CN101337882B - Method for purifying rupestonic acid and chrysosplenetin B by high speed reversed flow chromatography - Google Patents
Method for purifying rupestonic acid and chrysosplenetin B by high speed reversed flow chromatography Download PDFInfo
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Abstract
The invention relates to a method for purifying rupestonic acid and chrysosplenetin B by using high-speed counter-current chromatography, which comprises the following steps: disposing a separation solvent system in a tap funnel, fully shaking, standing for layering, selecting the upper phase as the fixed phase and the lower phase as the mobile phase, dissolving Radix Aconiti Brachypodi extract in the mixed solution of the upper phase and the lower phase, filling a chromatographic separation column with the fixed phase by a pump, connecting the head end of the column with a sampling valve, collecting the separated component when the mobile phase starts to flow out of the chromatographic column, respectively collecting different components, concentrating, drying, and separating to obtain monomer compounds of rupestonic acid and chrysosplenetin B above 95%.
Description
Technical field
The present invention relates to utilize high-speed countercurrent chromatography, from the natural phant that contains ketone acid and the plain second of golden waist, separate and prepare the method for high purity ketone acid and the plain second of golden waist.
Background technology
High-speed countercurrent chromatography (the High-speedcounter-current chromatography that grows up over nearly 20 years, HSCCC) be a kind of liquid luquid partition chromatography technology that does not have solid-state supporter, with light and handy polytetrafluoroethylhelix helix tube as separator column, stationary phase and moving phase all are liquid, in the time of the spiral tube rotation, do revolution motion around an outside axis that is parallel to the axis of rotation, so as to obtaining enough strong centrifuge field, under the prerequisite that is kept mutually in two-phase, the two-phase that forms stationary phase and moving phase is cut apart trend and convection current trend, carry out this cutting apart with the process of convection current continuously, if the inlet of wanting isolating sample from spiral tube is injected, successive distribution and transmission process will be carried out in tubing string, thereby realizes successive, liquid-liquid partition process efficiently.HSCCC separation efficiency height; Simple to operate, sample does not need strict pre-treatment; Can from extremely complicated rough sample, the separation of one step obtain highly purified component; The higher sample aspect with the absorption mutually that easily is fixed of analysis/separation viscosity has clear superiority; Can analyze/separating flavone, micromolecular compound such as alkaloid, quinones, lipoid, also can analyze/isolated polypeptide, macromolecular compound such as polysaccharide, protein, the scope of application is more extensive; Can clearly express the difference between different batches in this complex system of Chinese medicinal materials, stability and favorable reproducibility; Its instrument and consumptive material domesticize fully, and cost is starkly lower than high performance liquid chromatography.Along with the main research field of the flourish HSCCC of natural drug concentrates on the separation and purification of natural drug, from tens kinds of natural matters, separated obtaining hundreds of highly purified monomeric compounds.Be very suitable for tieing up the preparation of medicine chemical reference substance.
The dimension medicine is with a long history, in the process of its formation and development, adopt the chief of national medicines such as Arab, ancient Greek, and be subjected to the influence of traditional Chinese medicine and pharmacy, be the individual branches of China's national medicine, made significant contribution for the procreation and the prosperity of Western Regions people of all nationalities in history.There is kind surplus the dimension medicine 600 in Xinjiang autonomous region, and about 360 kinds commonly used, wherein this real estate resource is about 160 kinds, accounts for 27% of dimension medicine sum.Taking in " People's Republic of China (PRC) " officinal medicine has 202 kinds, 115 kinds of its Chinese medicinal materialss, and 87 kinds of prescribed preparations, what belong to national special use has 30 kinds approximately, and saussurea intybus and Herba Achilleae are very representational medicinal materials.
The dimension medicine has unique theory of curing the disease, and is the important component part of Chinese ethnic drug, but its fired basis originally is confined in the municipal scope in Xinjiang, in the whole country and promotion rate all over the world slow.The major cause that causes this situation: the one, also there is a big difference from the production requirement of modern medicine industry high quality for production technique; The 2nd, the main active ingredient of crude drug is a secondary metabolite, very strong to posteriori growing environment dependency, bulk drug is from natural, mostly being artificial dispersion again gathers, processes, it is very big influenced by weather, regional difference and human factor, raw material, intermediate and the finished product from standardization, also there is a big difference in standardized management, lacks strict quality monitoring standard and good monitoring method, is difficult to guarantee the stability of quality product; The 3rd, the curative effect of dimension medicine product lacks the science data explanation that scientific experiment methods such as the clinical study of employing modern medicines " random packet ", " contrast " commonly used, " double blinding ", " multiple spot observation " obtain, and embarrasses modern medicine worker and administration to convince.The modernization of dimension medicine is the basic outlet of dimension medicine industry Sustainable development.The quality monitoring of dimension medicine raw material and patent medicine (tablet, granule, oral liquid, injection) is to press for one of key issue of solution in the dimension medicine modernization.And standard reference material is the important substance basis of quality monitoring, can instruct plantation for the influence to quality of medicinal material such as crude drug planting base monitoring weather, region, collection period and human factor; Can instruct rationally blending of medicinal material for patent medicine manufacturer, the quality of control intermediate and the finished product is set up the relevant enterprise standard; Setting up for the demarcation of important indicator composition in the finger printing and spectrum effect relationship provides the basis.
And no matter be the dimension medicine or the research and development of Chinese medicine, highly purified standard reference materials of famine all, this is that the restriction standard is worked out and one of key factor of implementing.National Quality ﹠ Technology Inspection Bureau is setting up various national material standards at present, and the standard substance of natural drug are important contents wherein.
Artemisia rupestris L. (Artemisia rupestris L.) is a feverfew, originates in domestic Tianshan Mountains, Xinjiang Uygur Autonomous Regions and mountain range, Altai Mountains, is Xinjiang ethnic drug medicinal herbs most in use, has detoxifcation, antianaphylaxis, antibiotic, antiviral and effect such as protect the liver.The clinical treatment that is used for hepatitis, flu, pharyngitis, tonsillitis, ophthalmia.Contain compounds such as flavonoid, half times of terpene, amino acids, glucoside, polyose, volatile oil, polypeptide, alkaloid in the Herba Achilleae.
The present invention is directed to the problem of chemical reference substance famine, on existing dimension medicine pharmacodynamic study basis, adopt development in recent years high-speed countercurrent chromatography rapidly, prepare the method for two kinds of representative chemical ingredientss of the plain the second grade of rupestonic acid and golden waist simultaneously, and adopt this method to obtain the rupestonic acid of high purity (on the purity 95%) and the monomeric compound of the plain second of golden waist; And to set up with the high speed adverse current chromatogram be that the standard reference material of core technology prepares platform, for the foundation of Wei Yaohuahewuku and the application of national material standard lay the first stone.
Summary of the invention
The objective of the invention is to, a kind of method of utilizing high-speed countercurrent chromatography purifying rupestonic acid and the plain second of golden waist is provided, this method is that the separation solvent systems is placed standing demix behind the separating funnel shake well, get as stationary phase, following to moving phase, get in the mixing solutions that Herba Achilleae extract is dissolved in upper and lower phase, again stationary phase is filled chromatography column with pump, head end and sampling valve with post joins then, when treating that moving phase begins to flow out chromatographic column, collect isolate, heterogeneity is collected respectively, concentrate, drying is separated the ketone acid obtain 95% or more and the monomeric compound of golden waist element second.
The method of high-speed countercurrent chromatography purifying rupestonic acid and the plain second of golden waist of utilizing of the present invention is characterized in that following these steps to carrying out:
A, get the Herba Achilleae herb, pulverize the back and uses 70% alcohol reflux, united extraction liquid concentrating under reduced pressure becomes medicinal extract, and the water suspendible is used sherwood oil and ethyl acetate extraction respectively;
B, extraction liquid are concentrated into dried, ethyl acetate part dried powder, powder is dissolved in methylene dichloride, ultrasonic dissolution filters, and merges, concentrate crude samples;
C, employing high-speed counter-current chromatograph, chromatographic instrument separates solvent systems and is made up of normal hexane, ethyl acetate, first alcohol and water, and its volume ratio is 2-5: 4-6: 2-5: 4-6;
D, solvent systems is disposed in the separating funnel, standing demix behind the shake well, get as stationary phase, following to moving phase, get step b extraction crude samples and be dissolved in the mixed solvent of upper and lower phase, make in the high-speed counter-current chromatograph pillar to be full of stationary phase, its main frame is rotated, again moving phase is pumped into post, enter the mixed solvent that crude samples is dissolved in upper and lower phase by sampling valve again, according to detector spectrogram receiving target composition;
E, the sample that the high speed adverse current chromatogram separation is obtained utilize high performance liquid phase to detect, and chromatographic condition is: C18column (4.6mmI.D. * 250mm, 5 μ m), 35 ℃ of column temperatures, moving phase: A (methyl alcohol); B (0.2% formic acid); Gradient elution program: 0-40min, A: B (30: 70, v/v) to A: B (70: 30, v/v), during 60min, A: B (70: 30, v/v); Flow velocity: 1.0ml/min detects wavelength: 245nm.
The retention time of steps d rupestonic acid in high speed adverse current chromatogram is 260-400 minute, and the retention time of the plain second of Herba Achilleae gold waist is 560-720 minute.
The high-speed counter-current chromatograph flow rates that steps d is used is 1-3ml/min, and speed range is 750-900rpm/min.
Present method is applicable to the extraction to any one plant that contains the plain second of ketone acid and golden waist.
The method of utilizing high-speed countercurrent chromatography purifying rupestonic acid and the plain second of golden waist of the present invention, taked following design for realizing the present invention of this method:
The preparation of sample:
Artemisia rupestris L. herb 500g (dry weight) pulverizes the back and uses 70% alcohol reflux, and united extraction liquid concentrating under reduced pressure becomes medicinal extract, and the water suspendible is used sherwood oil, ethyl acetate extraction respectively.Extraction liquid is concentrated into dried, ethyl acetate part dried powder 95.394g, powder is dissolved in 1L methylene dichloride (5 times), ultrasonic dissolution filters, and merges, concentrate crude samples 5.1289g, this sample is used for the separation of high-speed countercurrent chromatography (HSCCC);
The screening of solvent systems:
Getting the 5mg crude samples is dissolved in the 10ml solvent systems, concussion is 5 minutes continuously, leave standstill, layering, separation time is less than 30 seconds, separately two-phase is placed in the test tube, again be dissolved in the 1ml methyl alcohol after the drying, by HPLC sample is analyzed, the phase peak area is the partition ratio (K) of this compound in two-phase solvent with the following ratio of peak area mutually on the target compound, solvent systems normal hexane-ethyl acetate-methanol-water 2-5: 4-6: 2-5: 4-6v/v, preferred volume ratio 3: 5: 3: 5, the K of the plain second of target compound rupestonic acid and golden waist is respectively 2.49,5.98;
The preparation of sample solution:
500 milligrams of crude samples are dissolved in the isopyknic phase up and down of the 10ml solution ultrasonic its dissolving, the elimination insolubles of making;
High speed adverse current chromatogram (HSCCC) sepn process:
At first be full of " chromatographic column " with stationary phase, open counter current chromatograph, revolution is 800rpm/min, after the flow velocity injection moving phase with 1.5ml/min, advances the 10ml sample solution.Effluent liquid detects by UV-detector, detects wavelength 254nm, according to peak type manual collection effluent liquid;
High performance liquid phase (HPLC) detects the raw material and the product of high speed adverse current chromatogram (HSCCC):
Crude samples separates the sample that obtains and detects by high performance liquid phase (HPLC) with high speed adverse current chromatogram (HSCCC), chromatographic condition is: C18column (4.6mmI.D. * 250mm, 5 μ m), 35 ℃ of column temperatures, moving phase: A (methyl alcohol); B (0.2% formic acid); Gradient elution program: 0-40min, A: B (30: 70, v/v) to A: B (70: 30, v/v), during 60min, and A: B (70: 30, v/v), flow velocity: 1.0ml/min detects wavelength: 245nm, and the retention time of the plain second of rupestonic acid and golden waist is respectively 30min, 37.5min.
High speed adverse current chromatogram (HSCCC) separates the monomer that obtains and identifies structure by MS, NMR.
Fig. 1 separates the figure of rupestonic acid and the plain second of golden waist for high speed adverse current chromatogram of the present invention, and wherein 1 is rupestonic acid, and 2 is the plain second of golden waist.
Fig. 2 separates preceding gross sample figure for high speed adverse current chromatogram of the present invention, and wherein 1 is rupestonic acid, and 2 is the plain second of golden waist.
Fig. 3 detects rupestonic acid figure for high performance liquid phase of the present invention, and wherein 1 is ketone acid.
Fig. 4 is the plain second figure of this school Liquid Detection gold waist of the present invention, and wherein 2 is the plain second of golden waist.
The preparation of sample:
A, get Artemisia rupestris L. herb 500g (dry weight), pulverize the back and uses 70% alcohol reflux, united extraction liquid concentrating under reduced pressure becomes medicinal extract, and the water suspendible is used sherwood oil and ethyl acetate extraction respectively;
B, extraction liquid are concentrated into dried, ethyl acetate part dried powder 95.394g, powder is dissolved in 1L methylene dichloride (5 times), ultrasonic dissolution filters, and merges, concentrate crude samples 5.1289g, this sample is used for the separation of high speed adverse current chromatogram (HSCCC);
C, employing high-speed counter-current chromatograph, chromatographic instrument separates solvent systems and is made up of normal hexane, ethyl acetate, first alcohol and water, and its volume ratio is 2: 6: 2: 6;
D, solvent systems is disposed at separatory to fall in the bucket, standing demix behind the shake well, get as stationary phase, following to moving phase, getting step b crude samples 150mg is dissolved in the mixed solvent of 3ml upper strata and 3ml lower floor, make in the high-speed counter-current chromatograph pillar and be full of stationary phase, head end and six-way injection valve with post joins then, the opening speed controller, its main frame is rotated, rotating speed reaches 750r/min, beginning is gone into moving phase with the 1.0ml/min flow pump, enter the mixed solvent of 150 milligrams of step b crude samples again by sampling valve, when treating that moving phase begins to flow out chromatographic column, collect isolate, the retention time of rupestonic acid is 260 minutes in high speed adverse current chromatogram, the retention time of the plain second of Herba Achilleae gold waist is 560 minutes, and from outer spectrogram receiving target composition, the purity of the plain second of ketone acid that separation obtains and golden waist is all more than 95% according to detector;
E, the sample that the high speed adverse current chromatogram separation is obtained utilize high performance liquid phase to detect, and chromatographic condition is: C18column (4.6mmI.D. * 250mm, 5 μ m), 35 ℃ of column temperatures, moving phase: A (methyl alcohol); B (0.2% formic acid); Gradient elution program: 0-40min, (30: 70, v/v) (70: 30, v/v), during 60min, (70: 30, v/v), flow velocity: 1.0ml/min detected wavelength: 245nm to A: B to A: B to A: B.
A, get Artemisia rupestris L. herb 500g (dry weight), pulverize the back and uses 70% alcohol reflux, united extraction liquid concentrating under reduced pressure becomes medicinal extract, and the water suspendible is used sherwood oil and ethyl acetate extraction respectively;
B, extraction liquid are concentrated into dried, ethyl acetate part dried powder 95.394g, powder is dissolved in 1L methylene dichloride (5 times), ultrasonic dissolution filters, and merges, concentrate crude samples 5.1289g, this sample is used for the separation of high speed adverse current chromatogram (HSCCC);
C, employing high-speed counter-current chromatograph, chromatographic instrument separates solvent systems and is made up of normal hexane, ethyl acetate, first alcohol and water, and its volume ratio is 3: 6: 3: 6;
D, solvent systems is disposed in the separating funnel, standing demix behind the shake well, get as stationary phase, following to moving phase, getting step b extraction crude samples 500mg is dissolved in the mixed solvent of 10ml upper strata and 10ml lower floor, make in the high-speed counter-current chromatograph pillar and be full of stationary phase, head end and six-way injection valve with post joins then, the opening speed controller, its main frame is rotated, rotating speed reaches 900r/min, beginning is gone into moving phase with the 3ml/min flow pump, enter the mixed solvent that step b extracts crude samples 500mg by sampling valve, when treating that moving phase begins to flow out chromatographic column, collect isolate, the retention time of rupestonic acid is 320 minutes in high speed adverse current chromatogram, the retention time of the plain second of Herba Achilleae gold waist is 600 minutes, and from outer spectrogram receiving target composition, the purity of the plain second of ketone acid that separation obtains and golden waist is all more than 95% according to detector;
E, the sample that the high speed adverse current chromatogram separation is obtained utilize high performance liquid phase to detect, and chromatographic condition is: C18column (4.6mmI.D. * 250mm, 5 μ m), 35 ℃ of column temperatures, moving phase: A (methyl alcohol); B (0.2% formic acid); Gradient elution program: 0-40min, A: B (30: 70, v/v) to A: B (70: 30, v/v), during 60min, A: B (70: 30, v/v).Flow velocity: 1.0ml/min detects wavelength: 245nm.
Present method is applicable to the extraction to any one plant that contains the plain second of ketone acid and golden waist.
Embodiment 3
A, get Artemisia rupestris L. herb 500g (dry weight), pulverize the back and uses 70% alcohol reflux, united extraction liquid concentrating under reduced pressure becomes medicinal extract, and the water suspendible is used sherwood oil and ethyl acetate extraction respectively;
B, extraction liquid are concentrated into dried, ethyl acetate part dried powder 95.394g, powder is dissolved in 1L methylene dichloride (5 times), ultrasonic dissolution filters, and merges, concentrate crude samples 5.1289g, this sample is used for the separation of high speed adverse current chromatogram (HSCCC);
C, employing high-speed counter-current chromatograph, chromatographic instrument separates solvent systems and is made up of normal hexane, ethyl acetate, first alcohol and water, and its volume ratio is 4: 5: 4: 5;
D, solvent systems is disposed in the separating funnel, standing demix behind the shake well, get as stationary phase, following to moving phase, getting step b extraction crude samples 200mg is dissolved in the mixed solvent of 5ml upper strata and 5ml lower floor, make in the high-speed counter-current chromatograph pillar and be full of stationary phase, head end and six-way injection valve with post joins then, the opening speed controller, its main frame is rotated, rotating speed reaches 800r/min, beginning is gone into moving phase with the 2.0ml/min flow pump, again moving phase is pumped into post, enters the mixed solvent that step b extracts crude samples 200mg by sampling valve, when treating that moving phase begins to flow out chromatographic column, collect isolate, the retention time of rupestonic acid is 300 minutes in high speed adverse current chromatogram, and the retention time of the plain second of Herba Achilleae gold waist is 620 minutes, according to detector spectrogram receiving target composition, the purity of the plain second of ketone acid that separation obtains and golden waist is all more than 95%;
E, the sample that the high speed adverse current chromatogram separation is obtained utilize high performance liquid phase to detect, and chromatographic condition is: C18column (4.6mmI.D. * 250mm, 5 μ m), 35 ℃ of column temperatures, moving phase: A (methyl alcohol); B (0.2% formic acid); Gradient elution program: 0-40min, (30: 70, v/v) (70: 30, v/v), during 60min, (70: 30, v/v), flow velocity: 1.0ml/min detected wavelength: 245nm to A: B to A: B to A: B.
Present method is applicable to the extraction to any one plant that contains the plain second of ketone acid and golden waist.
Embodiment 4
A, get Artemisia rupestris L. herb 500g (dry weight), pulverize the back and uses 70% alcohol reflux, united extraction liquid concentrating under reduced pressure becomes medicinal extract, and the water suspendible is used sherwood oil and ethyl acetate extraction respectively;
B, extraction liquid are concentrated into dried, ethyl acetate part dried powder 95.394g, powder is dissolved in 1L methylene dichloride (5 times), ultrasonic dissolution filters, and merges, concentrate crude samples 5.1289g, this sample is used for the separation of high speed adverse current chromatogram (HSCCC);
C, employing high-speed counter-current chromatograph, chromatographic instrument separates solvent systems and is made up of normal hexane, ethyl acetate, first alcohol and water, and its volume ratio is 3: 5: 3: 5;
D, solvent systems is disposed in the separating funnel, standing demix behind the shake well, get as stationary phase, following to moving phase, getting step b extraction crude samples 300mg is dissolved in the mixed solvent of 4ml upper strata and 4ml lower floor, make in the high-speed counter-current chromatograph pillar and be full of stationary phase, head end and six-way injection valve with post joins then, the opening speed controller, its main frame is rotated, rotating speed reaches 880r/min, beginning is gone into moving phase with the 2.5ml/min flow pump, again moving phase is pumped into post, enters the mixed solvent that step b extracts crude samples 300mg by sampling valve, when treating that moving phase begins to flow out chromatographic column, collect isolate, the retention time of rupestonic acid is 380 minutes in high speed adverse current chromatogram, and the retention time of the plain second of Herba Achilleae gold waist is 700 minutes, according to detector spectrogram receiving target composition, the purity of the plain second of ketone acid that separation obtains and golden waist is all more than 95%;
E, the sample that the high speed adverse current chromatogram separation is obtained utilize high performance liquid phase to detect, and chromatographic condition is: C18column (4.6mmI.D. * 250mm, 5 μ m), 35 ℃ of column temperatures, moving phase: A (methyl alcohol); B (0.2% formic acid); Gradient elution program: 0-40min, A: B (30: 70, v/v) to A: B (70: 30, v/v), during 60min, A: B (70: 30, v/v).Flow velocity: 1.0ml/min detects wavelength: 245nm.
Present method is applicable to the extraction to any one plant that contains the plain second of ketone acid and golden waist.
Embodiment 5
A, get Artemisia rupestris L. herb 500g (dry weight), pulverize the back and uses 70% alcohol reflux, united extraction liquid concentrating under reduced pressure becomes medicinal extract, and the water suspendible is used sherwood oil and ethyl acetate extraction respectively;
B, extraction liquid are concentrated into dried, ethyl acetate part dried powder 95.394g, powder is dissolved in 1L methylene dichloride (5 times), ultrasonic dissolution filters, and merges, concentrate crude samples 5.1289g, this sample is used for the separation of high speed adverse current chromatogram (HSCCC);
C, employing high-speed counter-current chromatograph, chromatographic instrument separates solvent systems and is made up of normal hexane, ethyl acetate, first alcohol and water, and its volume ratio is 5: 6: 5: 6;
D, solvent systems is disposed in the separating funnel, standing demix behind the shake well, get as stationary phase, following to moving phase, getting step b extraction crude samples 350mg is dissolved in the mixed solvent of 6ml upper strata and 6ml lower floor, make in the high-speed counter-current chromatograph pillar and be full of stationary phase, head end and six-way injection valve with post joins then, the opening speed controller, its main frame is rotated, rotating speed reaches 850r/min, beginning is gone into moving phase with the 1.5ml/min flow pump, again moving phase is pumped into post, enters the mixed solvent that step b extracts crude samples 350mg by sampling valve, when treating that moving phase begins to flow out chromatographic column, collect isolate, the retention time of rupestonic acid is 400 minutes in high speed adverse current chromatogram, and the retention time of the plain second of Herba Achilleae gold waist is 720 minutes, according to detector spectrogram receiving target composition, the purity of the plain second of ketone acid that separation obtains and golden waist is all more than 95%;
E, the sample that the high speed adverse current chromatogram separation is obtained utilize high performance liquid phase to detect, and chromatographic condition is: C18column (4.6mmI.D. * 250mm, 5 μ m), 35 ℃ of column temperatures, moving phase: A (methyl alcohol); B (0.2% formic acid); Gradient elution program: 0-40min, A: B (30: 70, v/v) to A: B (70: 30, v/v), during 60min, A: B (70: 30, v/v).Flow velocity: 1.0ml/min detects wavelength: 245nm.
Present method is applicable to the extraction to any one plant that contains the plain second of ketone acid and golden waist.
[0069] the invention provides a kind of high speed adverse current chromatogram method (HSCCC) that obtains two kinds of compounds of from the natural product crude extract, separating simultaneously, this method is mainly used in the preparation at preparation high-purity chemical reference substance and chemical example, have efficient, low-cost, the sample free of losses, characteristics such as the sample separation amount is big.
Claims (1)
1. method of utilizing the plain second of high-speed countercurrent chromatography purifying rupestonic acid and golden waist is characterized in that following these steps to carrying out:
A, get the Herba Achilleae herb, pulverize the back and uses 70% alcohol reflux, united extraction liquid concentrating under reduced pressure becomes medicinal extract, and the water suspendible is used sherwood oil and ethyl acetate extraction respectively;
B, extraction liquid are concentrated into dried, ethyl acetate part dried powder, powder is dissolved in methylene dichloride, ultrasonic dissolution filters, and merges, concentrate crude samples;
C, employing high-speed counter-current chromatograph, chromatographic instrument separates solvent systems and is made up of normal hexane, ethyl acetate, first alcohol and water, and its volume ratio is 2-5: 4-6: 2-5: 4-6;
D, solvent systems is disposed in the separating funnel, standing demix behind the shake well, get as stationary phase, following to moving phase, get step b extraction crude samples and be dissolved in the mixed solvent of upper and lower phase, make in the high-speed counter-current chromatograph pillar to be full of stationary phase, its main frame is rotated, again moving phase is pumped into post, enter the mixed solvent that crude samples is dissolved in upper and lower phase by sampling valve again, according to detector spectrogram receiving target composition; The retention time of steps d rupestonic acid in high speed adverse current chromatogram is 260-400 minute, and the retention time of the plain second of Herba Achilleae gold waist is 560-720 minute; The high-speed counter-current chromatograph flow rates of using is 1-3ml/min, and speed range is 750-900r/min;
E, the sample that the high speed adverse current chromatogram separation is obtained utilize high performance liquid phase to detect, and chromatographic condition is: C18 post, 4.6mmI.D. * 250mm, 5 μ m; 35 ℃ of column temperatures, moving phase: A methyl alcohol; B 0.2% formic acid; Gradient elution program: 0-40min, A: B=30: 70-70: 30, v/v, during 60min, A: B=70: 30, v/v, flow velocity: 1.0ml/min detects wavelength: 245nm.
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| CN101898966B (en) * | 2010-07-15 | 2013-04-17 | 中国科学院新疆理化技术研究所 | Piperazine ring-containing Artemisia rupestris L. keto ester derivative as well as preparation method thereof |
| CN105168042B (en) * | 2015-09-02 | 2017-10-27 | 集美大学 | A kind of method of the quick separating tyrosinase inhibitor from camellia pollen |
| CN109771463A (en) * | 2017-11-15 | 2019-05-21 | 中央民族大学 | Artemisia rupestris extract and its preparation method and application |
| CN111060626B (en) * | 2019-12-31 | 2022-06-14 | 中央民族大学 | Artemisia rupestris plant producing area/variety marker based on plant metabonomics and discrimination method |
| CN112961203B (en) * | 2021-05-18 | 2021-07-30 | 江西中医药大学 | Flavonol glycoside derivative and its use and preparation method |
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| CN1754871A (en) * | 2004-10-01 | 2006-04-05 | 斯拉甫·艾白 | Preparation method and use of alpine yarrow herb ketoacid |
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| CN1754871A (en) * | 2004-10-01 | 2006-04-05 | 斯拉甫·艾白 | Preparation method and use of alpine yarrow herb ketoacid |
Non-Patent Citations (1)
| Title |
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| Yanming Ma,et al..Preparative isolation and purification of rupestonic acid from theChinese medicinal plant Artemisia rupestris L.by high-speed counter-current chromatography..《Journal of Chromatography A》.2005,第1076卷198-201. * |
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