetate, propyl acetate or isopropyl acetate; the composition b is normal butanol, isobutyl alcohol or pentanol; the composition c is water or a hartshorn; a normal butanol extractant of the Acanthus ilicifolius L is dissolved in the solvent system to prepare a sample solution; flow fractions are detected by an ultraviolet spectrograph through a high-speed countercurrent chromatography and collected according to a chromatographic peak, and the compound with the formula (1) is obtained, wherein R represents H or OH. The method can save mass organic solvents; a separation processonly needs 3 to 6 hours; the produced compound has a purity of over 80 percent; and the product can be directly used for standard chromatograpical samples, pharmacological experiments or business applications.
Description
Technical field
The present invention relates to a kind of method of from Lao Shu le (Acanthus ilicifolius L.), isolating benzoxazole zionoes compound, relate to a kind of (2R)-2-O-β-D-glucosyl group-2H-1 that utilizes high-speed countercurrent chromatography from Lao Shu le, to isolate following formula (1) expression specifically, 4-benzoxazole piperazine-3 (4H)-ketone and (2R)-2-O-β-D-glucosyl group-4-hydroxyl-2H-1,4-benzoxazole piperazine-3 (4H)-ketone.
Background technology
Lao Shu le is a per nnial herb, and complete stool all can be used as medicine, and cures mainly lymphadenitis, hepatitis, asthma etc.Pharmaceutical research shows, that the extract of Lao Shu le has is antiviral, anticancer, anti-oxidant, anti-mutation, protect the liver and antiphlogistic activity.From Lao Shu le, extract 6 kinds of benzoxazole zionoes compounds at present.This compounds is mainly played the part of in plant and is resisted disease and pest, by allelopathy and other plant competition, and the role of tethelin.Through research, these secondary metabolites can be made natural insecticide.The traditional column chromatography of main at present employing is separated benzoxazole zionoes compound from old mouse le, comprising the compound of following formula (1) expression, need relate to a plurality of column chromatographys and separate, (complex operation sees reference document: 1.Wu J, Zhang S, Xiao Q, Li Q, Huang J, Long L, Huang L.Phenylethanoid and aliphatic alcohol glycosidesfrom Acanthus ilicifolius.Phytochemistry, 2003,63 (4): 491-495.; 2.Kanchanapoom T, Kamel M S, Kasai R, Picheansoonthon C, Hiraga Y, Yamasaki K.Benzoxazinoid glucosidesfrom Acanthus ilicifolius.Phytochemistry, 2001,58 (4): 637-640; 3.Huo C-H, WangB, Lin W-H, Zhao Y-Y.Benzoxazinones from Acanthus ilicifolius.BiochemicalSystematics and Ecology, 2005,33 (6): 643-645.; 4.Francisco A, Mac í as D M, AlbertoO-B, David C, Simonet A M, Jos é M G M.Isolation and Synthesis of Allelochemicalsfrom Gramineae:Benzoxazinones and Related Compounds.J Agric Food Chem, 2006,54 (4): 991-1000.).
Formula (1)
Wherein R represents H or OH.
High-speed countercurrent chromatography has been widely used in the separation of crude substance, this technology does not need solid-state carrier, and the stationary phase of solvent systems can not produce irreversible adsorption to sample in the process, therefore can be used for the direct separation of crude extract, and obtains very high sample recovery rate.But the correlative study that also high-speed countercurrent chromatography is not used at present separating the benzimidazole dihydrochloride ketone compounds of Lao Shu le appears in the newspapers.
Summary of the invention
The objective of the invention is to develop a kind of method of from Lao Shu le, isolating benzoxazole zionoes compound quickly and easily.
We are by using sherwood oil successively with Lao Shu le, behind ethyl acetate and the n-butanol extraction, n-butyl alcohol extract is dissolved in by the ester class, in the upper and lower phase of the solvent systems that alcohols and water or ammoniacal liquor are formed, the last stationary phase of solvent systems that will be identical with above-mentioned composition as high-speed counter-current chromatograph, following to moving phase, detect (wavelength 254nm) with ultraviolet spectrometer, collect target product according to chromatographic peak, not only simple to operate, and compound (the 2R)-2-O-β-D-glucosyl group-2H-1 that separates by formula (1) expression, 4-benzoxazole piperazine-3 (4H)-ketone and (2R)-2-O-β-D-glucosyl group-4-hydroxyl-2H-1, the purity of 4-benzoxazole piperazine-3 (4H)-ketone is all more than 80%, even can be up to 97%, thereby realized purpose of the present invention.
A kind of method of from Lao Shu le, isolating benzoxazole oxazinone glycoside compounds of the present invention, to be full of the pipeline of high-speed counter-current chromatograph on the solvent systems mutually as stationary phase, after the pipeline rotation again with the following pipeline that pumps into mutually of solvent systems, after clarifying moving phase flows out from the pipeline tail end, inject sample solution by sampling valve, receive the effusive elutriant of pipeline tail end, detect with ultraviolet spectrometer, collect product according to chromatographic peak, it is characterized in that described solvent systems is by a, b, c three components are 2～5: 3～6: 5～10 compositions by volume, wherein a component is ethyl acetate or propyl acetate or isopropyl acetate, the b component is propyl carbinol or isopropylcarbinol or amylalcohol, the c component is water or volume fraction 0.5%～1.0% ammonia soln, described sample solution be with Lao Shu le with extraction using alcohol after, the evaporate to dryness extracting solution, extract mixture is suspended from the water, use sherwood oil successively, ethyl acetate and n-butanol extraction, with the n-butyl alcohol extract that obtains be dissolved in above-mentioned pipeline in solvent systems form in the identical solvent systems and obtain, the wavelength that described ultraviolet spectrometer detects is 254nm, described product is the compound of formula (1) expression, wherein R represents H or OH, when the c of described solvent systems component is ammonia soln, the product that obtains is isolating two compounds, wherein the retention time of the compound of R=H is 125～145min, and the retention time of the compound of R=OH is 100～105min; When the c of solvent systems component is water, the product that obtains is the mixture of two compounds, retention time is 115～130min, repeat once by above-mentioned step again, the c component that is described solvent systems is volume fraction 0.5%～1.0% ammoniacal liquor, the mixture of above-mentioned two compounds that obtain is dissolved in the described solvent systems makes sample solution, collect isolating two compounds at last, wherein the retention time of the compound of R=H is 130～145min, and the retention time of the compound of R=OH is 103～112min.The above high speed adverse current chromatogram process operation is all finished under 25～35 ℃ of temperature.
The column volume of the high-speed counter-current chromatograph that the present invention is used can be 250～5000mL.
Among the present invention with the c component of ammoniacal liquor as solvent systems, can isolate (2R)-2-O-β-D-glucosyl group-2H-1 with settling at one go, 4-benzoxazole piperazine-3 (4H)-ketone (claiming HBOA-Glc again) and (2R)-2-O-β-D-glucosyl group-4-hydroxyl-2H-1,4-benzoxazole piperazine-3 (4H)-ketone (claiming DIBOA-Glc again), velocity of separation is fast, the consumption solvent is few, and resulting HBOA-Glc purity is 90～95%, and DIBOA-Glc purity is 80～85%.And with the c component of water as solvent systems, could be though will go on foot fully with two compound separation by two, the purity of the HBOA-Glc that obtains can reach 95%～97%, and the purity of DIBOA-Glc can reach 93%～95%.Compare with traditional preparation method, the present invention can save a large amount of organic solvents, simultaneously preparation time was extremely shortened to 3～6 hours from original several days in several weeks, and HBOA-Glc that obtains and DIBOA-Glc purity height, can directly apply to chromatogram standard specimen, pharmacological evaluation or commercial use.
Description of drawings
The color atlas of two compounds of Fig. 1: embodiment 1 isolated formula (1) expression, wherein HBOA-Glc is represented at peak 1, peak 2 expression DIBOA-Glc.
The color atlas of the mixture of two compounds of the 1st isolated formula of step (1) expression of Fig. 2: embodiment 2, the wherein mixture of peak 1 expression HBOA-Glc and DIBOA-Glc.
The color atlas of two compounds of the 2nd isolated formula of step (1) expression of Fig. 3: embodiment 2, wherein HBOA-Glc is represented at peak 1, peak 2 expression DIBOA-Glc.
Fig. 4: (condition of gradient elution: eluting solvent is mixed by acetonitrile and water and forms the color atlas of the purity of the compound that the embodiment 1 and 2 that measures with HPLC obtains; 0～15 minute, volume fraction 10% acetonitrile; 15～35 minutes, 10%～70% acetonitrile; 35～40 minutes, 70%～10% acetonitrile, the UV-detector wavelength: 254nm), wherein peak 1 is HBOA-Glc, peak 2 is DIBOA-Glc; (1) the HPLC analysis of spectra of the n-butyl alcohol extract (not sample separation) of the Lao Shu le ethanol extraction of expression: the purity test result of the DIBOA-Glc that (2) expression embodiment 1 obtains; (3) the purity test result of the HBOA-Glc that obtains of expression embodiment 1; (4) the HPLC color atlas of two kinds of compounds in the mixture that obtains of expression embodiment 2 the first steps; (5) the purity test result of the DIBOA-Glc that obtains of expression embodiment 2; (6) the purity test result of the HBOA-Glc that obtains of expression embodiment 2.
Embodiment
Following examples are to further specify of the present invention, are not limitations of the present invention.
The organic reagent that is adopted among the embodiment is Tianjin company's all generations and produces, and analytical pure, HPLC are that Merck ﹠ Co., Inc. produces (Germany) with methyl alcohol, acetonitrile, and ammoniacal liquor (25～28% ammonia solution) is available from Guangzhou reagent company.
Embodiment 1:
With Lao Shu le over-ground part 1kg crushed after being dried, use under volume fraction 95% ethanol (5000mL) room temperature and extract 3 times, be suspended in the water behind the extracting solution evaporate to dryness, add petroleum ether extraction, separate organic phase (repeating 3 times) after the layering, add ethyl acetate extraction at aqueous phase, after the layering, separate organic phase (repeating 3 times), add n-butanol extraction at aqueous phase, separate organic phase (repeating 3 times), organic phase boils off solvent under vacuum after n-butyl alcohol extract 40g be stored in 4 ℃ standby, the HPLC analysis color atlas of this n-butyl alcohol extract is seen Fig. 4 (1).
With ethyl acetate, propyl carbinol and 0.5% ammoniacal liquor mixed in 2: 3: 5 by volume, obtained solvent systems 1000mL, and solvent is at room temperature abundant balance after mixing, and two-phase is separated before use.Get 100mg n-butyl alcohol extract sample dissolution in the above-mentioned solvent systems of 8mL, each 4mL of phase makes sample solution up and down.
(internal diameter of the tetrafluoroethylene pipeline of pipeline is 2mm with the spiral pipeline of self-equilibrating that is full of TBE-300A type high-speed counter-current chromatograph (with field biochemistry) on the remaining solvent systems mutually, column volume is 250mL, the axis of rotation of pipeline and the distance between the hollow shaft (R) are 9.5cm, chromatographic β value from the pipeline internal layer 0.46 to pipeline outer field 0.73, β=r/R, r are the distance of pipeline to the axis of rotation.System also is equipped with a TBP-50 type constant flow pump (with field biochemistry); LC757UV type UV-detector and flow-through cell (Shanghai converge emerging instrument company); HX-1050 type radiator valve (moral sky, Beijing friend instrument company) and N2010 chromatographic working station (Zhejiang University).
After spiral pipeline speed of rotation reaches 850r/min, inject the following phase of remaining solvent systems with the flow pump of 2.0mL/min from the head end of spiral pipeline.After clarifying moving phase flows out from tail end, and sign reaches the hydromeehanics balance, inject sample from spiral pipeline head end by sampling valve.The effusive elutriant of tail end detects with the wavelength of ultraviolet spectrometer at 254nm, collects compound (1) 6.3mg at retention time 125～145min, purity 92%, and retention time 100～105min. collects compound (2) 6.8mg, purity 80%.The compound spectral data is as follows after testing:
Compound (1): UV (MeOH) λ
Max251.1nm;
1H NMR (500MHz, MeOH-d
4): δ 5.77 (s, H-2), 6.95 (m, H-5), 7.05 (m, H-6,7), 7.11 (m, H-8), (3.21 dd, J=8.2,8.3Hz, H-2 '), 3.32-3.40 (m, H-3 ', 4 ', 5 '), (3.87 dd, J=12.0,2.5Hz, H-6 '), 3.70 (dd, J=11.9,4.7Hz, H-6 ');
13C NMR (125MHz, MeOH-d
4): δ 96.5 (C-2), 163.2 (C-3), 116.9 (C-5), 125.1 (C-6), 124.2 (C-7), 119.1 (C-8), 142.2 (C-9), 127.2 (C-10), 104.0 (C-1 '), 74.9 (C-2 '), 78.5 (C-3 '), 71.2 (C-4 '), 78.0 (C-5 '), 62.4 (C-6 ');
Compound (2): UV (MeOH) λ
Max254.6nm;
1H NMR (500MHz, MeOH-d
4): δ 5.96 (s, H-2), 7.38 (m, H-5), 709-7.16 (m, H-6,7,8), (3.20 dd, J=8.2,8.2Hz, H-2 '), 3.32-3.40 (m, H-3 ', 4 ', 5 '), (3.87 dd, J=12.0,2.5Hz, H-6 '), 3.69 (dd, J=11.9,4.7Hz, H-6 ');
13C NMR (125MHz, MeOH-d
4): δ 97.8 (C-2), 158.3 (C-3), 114.3 (C-5), 125.8 (C-6), 124.3 (C-7), 118.7 (C-8), 142.5 (C-9), 129.3 (C-10), 103.8 (C-1 '), 74.9 (C-2 '), 78.6 (C-3 '), 71.1 (C-4 '), 78.0 (C-5 '), 62.6 (C-6 ').
Above-mentioned data conform to bibliographical information, can confirm the compound of product compound 1 for formula (1) expression, wherein R represents H, i.e. (2R)-2-O-β-D-glucosyl group-2H-1,4-benzoxazole piperazine-3 (4H)-ketone (HBOA-Glc) and compound 2 are the compound of formula (1) expression, wherein R represents OH, i.e. (2R)-2-O-β-D-glucosyl group-4-hydroxyl-2H-1,4-benzoxazole piperazine-3 (4H)-ketone (DIBOA-Glc).The high speed adverse current chromatogram figure of above-mentioned sepn process record sees Fig. 3, and the HPLC spectrogram of analysis of compounds (1) and (2) purity is seen and seen Fig. 4 (3) and (2) respectively.
Embodiment 2:
With Lao Shu le over-ground part 1kg crushed after being dried, use under volume fraction 95% ethanol (5000mL) room temperature and extract 3 times, be suspended in the water behind the extracting solution evaporate to dryness, add petroleum ether extraction, separate organic phase (repeating 3 times) after the layering, add ethyl acetate extraction at aqueous phase, after the layering, separate organic phase (repeating 3 times), add n-butanol extraction at aqueous phase, separate organic phase (repeating 3 times), must n-butyl alcohol extract 40g organic phase boils off solvent under vacuum after be stored in 4 ℃ standby, the HPLC analysis chart of this n-butyl alcohol extract is seen Fig. 4 (1).
The first step: with ethyl acetate, propyl carbinol and water mixed in 2: 3: 5 by volume, obtained solvent systems 10000mL, and solvent is at room temperature abundant balance after mixing, and two-phase is separated before use.Get 1600mg n-butyl alcohol extract sample dissolution in the 120mL solvent systems, each 60mL of phase makes sample solution up and down.
The spiral pipeline (total column volume is 5000mL, and maximum sample size is 200mL) of the self-equilibrating of TBE-5000A type high-speed counter-current chromatograph (with field biochemistry) will be full of on the remaining solvent systems mutually.System also is equipped with a TBP-50 type constant flow pump (with field biochemistry); LC757UV type UV-detector and flow-through cell (Shanghai converge emerging instrument company); HX-1050 type radiator valve (moral sky, Beijing friend instrument company) and N2010 chromatographic working station (Zhejiang University).
After spiral pipeline speed of rotation reaches 550rpm, inject the following phase of remaining solvent systems with the flow pump of 20.0mL/min from the head end of spiral pipeline.After clarifying moving phase flows out from tail end, and sign reaches the hydromeehanics balance, inject sample by sampling valve.The effusive elutriant of tail end detects with the wavelength of ultraviolet spectrometer at 254nm, collect the target flow point at 115～130min, obtain the mixture of compound 1 and compound 2, the high speed adverse current chromatogram figure of above-mentioned sepn process record sees Fig. 2, and the HPLC of the mixture of two compounds of gained analyzes collection of illustrative plates and sees Fig. 4 (4).
Second step: with ethyl acetate, propyl carbinol and 1% ammonia soln mixed in 5: 6: 10 by volume, obtained solvent systems 10000mL, and solvent is at room temperature abundant balance after mixing, and two-phase is separated before use.Be dissolved in the 120mL solvent systems behind the mixture evaporate to dryness that the first step is obtained, each 60mL mutually makes sample solution up and down.
With being full of the spiral pipeline of the self-equilibrating of high-speed counter-current chromatograph on the remaining solvent systems mutually, after spiral pipeline speed of rotation reaches 850r/min, inject the following phase of remaining solvent systems with the flow pump of 20.0mL/min from the head end of spiral pipeline.After clarifying moving phase flows out from tail end, and sign reaches the hydromeehanics balance, inject sample by sampling valve.The effusive elutriant of tail end detects with the wavelength of ultraviolet spectrometer at 254nm, and collect at retention time 130～145min and obtain compound 1116mg, purity 97.1%, retention time 103～112min collects compound 2,86mg, purity 94.8%.The compound spectral data confirms that with embodiment 1 product compound 1 is the compound of formula (1) expression after testing, and wherein R represents H, and compound 2 is the compound of formula (1) expression, and wherein R represents OH.The high speed adverse current chromatogram figure of above-mentioned sepn process record sees Fig. 3, and the HPLC spectrogram of analysis of compounds (1) and (2) purity is seen Fig. 4 (6) and (5) respectively.
Claims (2)
1. method of from old mouse le (Acanthus ilicifolius L.), isolating the compound of following formula (1) expression,
Formula (1)
R represents H or OH in the formula, to be full of the pipeline of high-speed counter-current chromatograph on the solvent systems mutually, after the pipeline rotation again with the following pipeline that pumps into mutually of solvent systems, after clarifying moving phase flows out from the pipeline tail end, inject sample solution by sampling valve, receive the effusive elutriant of pipeline tail end, detect with ultraviolet spectrometer, collect product according to chromatographic peak, it is characterized in that described solvent systems is by a, b, c three components are 2～5: 3～6: 5～10 compositions by volume, wherein a component is ethyl acetate or propyl acetate or isopropyl acetate, the b component is propyl carbinol or isopropylcarbinol or amylalcohol, the c component is water or volume fraction 0.5%～1.0% ammonia soln, described sample solution be with Lao Shu le with extraction using alcohol after, evaporate to dryness extracting solution under the vacuum condition, extract mixture is suspended from the water, use sherwood oil successively, ethyl acetate and n-butanol extraction, with the n-butyl alcohol extract that obtains be dissolved in above-mentioned pipeline in solvent systems form in the identical solvent systems and obtain, the wavelength that described ultraviolet spectrometer detects is 254nm, described product is the compound of formula (1) expression, wherein R represents H or OH, when the c of described solvent systems component is volume fraction 0.5%～1.0% ammonia soln, the product that obtains is isolating two compounds, wherein the retention time of the compound of R=H is 125～145min, and the retention time of the compound of R=OH is 100～105min; When the c of solvent systems component is water, the product that obtains is the mixture of two compounds, retention time is 115～130min, repeat once by above-mentioned step again, the c component that is described solvent systems is volume fraction 0.5%～1.0% ammoniacal liquor, the mixture of above-mentioned two compounds that obtain is dissolved in the described solvent systems makes sample solution, collect isolating two compounds at last, wherein the retention time of the compound of R=H is 130～145min, and the retention time of the compound of R=OH is 103～112min.
2. method according to claim 1, the column volume that it is characterized in that described high-speed counter-current chromatograph is 250～5000mL.
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| Title |
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| Chang-Hong Huo et al..Benzoxazinones from Acanthus ilicifolius.《Biochemical Systematics and Ecology》.2005,第33卷 * |
| Tripetch Kanchanapoom et al..Benzoxazinoid glucosides from Acanthus ilicifolius.《Phytochemistry》.2001,第58卷 * |
| Tripetch Kanchanapoom et al..Megastigmane, aliphatic alcohol and benzoxazinoid glycosides from Acanthus ebracteatus.《Phytochemistry》.2001,第58卷 * |
| TripetchKanchanapoometal..Megastigmane aliphatic alcohol and benzoxazinoid glycosides from Acanthus ebracteatus.《Phytochemistry》.2001 |
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