CN106957884A - A kind of theaflavin product rich in tea catechin - Google Patents
A kind of theaflavin product rich in tea catechin Download PDFInfo
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- CN106957884A CN106957884A CN201610017472.7A CN201610017472A CN106957884A CN 106957884 A CN106957884 A CN 106957884A CN 201610017472 A CN201610017472 A CN 201610017472A CN 106957884 A CN106957884 A CN 106957884A
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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Abstract
The invention discloses a kind of theaflavin product rich in tea catechin, relate generally to it is a kind of by the tea tree being fully ground after crushing (Camellia sinensis) fresh leaf or tender stem mix with the specific water saturated ethyl acetate of certain proportion, and water-bath vibration theaflavin product rich in tea catechin prepared by carry out enzymatic reaction.
Description
Technical field
The present invention relates to a kind of theaflavin product, more particularly to a kind of theaflavin product rich in tea catechin.
Background technology
Fresh leaves of tea plant (Camellia sinensis
(L.) O. Ktze) contain tea catechin class polyphenols and its corresponding polyphenol oxidase, under temperature and the oxygen effect of appropriateness, haptoreaction can generate theaflavins (Theaflavins) polyphenol compound.I.e. epicatechin (Epi-Catechin,
EC) joint epigallocatechin (Epigallo-Catechin,
EGC), EC combines Epigallo-catechin gallate (EGCG) (Epigallo-Catechin
Gallate, EGCG), L-Epicatechin gallate (Epi-Catechin
Gallate, ECG) combine EGC, ECG joint EGCG compositions through polyphenol oxidase (teapolyphenol
Oxidase, TPPO) enzymatic oxidation polycondensation form theaflavins compound.Theaflavins polyphenol compound generally includes simple theaflavin (TF) and ester type theaflavin, the composition that wherein ester type theaflavin is related generally to is TF-3-G (TF-3-G), theaflavin -3 '-mono- gallate (TF-3 '-G) and theaflavin -3,3 '-digallic acid ester (TF-3,3 '-G).Theaflavins polyphenol compound has unique effects such as antioxidation in vitro, reduction cardiovascular and cerebrovascular disease, inducing apoptosis of tumour cell, regulation immune cell function, resisiting influenza virus and inhibition of HIV.
The enzyme' s catalysis of theaflavin can both be carried out in pure water phase, can also be carried out in ester water Two Liquid Phases.Tea Polyphenols enzymatic oxidation is under the not miscible organic solvent composition biliquid phase system reaction condition of water and water, theaflavin content (Xiao Weixiang can be improved, Zhong Jin, Hu Yaowu, Xiao Hui Study On The Formation of The Tea Pigments With Two-liquid Phase Chemzyme Technology research and development of natural products, 2001,13 (5):49-52. | | Tea Pigment parameter optimization tea sciences, 2002,22 (2) are produced in the auspicious Tea Polyphenols biliquid phase oxidation of Li Lixiang, Xiao Wei:119-124. | | Tea Pigment technical study, Agricultural University Of Nanjing's journal, 2004,27 (2) are produced in the auspicious Tea Polyphenols biliquid phase oxidation of Li Lixiang, Xiao Wei: 99-104.).
Pertinent literature has carried out more detailed parameter optimization research under conditions of clearly being constituted using ethyl acetate and water as reaction system.Result of study shows, appropriate ethyl acetate is added in reaction system, can improve theaflavin growing amount (fresh different tea leaf homogenates Suspension fermentation high theaflavin content Tea Pigment preparation in application study Agricultural University Of Anhui master thesis, 2012. | | the optimization Southwestern University master thesis .2013. of all clean theaflavin enzymatic oxidation synthesis conditions).
Chinese patent CN200810023581.5 discloses the new technology for preparing theaflavin, it is to extract Tea Polyphenols using ethyl acetate from green tea to be used as raw material that it, which is prepared, and using the fresh tea leaf fully crushed as fermenting enzyme, after being passed through and being stirred fermentation under pure oxygen, again by separation, concentration, washing, etc. step, obtain qualified theaflavin product.Meanwhile, Chinese patent CN200910153520.5 also discloses that a kind of industrialization preparation method of theaflavin, and what the patent was used is respectively the tea fresh leaves of crushing using ethyl acetate and water composition as each component in reaction system:Ethyl acetate solution rich in Tea Polyphenols：Ethyl acetate：The amount ratio of cushioning liquid is equal to 800 kg：600-900 kg ：15-25 L ：1-2 L.
The present inventor has found during above-mentioned experiment is repeated, and the Two Liquid Phases constituted using ethyl acetate and water are as under conditions of reaction system, although can preferably synthesize theaflavin, but tea catechin is consumed excessively, and diester-type theaflavin cannot be effectively synthesized.
The content of the invention
It is an object of the invention to provide a kind of tea catechin using contained by fresh leaves of tea plant itself and Tea Polyphenols oxidizing ferment theaflavin product is prepared come enzyme' s catalysis.Another object of the present invention is farthest to retain the tea catechin not being converted on the premise of theaflavin yield is improved.A kind of theaflavin product rich in tea catechin that the present invention is provided, is achieved by the steps of：
(1) by tea tree (Camellia sinensis) fresh leaf or tender stem be fully ground crushing；
(2) normal hexane or petroleum ether are added in water saturated ethyl acetate by a certain percentage, specific water saturated ethyl acetate mixed solvent is made；
(3) fresh leaves of tea plant or tender stem crushed step (1) are mixed by a certain percentage with the specific water saturated ethyl acetate mixed solvent in step (2) and water-bath vibration carries out enzymatic reaction；
(4) reaction solution of step (3) is filtered with filter paper at reduced pressure conditions, obtains filtered fluid；
(5) filtered fluid of abundant concentration step (4) is depressurized, concentrated extract is obtained；
(6) added into the concentrated extract of step (5) 5% ethanol water, abundant mechanical shaking extraction object obtains extract solution；
(7) extract solution of concentration step (6)；With
(8) the optionally product of drying steps (7), obtains the theaflavin product rich in tea catechin.
Preferably, the normal hexane or petroleum ether wherein described in step (2) account for the 2.5-35 % (v/v) of specific water saturated ethyl acetate mixed solvent.
Preferably, the fresh leaves of tea plant wherein described in step (3) and specific water saturated ethyl acetate mixed solvent solid-to-liquid ratio are 1:20~1:40.
Preferably, the time of enzymatic reacting wherein described in step (3) is in 5~120 min, and bath temperature is at 15~50 DEG C.
Preferably, wherein step (5) and (7) are carried out below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature.
Preferably, in the theaflavin product rich in tea catechin wherein involved by step (8), tea catechin refers to epicatechin (EC), epigallocatechin (EGC), Epigallo-catechin gallate (EGCG) (EGCG) and L-Epicatechin gallate (ECG).
Preferably, in the theaflavin product rich in tea catechin wherein involved by step (8), theaflavin refers to simple theaflavin (TF), TF-3-G (TF-3-G), theaflavin -3 '-mono- gallate (TF-3 '-G) and theaflavin -3,3 '-digallic acid ester (TF-3,3 '-G).
Preferably, the wherein extract solution of step (6) is with ISO14502-2:2005 (E) or by adjusted ISO14502-2 described in embodiment:In the relation of each composition integral area of high performance liquid chromatography detection as defined in 2005 (E) methods, EGCG>TF-3,3 '-G, 0.7< (TF-3, 3′-G / ECG) <1, and TF-3,3 '-G are more than both sums between remaining each kinds of theaflavin monomer.
Beneficial effect
On the one hand the scheme implemented using the present invention, can reduce the proportion of goods damageds of tea catechin；On the other hand diester-type theaflavin can be improved is converted into rate, so as to realize preferable tea catechin utilization rate and preferably economic benefit.
Brief description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram that embodiment 1-3 is analyzed.
Fig. 2 is the high-efficient liquid phase chromatogram that embodiment 4-5 is analyzed.
Fig. 3 is the high-efficient liquid phase chromatogram that embodiment 6-7 is analyzed.
Fig. 4 is the high-efficient liquid phase chromatogram that embodiment 8-10 is analyzed.
Fig. 5 is the high-efficient liquid phase chromatogram that embodiment 11-13 is analyzed.
Fig. 6 is the high-efficient liquid phase chromatogram that embodiment 14-16 is analyzed.
Embodiment
With reference to embodiment, the purport of the present invention is further illustrated.
Following embodiments, which are used for explaining, to be not intended to limit the present invention, and under the premise of spirit and claims of the present invention, any modification that the present invention makes is all considered to fall into protection scope of the present invention.
The analysis of target product uses high performance liquid chromatography in following embodiments.Waters high performance liquid chromatographs (1525 pumps, column oven, 2487 UV, visible light double-channel detectors, 717-plus automatic samplers,BreezeControl software bag), chromatographic column is 5 μm of Phenomenex Luna
The mm of Phenyl-Hexyl, 250 mm × 4.6, column temperature is 35 DEG C, and Detection wavelength is 278 nm, the μ L of sample size 10.Efficient liquid phase chromatographic analysis is by the way of two-phase gradient elution, and wherein A phases are aqueous phase (acetonitrile of acetic acid and 9 % containing 2 %, v/v), and B phases are organic phase (acetonitrile of acetic acid and 80 % containing 2 %, v/v).Embodiment 1~10 uses ISO14502-2:The mode of 2005 (E) elution；Embodiment 11~13 is in ISO14502-2:0 to 10 min (100 % A) → 10 to 21.25 min (76 % A) → 21.25 to 29 min (76 % A) → 29 to 29.2 min (76 % are adjusted on the basis of 2005 (E) elution requirement
A, 1.3mi/min) → 29.2 to 39 min (76 % A, 1.3mi/min) → 39 to 39.2 min (100 % A) → 39.2 to 45 min (100 % A)；Embodiment 14~16 is in ISO14502-2:0 to 10 min (100 % A) → 10 to 21.25 min (76 % A) → 21.25 to 39 min (76 % A) → 39 to 41 min (100 % A) → 41 to 46 min (100 % A) is adjusted on the basis of 2005 (E) elution requirement.
Fresh leaves of tea plant or tender stem employed in following experimental examples are picked up from the first tenday period of a month in May, 2015, and it is standby to place freezing (- 20 DEG C) in refrigerator.Fresh leaves of tea plant or tender stem employed in following experimental examples are the fresh leaf or tender stem of the abundant attrition grinding in mortar.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following experimental examples.
Embodiment 1
The accurate tender stem for weighing 0.7257 g attrition grindings is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL, after (15 DEG C) 5 min of vibration of normal temperature, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -1 and table 1 in Fig. 1), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 1
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 2238262 | 157493 | 1370645 | 560727 | 257519 | 130192 | 77150 |
Embodiment 2
The accurate tender stem for weighing 0.7023 g attrition grindings is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL, after (35 DEG C) 120 min of vibration of water-bath, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -2 and table 2 in Fig. 1), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 2
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 2076222 | 43586 | 522939 | 399851 | 418620 | 412256 | 156880 |
Embodiment 3
The fresh leaves of tea plant of the accurate tender stem and 0.2170 g attrition grindings for weighing 0.6411 g attrition grindings is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL, after (35 DEG C) 120 min of vibration of water-bath, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -3 and table 3 in Fig. 1), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 3
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 2749060 | Do not detect | 140670 | 265097 | 502498 | 556447 | 697563 |
Embodiment 4
The accurate fresh leaves of tea plant for weighing 0.5 g attrition grindings, the high fire of microwave is after 40 seconds, it is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL, after mechanical shaking extraction, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -4 and table 4 in Fig. 2), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 4
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 2839859 | 48215 | 2485347 | 983214 | 92182 | 64198 | 143545 |
Embodiment 5
The accurate fresh leaves of tea plant for weighing 0.5 g attrition grindings (as the Duplicate Samples of embodiment 4), the high fire of microwave is after 40 seconds, it is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL, after mechanical shaking extraction, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -5 and table 5 in Fig. 2), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 5
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 2963288 | 53784 | 2827240 | 1097723 | 76167 | 48375 | 87189 |
Embodiment 6
The accurate fresh leaves of tea plant for weighing 0.5 g attrition grindings, it is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL, after the min of water-bath (35 DEG C) oscillating reactions 5, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -6 and table 6 in Fig. 3), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 6
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 2811228 | 8715 | 1349179 | 600956 | 135093 | 150445 | 393842 |
Embodiment 7
The accurate fresh leaves of tea plant for weighing 0.5 g attrition grindings, it is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL, after the min of water-bath (35 DEG C) oscillating reactions 30, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -7 and table 7 in Fig. 3), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 7
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 2780535 | Do not detect | 829488 | 455256 | 123762 | 170850 | 528947 |
Embodiment 8
The accurate fresh leaves of tea plant for weighing 0.2481 g attrition grindings, it is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL, after the min of water-bath (15 DEG C) oscillating reactions 120, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -8 and table 8 in Fig. 4), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 8
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 1820408 | Do not detect | 1538888 | 658108 | 142926 | 108894 | 171845 |
Embodiment 9
The accurate fresh leaves of tea plant for weighing 0.525 g attrition grindings, is placed in the flat conical flasks of 100 mL, and adding the specific water saturated ethyl acetate of 20 mL, (the ratio between water saturated ethyl acetate and petroleum ether volume is 40:1), after the min of water-bath (35 DEG C) oscillating reactions 30, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -9 and table 9 in Fig. 4), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 9
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 2960583 | Do not detect | 782014 | 493280 | 134249 | 177667 | 608392 |
Embodiment 10
The accurate fresh leaves of tea plant for weighing 0.5025 g attrition grindings, is placed in the flat conical flasks of 100 mL, and adding the specific water saturated ethyl acetate of 20 mL, (the ratio between water saturated ethyl acetate and petroleum ether volume is 20:1), after the min of water-bath (35 DEG C) oscillating reactions 30, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -10 and table 10 in Fig. 4), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 10
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G + TF-3, 3′-G |
| Integral area | 3253717 | Do not detect | 742215 | 472706 | 136650 | 192368 | 703953 |
Embodiment 11
The accurate fresh leaves of tea plant for weighing 0.5115 g attrition grindings, is placed in the flat conical flasks of 100 mL, and adding the specific water saturated ethyl acetate of 20 mL, (the ratio between water saturated ethyl acetate and normal hexane volume is 8:1), after the min of water-bath (45 DEG C) oscillating reactions 30, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -11 and table 11 in Fig. 5), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 11
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G | TF-3, 3′-G |
| Integral area | 3383546 | Do not detect | 717187 | 586483 | 135748 | 197222 | 159649 | 457178 |
Embodiment 12
The accurate fresh leaves of tea plant for weighing 0.5391 g attrition grindings, is placed in the flat conical flasks of 100 mL, and adding the specific water saturated ethyl acetate of 20 mL, (the ratio between water saturated ethyl acetate and petroleum ether volume is 8:1), after the min of water-bath (18 DEG C) oscillating reactions 60, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -12 and table 12 in Fig. 5), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 12
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G | TF-3, 3′-G |
| Integral area | 3110159 | 2957 | 963873 | 573040 | 133102 | 182781 | 152277 | 441419 |
Embodiment 13
The accurate fresh leaves of tea plant for weighing 0.5146 g attrition grindings, is placed in the flat conical flasks of 100 mL, and adding the specific water saturated ethyl acetate of 20 mL, (the ratio between water saturated ethyl acetate and petroleum ether volume is 8:1), after the min of water-bath (18 DEG C) oscillating reactions 120, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -13 and table 13 in Fig. 5), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 13
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G | TF-3, 3′-G |
| Integral area | 3080455 | Do not detect | 512429 | 486095 | 129237 | 193550 | 153854 | 438713 |
Embodiment 14
The accurate fresh leaves of tea plant for weighing 0.5082 g attrition grindings, it is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL and 5 mL petroleum ethers, it is sufficiently mixed and after the min of water-bath (35 DEG C) oscillating reactions 30, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -14 and table 14 in Fig. 6), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 14
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G | TF-3, 3′-G |
| Integral area | 3239301 | Do not detect | 61771 | 224738 | 140088 | 243423 | 193513 | 757353 |
Embodiment 15
The accurate fresh leaves of tea plant for weighing 0.5261 g attrition grindings, it is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 20 mL and 10 mL petroleum ethers, it is sufficiently mixed and after the min of water-bath (35 DEG C) oscillating reactions 30, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -15 and table 15 in Fig. 6), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 15
| Composition | Caffeine | EC | EGCG | ECG | TF | TF-3-G | TF-3′-G | TF-3, 3′-G |
| Integral area | 3395020 | Do not detect | 4157 | 56580 | 83387 | 182871 | 130266 | 551148 |
Embodiment 16
The accurate fresh leaves of tea plant for weighing 0.5479 g attrition grindings, it is placed in the flat conical flasks of 100 mL, add the water saturated ethyl acetate of 10 mL and 10 mL petroleum ethers, it is sufficiently mixed and after the min of water-bath (35 DEG C) oscillating reactions 30, it is filtered under diminished pressure with filter paper rapidly, obtain filtered fluid, after filtered fluid below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature fully concentrate, add the 5 % mL of ethanol water 20, abundant mechanical shaking extraction object and constant volume is in 25 mL volumetric flasks, shake up the rear μ L of sample introduction 10 and carry out efficient liquid phase chromatographic analysis (result is shown in embodiment -16 and table 16 in Fig. 6), remaining solution concentrate drying must be rich in the theaflavin product of tea catechin.
Each composition integral area in the efficient liquid phase chromatographic analysis figure of table 16
| Composition | EGCG | ECG | TF | TF-3-G | TF-3′-G | TF-3, 3′-G |
| Integral area | 5910 | 7804 | 25795 | 50464 | 36421 | 161471 |
Claims (8)
1. a kind of theaflavin product rich in tea catechin, including：
(1) by tea tree (Camellia sinensis) fresh leaf or tender stem be fully ground crushing；
(2) normal hexane or petroleum ether are added in water saturated ethyl acetate by a certain percentage, specific water saturated ethyl acetate mixed solvent is made；
(3) fresh leaves of tea plant or tender stem crushed step (1) are mixed by a certain percentage with the specific water saturated ethyl acetate mixed solvent in step (2) and water-bath vibration carries out enzymatic reaction；
(4) reaction solution of step (3) is filtered with filter paper at reduced pressure conditions, obtains filtered fluid；
(5) filtered fluid of abundant concentration step (4) is depressurized, concentrated extract is obtained；
(6) added into the concentrated extract of step (5) 5% ethanol water, abundant mechanical shaking extraction object obtains extract solution；
(7) extract solution of concentration step (6)；With
(8) the optionally product of drying steps (7), obtains the theaflavin product rich in tea catechin.
2. the normal hexane or petroleum ether described in product as claimed in claim 1, wherein step (2) account for 2.5~35 % (v/v) of specific water saturated ethyl acetate mixed solvent.
3. the fresh leaves of tea plant and specific water saturated ethyl acetate mixed solvent solid-to-liquid ratio described in product as claimed in claim 1, wherein step (3) are 1:20~1:40.
4. the time of enzymatic reacting described in product as claimed in claim 1, wherein step (3) is in 5~120 min, bath temperature is at 15~50 DEG C.
5. product as claimed in claim 1, wherein step (5) and (7) are carried out below 50 DEG C of the MPa of the MPa of vacuum -0.07~-0.1 and temperature.
6. product as claimed in claim 1, in the theaflavin product rich in tea catechin wherein involved by step (8), tea catechin refers to epicatechin (EC), epigallocatechin (EGC), Epigallo-catechin gallate (EGCG) (EGCG) and L-Epicatechin gallate (ECG).
7. product as claimed in claim 1, in the theaflavin product rich in tea catechin wherein involved by step (8), theaflavin refers to simple theaflavin (TF), TF-3-G (TF-3-G), theaflavin -3 '-mono- gallate (TF-3 '-G) and theaflavin -3,3 '-digallic acid ester (TF-3,3 '-G).
8. the extract solution of product as claimed in claim 1, wherein step (6) is with ISO14502-2:2005 (E) or by adjusted ISO14502-2 described in embodiment:In each composition integral area of high performance liquid chromatography detection as defined in 2005 (E) methods, EGCG>
TF-3,3 '-G, 0.7< (TF-3, 3′-G / ECG) <1, and TF-3,3 '-G are more than both sums between remaining each kinds of theaflavin monomer.
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