CN101081844B - Method for large-batch separating preparation of high-purity theaflavine monomer - Google Patents
Method for large-batch separating preparation of high-purity theaflavine monomer Download PDFInfo
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Abstract
The present invention discloses process of separating black tea extract to prepare four kinds of high purity theaflavin and its derivatives in batch. By means of combining gel column chromatographic separation with great treating quantity and high speed countercurrent chromatographic separation with high separating efficiency, four kinds of theaflavin and its derivatives, including theaflavin (TF), theaflavin-3-gallate (TF-3-MG),theaflavin-3'-gallate (TF-3'-MG) and theaflavin-3, 3'-gallate (TFDG), in high purity are prepared. The process has great preparation amount, high product purity, short period, low solvent consumption and other features.
Description
Technical field
The present invention relates to the method for large-batch separating preparation of high-purity theaflavine monomer from black tea extract.
Technical background
(Theaflavins is a distinctive class Polyphenols polymkeric substance in the black tea TFs) to theoflavin, and they are to be formed by a pair of suitable catechin oxypolymerization in the fermenting process of black tea.The content of theoflavin in black tea is very low, accounts for about 2% of dry weight greatly, and still, they have very significant effects to the color and luster and the mouthfeel of black tea.Studies show that in recent years, theoflavin not only have very big influence to the quality of black tea, the more important thing is that it has multiple and the closely-related biological activity of human health, as anti-cardiovascular disease, antiviral, anti-inflammatory, anti-oxidant, antitumor action etc.The theoflavin composition of finding in black tea at present has tens kinds, wherein main has following four kinds: the single gallic acid ester (TF-3-MG) of theoflavin (TF), theoflavin-3-, theoflavin-3 '-single gallic acid ester (TF-3 '-MG) and theoflavin-3,3 '-digallic acid ester (TFDG).Wherein theoflavin-3-gallic acid ester (TF-3-MG) and theoflavin-3 '-gallic acid ester (TF-3 '-MG) be two kinds of isomerss.Its structure as shown in Equation 1.
Theaflavin?R＝R′＝H
Theaflavin-3-O-gallate?R＝gallate，R′＝H
Theaflavin-3′-O-gallate?R′＝gallate，R＝H
Theaflavin-3，3′-di-O-gallate?R＝R′＝gallate
A: theoflavin (theaflavin, TF)
B: the single gallic acid ester of theoflavin-3-(theaflavin-3-O-monogallate, TF-3-MG)
C: theoflavin-3 '-single gallic acid ester (theaflavin-3 '-O-monogallate, TF-3 '-MG)
D: theoflavin-3,3 '-digallic acid ester (theaflavin-3,3 '-O-digallate, TFDG)
Further investigation to the pharmacologically active of kinds of theaflavin monomer composition is had higher requirement to the method for separating and preparing of theoflavin, and the wherein excellent difficulty the most of separating with TF-3-MG and TF-3 '-MG yet there are no specific separation method report.The method for separating and preparing of the theoflavin of bibliographical information mainly contains two kinds, i.e. SephadexLH-20 gel filtration chromatography method or high-speed countercurrent chromatography.With regard to these two kinds of methods, gel filtration chromatography method preparation amount is bigger, but required time is longer, and solvent-oil ratio is big; And the high-speed countercurrent chromatography separation efficiency is higher, but preparation amount is not as good as column chromatography.These two kinds of methods of reporting in the document mainly are to be used for little preparation amount to separate (hundreds of milligram applied sample amount) at present.For above-mentioned four kinds of main theoflavin, generally speaking, TF and TFDG ratio are easier to and other theoflavin component separating, and the separation between two kinds of TF-MG has very big challenge all the time, use above-mentioned any method can only realize that all its part separates merely.When being separated into purpose with a large amount of preparations, along with the increase of applied sample amount, both separation selectivities are poorer, and the purifying difficulty is bigger.Therefore, the monomeric big preparation amount separation of high-purity theaflavin has big difficulty all the time.
Summary of the invention
The purpose of this invention is to provide and prepare the method for ten grams to the highly purified kinds of theaflavin monomer of tens grams a kind of the separation from the theoflavin crude extract, this method preparation amount is big, the product purity height, and the time is short, and solvent-oil ratio is few.
For achieving the above object, separation method and step that the present invention adopts are as follows:
1, at first according to post bed height and the diameter of intending filling, calculates and take by weighing a certain amount of gel column filler, carry out swelling with elutriant.Adopt wet method dress post, the gel column that installs should be evenly, no bubble.The preferred Sephadex LH-20 of gel filler in present method; Elutriant can adopt the aqueous acetone solution of dehydrated alcohol, 50-95% aqueous ethanolic solution or 30-70%.
2, get a certain amount of black tea crude extract, go up sample with elutriant dissolving back.According to the color segment on the gel column behind the last sample, Fractional Collections.Collected flow point after HPLC detects, is collected the final stage Fn+1 that wherein only contains first section F0 of TF and only contain TFDG respectively, used gel filtration chromatography and high speed adverse current chromatogram purifying behind the concentrate drying again.And the middle shade section that contains TF-3-MG and TF-3 '-MG different according to the relative content of wherein TF-3-MG and TF-3 '-MG are divided five part collections, be respectively F1, F2, F3, F4 ... Fn (successively arranging) by elution order.Repeat sample repeatedly, make F1, F2, F3, F4 ... Fn obtains enrichment, concentrate drying.
Wherein, the quantity of n can adjust according to the experiment needs, and when n was big, segmentation was thinner, but the follow-up work amount strengthens.
3, will go up gel column respectively again in the fraction more than 50% by the separating obtained TF-3 ' of study-MG relative content separates, TF-3 '-MG relative content that separation and concentration is repeatedly obtained is further purified with HSCCC in the fraction more than 90%, removes wherein a spot of TF-3-MG and other impurity.
4, will merge with above-mentioned similar fraction after separating TF-3 '-MG in the fraction more than 50% by the separating obtained TF-3-MG relative content of study, going up gel column more respectively separates, separation and concentration is repeatedly obtained the TF-3-MG relative content be further purified with HSCCC, remove polar impurity wherein in the fraction more than 90%.
5, fraction that mainly contains TF-3 '-MG that above-mentioned two steps gel filtration chromatographies separation is obtained and the fraction that mainly contains TF-3-MG are carried out the HSCCC purifying respectively to remove wherein a spot of impurity, reach the purified purpose.The solvent system that this step preferably is made up of alkane-fatty acid ester-Fatty Alcohol(C12-C14 and C12-C18)-water carries out high speed adverse current chromatogram to be separated; Wherein alkane can be normal hexane, normal heptane, sherwood oil etc., preferred normal hexane; Fatty acid ester can be ethyl acetate, methyl acetate etc., ethyl acetate; Fatty Alcohol(C12-C14 and C12-C18) can be methyl alcohol or ethanol.Moving phase is preferably descended phase.
6, the first step is separated TF and two sections fractions of TFDG of obtaining and remove some impurity components with gel filtration chromatography respectively, obtain two fractions of C and D respectively, and then obtain high TF of purity and TFDG with HSCCC method purifying respectively.Wherein the purifying of TFDG can adopt solvent system identical in the step 5 and separate mode.And the purifying of TF need to adjust and adopt upper and lower phase respectively the solvent system proportioning be that positive and anti-phase two kinds of separate modes of moving phase could finally be removed some polar impurities.
7, to above-mentioned separation prepare the gained kinds of theaflavin monomer through rotary evaporation concentrate, carry out after the vacuum-drying, lyophilize HPLC purity check and MS,
1HNMR and
13The CNMR structural confirmation.
Advantage of the present invention is: the separation preparation amount of kinds of theaflavin monomer big (tens grams), purity height.Efficient liquid phase chromatographic analysis shows that the kinds of theaflavin monomer purity that adopts this method to separate preparation can reach more than 97%.
Description of drawings
Fig. 1 is the separation preparation flow figure of kinds of theaflavin monomer among the embodiment 1
Fig. 2 is the HPLC spectrogram that theoflavin mixes standard specimen (A) and crude extract (B) among the embodiment 1
Fig. 3 is TF level segmentation (F0) among the embodiment 1, the segmentation of TFMG level (F1-F5) and the HPLC analysis of spectra of TFDG level segmentation (F6)
Fig. 4 is rich in A1, the A2 of TF-3 '-MG and the HPLC analysis of spectra of A3 fraction among the embodiment 2
Fig. 5 is for being rich in A1, the A2 of TF-3 '-MG among the embodiment 2 and the HSCCC of A3 fraction separates spectrogram
Fig. 6 is through the HPLC purity check figure of HSCCC purifying gained TF-3 '-MG among the embodiment 2
Fig. 7 is rich in the B1 of TF-3-MG, the HPLC analysis of spectra of B2 fraction among the embodiment 3
Fig. 8 separates the HPLC purity check figure of spectrogram and gained TF-3-MG for the HSCCC of the B2 fraction that is rich in TF-3-MG among the embodiment 3
Fig. 9 separates the HPLC purity check spectrogram (b) of spectrogram (a) and separating obtained TFDG for the HSCCC of the D fraction that obtains from TFDG level segmentation (F6) among the embodiment 4
Figure 10 for the HPLC spectrogram (a) of the C fraction that obtains from TF level segmentation (F0) among the embodiment 4, HSCCC purifying spectrogram (b, c) and the HPLC purity check figure (d) of gained TF
The separating obtained four kinds of kinds of theaflavin monomer of Figure 11 the evaluation structure
Embodiment
Embodiment 1: the first step column chromatography for separation of theoflavin crude extract (referring to the schema of Fig. 1)
Get black tea crude extract 40g, go up gel chromatographic columns 1 (Sephadex LH-20,900g, 100 * 10cm i.d.) with 70ml elutriant dissolving back and separate.Dehydrated alcohol is an elutriant, and mean flow rate is 18ml/min.According to the color segment on the gel column behind the last sample, Fractional Collections.After HPLC detects, collected flow point is merged into three sections 7 fractions, be respectively the F0 that mainly contains TF, contain F1, F2, F3, F4, the F5 (successively arranging) of TF-3-MG and TF-3 '-MG and mainly contain the F6 fraction of TFDG by elution order.Mixed standard specimen of Sigma and the HPLC analysis of spectra of theoflavin crude extract under two wavelength have been provided among Fig. 2.Analysis condition: chromatographic column: Waters Xterra RP-C18 post (5 μ m, 150 * 4.6mmi.d.); Moving phase: acetonitrile-water (28:72) (in acetonitrile and water, adding 2% acetate respectively); Flow velocity: 1mL/min; Detect wavelength: 280nm, 374nm.By the UV spectrogram at contrast retention time and each peak, determined the target components of the theoflavin in the crude extract, as can be seen in the crude extract except that four kinds of main theoflavin compositions, also have a large amount of impurity and its coexistence.The HPLC analysis of spectra of three sections 7 fractions that obtain after gel column 1 chromatographic separation as shown in Figure 3.By more as can be seen, through a step column chromatography, TF can separate with the TFMG in second section basically fully with the TFDG section in the crude extract, just wherein still contains other impurity, need be further purified.Separate and the TF in second section-3-MG and TF-3 '-MG can only realize part, also need by further column chromatography for separation.Handle black tea crude extract 480 grams, obtain F altogether
0(70 gram), F
1(7.5 gram), F
2(29.17 gram), F
3(32.72 gram), F
4(21.32 gram), F
5(10.33 gram) and F
6(80 gram).
The further separation and the purifying of embodiment 2:TF-3 '-MG fraction
To go up gel column respectively again in the F2 more than 50% and F3 fraction by the separating obtained TF-3 ' of study-MG relative content and carry out secondary separation, the column chromatography condition that is adopted is identical with embodiment 1.TF-3 ' in the gained fraction-MG content carries out column chromatography for separation for the third time respectively in 50%-90% fraction, use 35%-aqueous acetone solution wash-out instead in this time separating, this moment, TF-3-MG was opposite with the alcoholic acid elution order with TF-3 '-MG elution order, TF-3-MG formerly, TF-3 '-MG after, can obtain the higher TF-3 ' of more purity-MG from hangover part like this.TF-3 '-MG relative content that column chromatography for separation enrichment is repeatedly obtained is at three the fraction A1 (7.83g) more than 90%, A2 (7.89g) and A3 (6.93g) (wherein F1 fraction and similar fraction being merged) are further purified with HSCCC respectively, remove wherein a spot of TF-3-MG and other impurity.
The HPLC of A1, A2 and three fractions of A3 analyzes as shown in Figure 4, as can be seen, wherein mainly contains TF-3 '-MG (90%) and a spot of TF-3-MG (10%) among the A1, contains the impurity of a large amount of 7.4min in addition; Composition and the A1 of A2 are basic identical, and just the content of the impurity of 7.4min decreases; The content of the impurity of 7.4min further reduces among the A3.
Fig. 5 has provided the spectrogram that separates that respectively three fractions of A1, A2 and A3 is carried out purifying with HSCCC.The separation condition that is adopted is: solvent system: normal hexane-ethyl acetate-methyl alcohol-water (1:5:1:5, v/v/v/v), on be stationary phase mutually, is moving phase mutually down.As can be seen, separate through HSCCC, the impurity of a spot of TF-3 '-MG, 7.4min and some a spot of other impurity can be removed simultaneously, and the applied sample amount of HSCCC has reached the gram magnitude, this reaches the preparation amount upper limit of present HSCCC instrument (column capacity 220mL, internal diameter 1.6mm) substantially.From the HPLC spectrogram of Fig. 6 as can be seen, all reach more than 97% in the HPLC purity (with the HPLC calculated by peak area) that difference detects (280nm and 374nm) under the wavelength through refining gained TF-3 '-MG.Obtain nearly 10 grams of high purity TF-3 '-MG altogether.
The further separation and the purifying of embodiment 3:TF-3-MG fraction
To merge with above-mentioned similar fraction after separating TF-3 '-MG at the F4 more than 50% and F5 by the separating obtained TF-3-MG relative content of study, go up gel column more respectively and carry out secondary separation, the TF-3-MG relative content is in 50%-90% fraction in the gained fraction, carry out column chromatography for separation for the third time respectively, the column chromatography condition that is adopted is with embodiment 1.Separation and concentration is repeatedly obtained the TF-3-MG relative content at the level B1 (0.52g) more than 90%, and B2 (17.27g) is further purified with HSCCC respectively, removes polar impurity wherein.
Analyze (as shown in Figure 7) as can be seen through HPLC, wherein B1 contains about 90% TF-3-MG and TF-3 '-MG of 10%; B2 then mainly contains TF-3-MG, and TF-3 '-MG content is very low, but all contains a large amount of polar impurities in each fraction.The HSCCC that has provided the B2 fraction on Fig. 8 separates spectrogram, and the separation condition that is adopted is with embodiment 2.HPLC analyzes (under Fig. 8) and shows, separates through HSCCC, and polar impurity can be removed fully, and the applied sample amount of HSCCC has also reached the gram magnitude.Also reached more than 97% in the HPLC purity (with the HPLC calculated by peak area) that difference detects under the wavelength (280nm and 374nm) through refining gained TF-3-MG.Obtain nearly 10 grams of high purity TF-3-MG altogether.
The HSCCC purifying of embodiment 4:TF and TFDG
The theoflavin crude extract is through mainly containing TF and TFDG respectively among step first section fraction F0 obtaining of column chromatography for separation and the 3rd section fraction F6, they have realized separating fully with other theoflavin composition substantially, but wherein still contain other impurity (shown in the HPLC analytical results of the F0 of Fig. 3 and F6).To their method that can adopt column chromatography that is further purified, but often still can residual small amount of polar impurity in the column chromatography gained fraction, therefore, need adopt HSCCC to be further purified.
Get 8 gram F6 fractions, last gel chromatographic columns 2 (Sephadex LH-20,400g, 70 * 5cm i.d.) is an elutriant with the dehydrated alcohol, separates.Obtain containing the fraction D (about 5 grams) of TFDG, adopt HSCCC that it is further purified.Fig. 9 has provided the HSCCC purifying spectrogram of D fraction and the HPLC analysis of spectra of final gained TFDG.The separation condition that is adopted is: solvent system: normal hexane-ethyl acetate-methyl alcohol-water (1:5:1:5, v/v/v/v), on be stationary phase mutually, be moving phase mutually down, applied sample amount is 1 to restrain.TFDG has also reached more than 98% in the HPLC purity (with the HPLC calculated by peak area) that difference detects under the wavelength (280nm and 374nm).From 8 gram F6 fractions, obtain about 2 grams of high purity TFDG altogether.
The C fraction that 8 gram F0 obtain behind same secondary column chromatography (about 5 grams), (Figure 10 a) shows that wherein containing retention time is difficult to remove at two polar impurities of 2.3min and 2.5min in the HPLC analysis.In the HSCCC purifying, at first adopt normal hexane-ethyl acetate-methyl alcohol-water (2:5:2:5, v/v/v/v), be stationary phase mutually down, on be moving phase mutually, applied sample amount is 0.9 to restrain, carry out the first step purifying (shown in Figure 10 b), and then adopt normal hexane-ethyl acetate-methyl alcohol-water (1:7:1:7, v/v/v/v), on be stationary phase mutually, be moving phase mutually down, applied sample amount is that 0.7 gram carries out secondary HSCCC purifying (shown in Figure 10 c).Combination by positive and anti-phase two kinds of HSCCC separate modes like this, two polar impurities are removed the most at last, obtain highly purified TF (shown in Figure 10 d), the HPLC purity (with the HPLC calculated by peak area) that detects under the wavelength (280nm and 374nm) in difference has also reached more than 98%.From 8 gram F0 fractions, obtain about 1 gram of high purity TF altogether.
Embodiment 5: the proton nmr spectra of separation and purification gained TF, TF-3-MG, TF-3 '-MG and TFDG (
1HNMR) and carbon spectrum (
13CNMR) analyze
To four kinds of separating obtained kinds of theaflavin monomer carried out proton nmr spectra (
1HNMR) and carbon spectrum (
13CNMR) analyze, adopt deuterated acetone (CD
3OCD
3) be deuterated reagent, the result shows coincide substantially with the data of bibliographical information (listed to table 4 as table 1).Wherein the pairing position of C and H as shown in figure 11.
Table 1TF-3 '-MG and TF-3-MG's
13The CNMR data
(CD
3OCD
3，600MHz)
*Davis，A.L.，Cai，Y.，Davies，A.P.(1995).Magn.Reson.Chem.，33，549-552.
Table 2TF-3 '-MG and TF-3-MG's
1The HNMR data
(CD
3OCD
3，600MHz)
*Davis，A.L.，Cai，Y.，Davies，A.P.(1995).Magn.Reson.Chem.，33，549-552.
Table 3TF and TFDG's
13The CNMR data
(CD
3COCD
3，600MHz)
*Davis，A.L.，Cai，Y.，Davies，A.P.(1995).Magn.Reson.Chem.，33，549-552.
Table 4TF and TFDG's
1The HNMR data
(CD
3COCD
3，600MHz)
*Davis，A.L.，Cai，Y.，Davies，A.P.(1995).Magn.Reson.Chem.，33，549-552
Claims (9)
1. the method for a large-batch separating preparation of high-purity theaflavine monomer, it is characterized in that: it may further comprise the steps:
(1), with elutriant the gel column column chromatography is gone up in black tea crude extract dissolving back, according to the color segment on the gel column behind the last sample, Fractional Collections, the final stage Fn+1 that wherein only contains first section F0 of TF and only contain TFDG is collected respectively, use gel filtration chromatography and high speed adverse current chromatogram purifying behind the concentrate drying again; And the middle shade section that contains TF-3-MG and TF-3 '-MG different according to the relative content of wherein TF-3-MG and TF-3 '-MG divide a plurality of fractions to collect, be respectively F1, F2, F3, F4 ... Fn, repeat sample repeatedly, make F1, F2, F3, F4 ... Fn obtains enrichment, concentrate drying;
(2), will go up gel column respectively again in the fraction more than 50% by the separating obtained TF-3 ' of study-MG relative content separates, TF-3 '-MG relative content that separation and concentration is repeatedly obtained separates with high speed adverse current chromatogram in the fraction more than 90% and is further purified, and removes wherein a spot of TF-3-MG and other impurity;
(3), merge with above-mentioned similar fraction after separating TF-3 '-MG in the fraction more than 50% by the separating obtained TF-3-MG relative content of study, going up gel column more respectively separates, separation and concentration is repeatedly obtained the TF-3-MG relative content separate with high speed adverse current chromatogram in the fraction more than 90% and be further purified, remove polar impurity wherein.
2. the method for large-batch separating preparation of high-purity theaflavine monomer according to claim 1, it is characterized in that: described gel filler is Sephadex LH-20, and described elutriant is the aqueous acetone solution of dehydrated alcohol, 50-95% aqueous ethanolic solution or 30-70%.
3. the method for large-batch separating preparation of high-purity theaflavine monomer according to claim 1 and 2 is characterized in that: described TF-3 '-MG and TF-3-MG relative content are the solvent system that alkane-fatty acid ester-Fatty Alcohol(C12-C14 and C12-C18)-water is formed at the isolating solvent system of the high speed adverse current chromatogram of the fraction more than 90%.
4. the method for large-batch separating preparation of high-purity theaflavine monomer according to claim 3, it is characterized in that: wherein alkane is normal hexane, normal heptane, sherwood oil; Fatty acid ester is ethyl acetate or methyl acetate; Fatty Alcohol(C12-C14 and C12-C18) can be methyl alcohol or ethanol; Its proportioning is 1: 5: 1: 5 or 1: 10: 1: 10.
5. the method for large-batch separating preparation of high-purity theaflavine monomer according to claim 4 is characterized in that: be moving phase down mutually.
6. the method for large-batch separating preparation of high-purity theaflavine monomer according to claim 1 and 2 is characterized in that: wherein, the isolating solvent system of the high speed adverse current chromatogram of TF is the solvent system that alkane-fatty acid ester-Fatty Alcohol(C12-C14 and C12-C18)-water is formed.
7. the method for large-batch separating preparation of high-purity theaflavine monomer according to claim 6, it is characterized in that: wherein alkane is normal hexane, normal heptane, sherwood oil; Fatty acid ester is ethyl acetate or methyl acetate; Fatty Alcohol(C12-C14 and C12-C18) is methyl alcohol or ethanol; Its proportioning is 2: 5: 2: 5 or 1: 10: 1: 10; The positive or the anti-phase two kinds of separate modes that in the employing are moving phase mutually or are down mutually removed polar impurity.
8. the method for large-batch separating preparation of high-purity theaflavine monomer according to claim 1 and 2 is characterized in that: wherein, the isolating solvent system of the high speed adverse current chromatogram of TFDG is the solvent system that alkane-fatty acid ester-Fatty Alcohol(C12-C14 and C12-C18)-water is formed.
9. the method for large-batch separating preparation of high-purity theaflavine monomer according to claim 8, it is characterized in that: wherein alkane is normal hexane, normal heptane, sherwood oil; Fatty acid ester is ethyl acetate or methyl acetate; Fatty Alcohol(C12-C14 and C12-C18) is methyl alcohol or ethanol; Its proportioning is 1: 5: 1: 5 or 1: 10: 1: 10; Be moving phase mutually down.
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| CN101235025B (en) * | 2008-02-29 | 2011-11-02 | 天津科技大学 | Method for preparing theaflavin |
| CN101475556B (en) * | 2009-01-20 | 2011-09-14 | 南方医科大学 | 1',2',3',7'-tetrahydrotheaflavine, and preparation and use thereof |
| CN106957884B (en) * | 2016-01-12 | 2020-10-02 | 中华全国供销合作总社杭州茶叶研究所 | Theaflavin product rich in tea catechin |
| CN106432167B (en) * | 2016-07-19 | 2018-06-01 | 广东省农业科学院茶叶研究所 | A kind of method that theaflavin is extracted from black tea |
| CN107821687B (en) * | 2017-08-30 | 2021-06-04 | 中国农业科学院茶叶研究所 | Preparation method of thearubigin |
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