CN101805352B - Method for preparing eriocalyxin B - Google Patents
Method for preparing eriocalyxin B Download PDFInfo
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- CN101805352B CN101805352B CN2009100951839A CN200910095183A CN101805352B CN 101805352 B CN101805352 B CN 101805352B CN 2009100951839 A CN2009100951839 A CN 2009100951839A CN 200910095183 A CN200910095183 A CN 200910095183A CN 101805352 B CN101805352 B CN 101805352B
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Abstract
The invention relates to a method for preparing eriocalyxin B with high purity from crude extract of lamiaceae plectranthus genus plant flower thinning isodon eriocalyx by applying high speed counter-current chromatography, which is characterized in that two solvent systems which are not mixed and dissolved with each other are applied for separating and purifying natural products in a support tube in a way of high-speed planetary motion, so that irreversible adsorption is not available, the method has the advantages of no loss of sample, no pollution, high efficiency, high speed and large amount of separation, the obtained eriocalyxin B has the purity of more than 98%, and method is applicable to separating and preparing eriocalyxin B monomer from flower thinning isodon eriocalyx crude product prepared under various technical conditions and is suitable for separating and preparing the eriocalyxin B monomer by using counter current chromatographs with various models. The preparation method has simple operation, less separating steps, high efficiency, low loss of solvent and high product purity, and is suitable for industrialized production.
Description
Technical field
The present invention relates to field of pharmacology, particularly, relate to a kind of application high speed adverse current chromatogram (HSCCC) method prepares highly purified medical compounds eriocalyxin B from flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract method.
Background technology
Eriocalyxin B is a kind of mapping-kaurane compounds that is purified into from be distributed in China west and south distinctive Labiatae Rabdosia vegetable wool calyx Herba Rabdosiae glaucocalycis (Isodon eriocalyx (Dunn) Hara) and flower thinning hair calyx Herba Rabdosiae glaucocalycis (Isodon eriocalyx var.laxiflora C.Y.Wu et H.W.Li).At the anti-inflammatory and antibiotic that is used among the people.At Chinese patent application CN02134163.X, from hair calyx Herba Rabdosiae glaucocalycis, separated the medicine that the compound eriocalyxin B is used to prepare prevention or treats cancer.At Chinese application number CN200510110089.8, hair calyx Herba Rabdosiae glaucocalycis can also be used for the treatment of diseases such as acute leukemia and Burkitt lymphoma.The extraction separation of Bao Dao eriocalyxin B mainly was to adopt column chromatography in the past, comprised silicagel column and gel column.But these all column chromatographys all need to use solid packing, easily produce irreversible adsorption, have that sample easily loses, easily pollution, poor efficiency, a shortcoming such as speed is slow and separating step is loaded down with trivial details.High-speed countercurrent chromatography is different from traditional column chromatography, need not use solid packing, but use the separation and purification of carrying out natural product under the mode of the immiscible solvent systems high speed planetary motion in supporting tube of two-phase, thereby do not have irreversible adsorption, have sample free of losses, the isolating advantage of pollution-free, efficient, quick and big preparation amount.So far, there is not application high speed adverse current chromatogram (HSCCC) method to prepare the report of the method for highly purified medical compounds eriocalyxin B in the prior art.
Summary of the invention
The present invention aims to provide a kind of application high speed adverse current chromatogram (HSCCC) method prepares highly purified eriocalyxin B from flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract method.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme: prepare the method for eriocalyxin B, from flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract, prepare with high speed adverse current chromatogram HSCCC method.
The solvent system of high speed adverse current chromatogram stationary phase, moving phase is made up of normal hexane, ethyl acetate, methyl alcohol, water, and proportion of composing is 4-6: 4-6: 4-6: 4-6.
Preparation temperature is chosen in 18-35 ℃.
More specifically, the method that the present invention prepares eriocalyxin B is: under room temperature 18-35 ℃, at first with 4-6: 4-6: 4-6: the 4-6 volume ratio is disposed at n-hexane/ethyl acetate/methanol solvent system in the separating funnel, shake all back standing demix, ready to balance is after for some time, upper and lower phase is separated, get and be stationary phase mutually, be moving phase mutually down, get that flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract is dissolved in that 5mL goes up mutually and make sample solution usefulness to be separated in mutually the mixing solutions under the 5mL, adopt analysis mode TBE-20A high-speed counter-current chromatograph (multiple wraps tetrafluoroethylene column capacity is about 16mL), and be furnished with ram pump, sampling valve, UV-detector and registering instrument.Earlier be full of whole pillar, adjust engine speed, moving phase is pumped in the post with the flow velocity of 0.5mL/min to 1600rpm with stationary phase; After treating that whole system is set up running balance, by sampling valve sample introduction 0.1mL; According to detector uv atlas receiving target composition, get final product then through the HPLC detection.
The preparation of flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract, CN02134163.X is contained with reference to Chinese patent application, gets flower thinning hair calyx Herba Rabdosiae glaucocalycis medicinal material over-ground part that is:, pulverizes, and medicinal powder extracts three times with 70% acetone soaking at room temperature, and united extraction liquid is evaporated to dried.
This invention scheme is as follows: adopt a small amount of preparation and the relatively large preparation of half countercurrent chromatography instrument of analysis mode high-speed counter-current chromatograph, the solvent system of selecting for use is made up of normal hexane, ethyl acetate, methyl alcohol, water, proportion of composing is 4-6: 4-6: 4-6: 4-6, and step separation promptly can obtain eriocalyxin B.Room temperature has certain influence to resolution, and above separation condition is fit to temperature and uses in the time of 18-35 ℃.
Than prior art, the present invention uses high-speed countercurrent chromatography HSCCC prepares highly purified eriocalyxin B from flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract method, be different from traditional column chromatography, need not use solid packing but use the separation and purification of carrying out natural product under the mode of the immiscible solvent systems high speed planetary motion in supporting tube of two-phase, thereby there is not an irreversible adsorption, has the sample free of losses, pollution-free, efficiently, the isolating advantage of quick and big preparation amount, the eriocalyxin B purity that obtains can reach more than 98%, be applicable to that the counter current chromatograph with various models separates preparation eriocalyxin B monomer, as all can with a small amount of preparation of analysis mode high-speed counter-current chromatograph and the relatively large preparation of half countercurrent chromatography instrument.
Description of drawings
Fig. 1 analysis mode high-speed counter-current chromatograph TBE-20A is to the separating effect figure of eriocalyxin B (eriocalyxin B) in the flower thinning hair calyx Herba Rabdosiae glaucocalycis.
Fig. 2 half countercurrent chromatography instrument TBE-300B is to the separating effect figure of eriocalyxin B (eriocalyxin B) in the flower thinning hair calyx Herba Rabdosiae glaucocalycis.
Fig. 3 high speed adverse current chromatogram gained eriocalyxin B
1H NMR schemes (Bruker DRX500,500MHz, deuterated pyridine).
Fig. 4 high speed adverse current chromatogram gained eriocalyxin B
13C and DEPT NMR figure (BrukerDRX500,125MHz, deuterated pyridine).
The ESI (a) of Fig. 5 high speed adverse current chromatogram gained eriocalyxin B and HRESI (b) MS figure (API QSTAR).
Embodiment
In order to further specify the present invention, below in conjunction with accompanying drawing, provide specific embodiments of the invention, but do not limit the present invention with this.
Embodiment 1:
1, the preparation of eriocalyxin B crude product:
Get 1 kilogram of flower thinning hair calyx Herba Rabdosiae glaucocalycis over-ground part, pulverizing is placed on 10 liters diafiltration jar, extract 3 times (3 times shared 20 liters) with 10 liter of 95% ethanol room temperature diafiltration, merge diafiltration extracting solution (about 15 liters), in spherical concentration tank, concentrate to such an extent that medicinal extract 67.8 restrains, add 120mL water and stir, the ethyl acetate extraction (V: V=1: 1 of this water with equal volume, extract three times), get ethyl acetate extract 62.7 grams; This medicinal extract is dissolved in 95% ethanol, and after the filtration, last MCI post depigmentation towards post, after the flow point of waiting to contain eriocalyxin B washes, is washed post with acetone with 90% ethanol.Promptly get flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract 58.8 after the flow point that contains eriocalyxin B concentrates restrains.
2, the preparation of eriocalyxin B:
Adopt analysis mode high-speed counter-current chromatograph solvent to select for use n-hexane/ethyl acetate/methanol system from flower thinning hair calyx Herba Rabdosiae glaucocalycis crude product, to separate preparation eriocalyxin B monomer.At first with 4.5: 5: 4.5: 5 volume ratios are disposed at n-hexane/ethyl acetate/methanol solvent system in the separating funnel, shake all back standing demix, ready to balance is after for some time, upper and lower phase is separated, get and be stationary phase mutually, be moving phase mutually down, get that step 1 gained 500mg flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract is dissolved in that 5mL goes up mutually and make sample solution usefulness to be separated in mutually the mixing solutions under the 5mL, the analysis mode TBE-20A high-speed counter-current chromatograph that adopts Shanghai Tongtian Biotechnology Co., Ltd. to produce, and be furnished with ram pump, sampling valve, UV-detector and registering instrument, the column capacity of multiple wraps tetrafluoroethylene is about 16mL.Earlier be full of whole pillar with stationary phase; Adjust engine speed to 1600rpm, moving phase is pumped in the post with the flow velocity of 0.5mL/min; After treating that whole system is set up running balance, by sampling valve sample introduction 0.1mL; According to detector uv atlas receiving target composition (see figure 1), detect target compound eriocalyxin B purity all more than 98% then through HPLC.
The eriocalyxin B molecular structural formula is:
Embodiment 2:
Adopt half preparation type TBE-300B high-speed counter-current chromatograph, and the column capacity of being furnished with ram pump, sampling valve, UV-detector and registering instrument, multiple wraps tetrafluoroethylene is about 260mL.Solvent system was selected n-hexane/ethyl acetate/methanol for use 5: 5: 5: 5 separate preparation eriocalyxin B monomer.At first above-mentioned solvent system is disposed in the separating funnel, shakes all back standing demix.Ready to balance after for some time separates upper and lower phase, gets to be stationary phase mutually, and be moving phase mutually down.Get 500mg flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract be dissolved in 5mL go up mutually with 5mL under stand-by in mutually the mixture.Earlier be full of whole pillar with stationary phase; Adjust engine speed to 800rpm, moving phase is pumped in the post with the flow velocity of 2.0mL/min; After treating that whole system is set up running balance, by sampling valve sample introduction 10mL; Then according to detector uv atlas receiving target composition.The decompression rotary evaporation is to doing.Obtain eriocalyxin B monomer 95mg, detect target compound eriocalyxin B purity all in 98% above (see figure 2) through HPLC.Utilize NMR and MS (Fig. 3,4 and 5) to carry out structure elucidation to the gained compound, and with data in literature comparison, determine this compound be eriocalyxin B undoubtedly.
Claims (3)
1. method for preparing eriocalyxin B, prepare from flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract with high speed adverse current chromatogram HSCCC method, the solvent system that it is characterized in that high speed adverse current chromatogram stationary phase, moving phase is made up of normal hexane, ethyl acetate, methyl alcohol, water, and proportion of composing is 4-6: 4-6: 4-6: 4-6.
2. the method for preparing eriocalyxin B as claimed in claim 1 is characterized in that preparation temperature is chosen in 18-35 ℃.
3. the method for preparing eriocalyxin B as claimed in claim 1, it is characterized in that under room temperature 18-35 ℃, at first with 4-6: 4-6: 4-6: the 4-6 volume ratio is disposed at n-hexane/ethyl acetate/methanol solvent system in the separating funnel, shake all back standing demix, ready to balance is after for some time, upper and lower phase is separated, get and be stationary phase mutually, be moving phase mutually down, get that flower thinning hair calyx Herba Rabdosiae glaucocalycis crude extract is dissolved in that 5mL goes up mutually and make sample solution usefulness to be separated in mutually the mixing solutions under the 5mL, adopt analysis mode TBE-20A high-speed counter-current chromatograph, and is furnished with ram pump, sampling valve, UV-detector and registering instrument, multiple wraps tetrafluoroethylene column capacity is 15mL-16mL, is full of whole pillar with stationary phase earlier, adjust engine speed to 1600rpm, moving phase is pumped in the post with the flow velocity of 0.5mL/min; After treating that whole system is set up running balance, by sampling valve sample introduction 0.1mL; According to detector uv atlas receiving target composition, get final product then through the HPLC detection.
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| CN2009100951839A CN101805352B (en) | 2009-11-12 | 2009-11-12 | Method for preparing eriocalyxin B |
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| CN102372724A (en) * | 2010-08-26 | 2012-03-14 | 苏州宝泽堂医药科技有限公司 | Method for extracting eriocalyxin B |
| CN102557906A (en) * | 2010-12-24 | 2012-07-11 | 苏州宝泽堂医药科技有限公司 | Method for extracting and purifying amethystoidin from isodon amethystoides |
| US9408811B2 (en) | 2013-07-18 | 2016-08-09 | Hong Kong Baptist University | Pharmaceutical composition comprising guttiferone F for the treatment of prostate cancer |
| CN115894370A (en) * | 2023-03-03 | 2023-04-04 | 中国科学院昆明植物研究所 | Method for preparing bulleyaconitine A by high-speed countercurrent chromatography |
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