CN105820148B - Technology for purifying tea catechin from tea polyphenol - Google Patents
Technology for purifying tea catechin from tea polyphenol Download PDFInfo
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- CN105820148B CN105820148B CN201510001063.3A CN201510001063A CN105820148B CN 105820148 B CN105820148 B CN 105820148B CN 201510001063 A CN201510001063 A CN 201510001063A CN 105820148 B CN105820148 B CN 105820148B
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- tea
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- catechin
- water
- ethyl acetate
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- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 title claims abstract description 75
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 72
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 72
- 238000005516 engineering process Methods 0.000 title abstract description 14
- 241001122767 Theaceae Species 0.000 title abstract description 4
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 claims abstract description 20
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 13
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 claims abstract description 9
- 238000006467 substitution reaction Methods 0.000 claims abstract description 8
- 239000001863 hydroxypropyl cellulose Substances 0.000 claims abstract description 7
- 238000007670 refining Methods 0.000 claims abstract 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical class CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 91
- 235000013616 tea Nutrition 0.000 claims description 91
- 244000269722 Thea sinensis Species 0.000 claims description 79
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 53
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 30
- 229940030275 epigallocatechin gallate Drugs 0.000 claims description 27
- 239000003480 eluent Substances 0.000 claims description 25
- 239000006228 supernatant Substances 0.000 claims description 25
- 239000000843 powder Substances 0.000 claims description 19
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 16
- 239000000741 silica gel Substances 0.000 claims description 16
- 229910002027 silica gel Inorganic materials 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 14
- XMOCLSLCDHWDHP-UHFFFAOYSA-N L-Epigallocatechin Natural products OC1CC2=C(O)C=C(O)C=C2OC1C1=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-UHFFFAOYSA-N 0.000 claims description 13
- 150000002148 esters Chemical class 0.000 claims description 13
- 239000012141 concentrate Substances 0.000 claims description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 10
- 238000004090 dissolution Methods 0.000 claims description 9
- -1 tea catechin class compound Chemical class 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- VZOPRCCTKLAGPN-ZFJVMAEJSA-L potassium;sodium;(2r,3r)-2,3-dihydroxybutanedioate;tetrahydrate Chemical compound O.O.O.O.[Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O VZOPRCCTKLAGPN-ZFJVMAEJSA-L 0.000 claims description 6
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 claims description 5
- DZYNKLUGCOSVKS-UHFFFAOYSA-N epigallocatechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3cc(O)c(O)c(O)c3 DZYNKLUGCOSVKS-UHFFFAOYSA-N 0.000 claims description 5
- 230000008929 regeneration Effects 0.000 claims description 5
- 238000011069 regeneration method Methods 0.000 claims description 5
- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 claims description 4
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 4
- LPTRNLNOHUVQMS-UHFFFAOYSA-N epicatechin Natural products Cc1cc(O)cc2OC(C(O)Cc12)c1ccc(O)c(O)c1 LPTRNLNOHUVQMS-UHFFFAOYSA-N 0.000 claims description 4
- 235000012734 epicatechin Nutrition 0.000 claims description 4
- 229940074446 sodium potassium tartrate tetrahydrate Drugs 0.000 claims description 4
- 229910021536 Zeolite Inorganic materials 0.000 claims description 3
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000010457 zeolite Substances 0.000 claims description 3
- LSHVYAFMTMFKBA-PZJWPPBQSA-N (+)-catechin-3-O-gallate Chemical compound O([C@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-PZJWPPBQSA-N 0.000 claims description 2
- 235000006468 Thea sinensis Nutrition 0.000 claims description 2
- LVJJFMLUMNSUFN-UHFFFAOYSA-N gallocatechin gallate Natural products C1=C(O)C=C2OC(C=3C=C(O)C(O)=CC=3)C(O)CC2=C1OC(=O)C1=CC(O)=C(O)C(O)=C1 LVJJFMLUMNSUFN-UHFFFAOYSA-N 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 239000001476 sodium potassium tartrate Substances 0.000 claims description 2
- 235000011006 sodium potassium tartrate Nutrition 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 235000009569 green tea Nutrition 0.000 claims 1
- 235000020333 oolong tea Nutrition 0.000 claims 1
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 abstract description 20
- 235000005487 catechin Nutrition 0.000 abstract description 20
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 abstract description 18
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 abstract description 18
- 229950001002 cianidanol Drugs 0.000 abstract description 14
- 239000000049 pigment Substances 0.000 abstract description 14
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 abstract description 12
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 abstract description 9
- 229960001948 caffeine Drugs 0.000 abstract description 9
- 239000000945 filler Substances 0.000 abstract description 6
- 238000002360 preparation method Methods 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000001556 precipitation Methods 0.000 description 15
- 229960001866 silicon dioxide Drugs 0.000 description 15
- 239000000047 product Substances 0.000 description 14
- 238000000926 separation method Methods 0.000 description 13
- 239000004033 plastic Substances 0.000 description 12
- 229920003023 plastic Polymers 0.000 description 12
- 238000011160 research Methods 0.000 description 12
- 238000010521 absorption reaction Methods 0.000 description 9
- 238000004587 chromatography analysis Methods 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 9
- 238000005303 weighing Methods 0.000 description 9
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 8
- 239000004615 ingredient Substances 0.000 description 8
- 239000007791 liquid phase Substances 0.000 description 7
- 238000004440 column chromatography Methods 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 239000012264 purified product Substances 0.000 description 5
- 244000235603 Acacia catechu Species 0.000 description 4
- 235000006226 Areca catechu Nutrition 0.000 description 4
- 235000009024 Ceanothus sanguineus Nutrition 0.000 description 4
- 240000003553 Leptospermum scoparium Species 0.000 description 4
- 235000015459 Lycium barbarum Nutrition 0.000 description 4
- 150000001765 catechin Chemical class 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229930014626 natural product Natural products 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 244000170916 Paeonia officinalis Species 0.000 description 3
- 235000006484 Paeonia officinalis Nutrition 0.000 description 3
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 3
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 3
- 235000004515 gallic acid Nutrition 0.000 description 3
- 229940074391 gallic acid Drugs 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- DATAGRPVKZEWHA-YFKPBYRVSA-N N(5)-ethyl-L-glutamine Chemical compound CCNC(=O)CC[C@H]([NH3+])C([O-])=O DATAGRPVKZEWHA-YFKPBYRVSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000005377 adsorption chromatography Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 229940031703 low substituted hydroxypropyl cellulose Drugs 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000419 plant extract Substances 0.000 description 2
- 238000005498 polishing Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 238000013517 stratification Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000209507 Camellia Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 description 1
- CITFYDYEWQIEPX-UHFFFAOYSA-N Flavanol Natural products O1C2=CC(OCC=C(C)C)=CC(O)=C2C(=O)C(O)C1C1=CC=C(O)C=C1 CITFYDYEWQIEPX-UHFFFAOYSA-N 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- XABJJJZIQNZSIM-UHFFFAOYSA-N azane;phenol Chemical compound [NH4+].[O-]C1=CC=CC=C1 XABJJJZIQNZSIM-UHFFFAOYSA-N 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 239000012156 elution solvent Substances 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 150000002206 flavan-3-ols Chemical class 0.000 description 1
- 235000011987 flavanols Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 229920001002 functional polymer Polymers 0.000 description 1
- JLYXXMFPNIAWKQ-GNIYUCBRSA-N gamma-hexachlorocyclohexane Chemical compound Cl[C@H]1[C@H](Cl)[C@@H](Cl)[C@@H](Cl)[C@H](Cl)[C@H]1Cl JLYXXMFPNIAWKQ-GNIYUCBRSA-N 0.000 description 1
- JLYXXMFPNIAWKQ-UHFFFAOYSA-N gamma-hexachlorocyclohexane Natural products ClC1C(Cl)C(Cl)C(Cl)C(Cl)C1Cl JLYXXMFPNIAWKQ-UHFFFAOYSA-N 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
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- 230000007407 health benefit Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 235000020344 instant tea Nutrition 0.000 description 1
- 229960002809 lindane Drugs 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
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- 230000002265 prevention Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
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Landscapes
- Pyrane Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a technology for purifying tea catechin from tea polyphenol. Comprises enriching and refining catechin components by silica gel column chromatography and series connection of low-substitution hydroxypropyl cellulose, so as to remove water-soluble and fat-soluble pigments in tea polyphenol and completely remove caffeine components. The chromatographic and enriched filler can be recycled after being activated and regenerated, and reagents used in the preparation process of the tea catechin product are green and environment-friendly.
Description
Technical field
The invention belongs to Separation of Natural Products technical field of purification, and in particular to a kind of to utilize silica gel and low degree of substitution hydroxypropyl
The purifying from tea polyphenols of the mode of base cellulose tandem compound prepares the technology of tea catechin.
Background technology
Tea tree source originate in Chinese yunnan Xishuangbanna (old rafter again discuss tea tree source area Tea Industry be notified to, 1981,01:20-
25.), be under the jurisdiction of Theaceae (Theacea) Camellia (Camellia Sinensis(L.) O. Ktze.) plant (plant by China
Object will, the 49th (3) volume).The young leaves that tea tree is sprouted can be made into a kind of favorite beverage of consuming public, have in China thousands of
Year drinks history, and often drink can promote the production of body fluid to quench thirst, disease salubrity and improving eyesight benefit is gone to think, beneficial to physically and mentally healthy (the natural antioxygens of the roads Zhao Bao
The health benefit and its mechanism Acta Biophysica Sinicas of agent tea polyphenols, 2012,28 (5):426-437.||Chung S.Yang.
Cancer Prevention by Vitamin E and Tea Polyphenols:Lessons Learned from
Studies in Animal Models and humans.Progress in Chemistry, 2013,25(9):1492-
1500.)。
Tealeaves through separation identification about 700 kinds of compound (as if dawn spring tealeaves biochemistry, Chinese agriculture publishing house,
2003), including tea polyphenols, organic acid, theanine, alkaloid, Tea Saponin, aromatic substance, etc..Tea polyphenols are in tea tree
Polyphenols mixture, with flavylium ion mutter based on flavanols compounds-tea catechin of structure account for bulk composition.
Tea catechin generally comprises phenotype catechin (Epicatechin, EC), epi-nutgall type catechin
(Epigallocatechin, EGC), L-Epicatechin gallate (Epicatechin gallate, ECG), epi-nutgall
Catechin and gallate (Epigallocatechin gallate, EGCG), gross mass number are generally greater than in dry tea
7% (w/w) tea catechins have anti-oxidant, anti-inflammatory, antiviral and antitumor, etc. characteristics.
China's Resources of Tea Plant is abundant, has 2279.94 thousand hectares of tea place area in fact, can pick 1735.2 thousand hectare (2012
Annual national statistics office data), 192.45 ten thousand tons of tea yield (2013 annual national statistics office data).The fresh tea leaves of picking pass through
The dry tea being processed into generally comprises Famous High-quality Tea and large tea.By taking Zhejiang Province's tea industry as an example (Mao Zufa enumerates ten thousand, Lu Debiao,
The Famous High-quality Tea Industry Countermeasures for Sustainable Development research Chinese teas processing of the Zhejiang Province equal, 2012,2:9-12.), Famous High-quality Tea in 2010
Yield (6.5 ten thousand tons) and the output value (84.4 hundred million yuan) are the 39.2% and 93.3% of total output and the output value respectively.Therefore large Summer-autumn tea is deposited
In the problem that yield is big, the output value is low.Large tea phenol ammonia is than high, with deep processing means come to develop and use polyphenol therein will be its hair
One of exhibition approach.It is reported that current tea comprehensive processing enterprise of Zhejiang Province about 40, year about 8.2 ten thousand tons of dry tea is consumed, be mainly used to give birth to
Tea polyphenols, instant tea are produced, etc..Tea polyphenols are the main product of tea comprehensive processing enterprise at present, and product mainly applies to tea-drinking
Material, food additives and health-related product.But with the development of plant extracts, the progress of biochemistry polishing purification technology,
And people constantly pursue high-quality product, ingredient natural products product that is single, concentrating opposite with function will be following a kind of
Development trend.Therefore, a kind of health elements of the tea catechin as the mankind are purified purifying and meet current big health industry
Development.
Presently commercially available tea polyphenols product is mainly carried using water or the ethanol solution of water extracts, is concentrated and dried and obtains, powder
In yellowish-brown.Tea catechin typically results in preferable reservation, and the impurity only to accompany with it is more, especially fat-soluble and water
Dissolubility pigment composition influence product quality.Collect the tea polyphenols of the different content specification of market sale early period in our laboratories
Product is mainly derived from Zhejiang Orient Tea Development Co., Ltd., Sichuan Yujia Tea Industry Technology Co., Ltd., morning twilight biotechnology
Group Plc, Zhejiang Ta Ta Tea Industry Science and Technology Ltd., Zhejiang University of Technology, the Fujian sources Li Kang
Bioengineering Co., Ltd, Jiangxi Lv Kang natural products Co., Ltd, Hunan Jinnong Biological Resources Co., Ltd., peace
Emblem Red Star medicine company Co., Ltd, and the tea as contained by national standard man tealeaves standard GB/T 8313-2008 determine it is more
Phenol, tea catechin ingredient (being shown in Table 1).The result shows that tea catechin total amount accounts for tea polyphenols mass percent in 50 %-88.8 % models
It encloses.
Tea polyphenols, gallic acid and tea catechin content (%, w/w) in 1 commercially available tea polyphenols product of table
Note：Each ingredient is indicated with average ± standard deviation (n=3) in sample in table.
The purification process of tea catechin mainly have the precipitation method (ZL02139219.6), resin adsorption method (Zhang Sheng, Liu Zhonghua,
Huang Jianan waits adsorption resin methods to prepare the research tea sciences of high-purity catechin, 2002,22 (2): 125-130|
|Zhang Sheng, Liu Zhonghua, Huang Jianan wait journal (natural section of high ECG type catechin purifying process research Agricultural University Of Hunan
Learn version), 2003,29 (2):144-146.||Gong Yushun, Liu Zhonghua, Huang Jianan wait macroporous absorbent resins to detach tea catechu
Element and caffeine research Agricultural University Of Hunan's journals (natural science edition), 2005,31 (1):50-52||Li Huixing resin methods
Technical study HeFei University of Technologys prepared by extraction purification catechin and catechin monomers EGCG separation, 2006.||Zhong Shian,
He Guowen, Tu Qiuyun wait research ion exchanges and the absorption of Adsorption of Ester Catechins With Macroporous Resins,
2007, 23(5): 392-399.||The separation purifying technique research Dalian University of Technology of Zhang swallow Lai ester catechins,
2006||EP1767097, Process for preparing epigallocatechin gallate||Yang Xingmin, Liu Qing
Plum, Gao Haiyue, Ying Min, poplar stay in bright tea polyphenols EGCG separation purifying techniques to optimize Chinese food journals, and 2006,6
(5): 77-80.||Wang Chuanjin, Wei Yunyang, Zhu Guangjun wait the separation of polyamide chromatographies to prepare high-purity epigallocatechin gallate
Catechin gallate applied chemistries, 2007,24 (4): 443-447.||Gong Zhihua, Huang Tian, Pang Yuelan wait
HP-20 macroporous absorbent resins isolate and purify the effect Agricultural University Of Hunan journal (natural science edition) of catechin EGCG,
2010, 36(1): 87-90||Jianhan Huang, Kelong Huang, Suqin Liu, Qiong Luo, Mancai
Xu. Adsorption properties of tea polyphenols onto three polymeric adsorbents
with amide group. Journal of colloid and interface science.||Ruiying Zhao, Yu
Yan, Mingxian Li, Husheng Yan. Selective adsorption of tea polyphenols from
aqueous solution of the mixture with caffeine on macroporous crosslinked poly
(N-Vinyl-2-pyrrolidinone). Reactive & Functional Polymers 68 (2008) 768-774.|
|Yang Wenhong, Zhao Changqing, Gao Jie wait to be selectively adsorbing and separating tea polyphenols and coffee with macropore aminomethyl polystyrene resin
Because of ion exchanges and absorption, 2007,23 (6): 481-488.||Tang's text, Zhou Chunshan, Zhong Shian wait polyamide
Resin is to tea polyphenols and caffeine adsorptive selectivity research spectroscopy and spectrum analysis, 2003,23 (1): 143-145.),
18 filler column chromatography of reverse phase carbon (the natural ester catechin EGCG of Zhou Qingqiu isolate and purify and the Heilungkiang study of pharmacy is big
It learns, 2008.||Lindane, Li Chunmiao is fresh different, waits in and preparative liquid chromatography quick separating is pressed to prepare catechin monomers natural products
Research and development, 2013,25 (1):92-95,100.||Lai Ronghui, the research of yellow Asia brightness medium and low pressure column chromatography catechins
Ion exchange and absorption, 2012,28 (2):165-170.), (Zhang Jianyong, Jiang Yongwen, Jiang Heyuan wait UF membrane mistakes to membrane separation process
Catechin and caffeine content Changeement Agriculture of Anhui science, 2013,41 (2) in journey:493-496.), gel filtration chromatography method
(Huang Jing high-purity catechins monomer EGCGs and ECG separation and purifying process research HeFei University of Technologys, 2004), etc..
Silica gel has been widely used in the separation of active ingredient in plant extracts as a kind of Nantural non-toxic separation material
With purifying, the no exception (Shanxi Gong Zhengli, Pu Jian, the Liu Qin tea catechins extraction of preparation purifying of the tea catechin of tea polyphenols
With purifying research Agricultural University Of Southwest journal, 1995,17 (6):546-549||Yang Lei, high Yan China, Zu YuanGang are waited and are pressed in
The research chemistry of forest product of EGCG and ECG and industry, 2007,27 (2) in silica gel column chromatography continuous purification tealeaves: 100-
104.||Yuan Hua, Wu Li, Wu Yuanxin wait the research Central China Normal University journal of silica gel column chromatographies purification tea polyphenols (natural
Science version), 2007,41 (4):553-556..||Lu Tao, Lan Xianqiu, Zhu Bin, wait in difference tea polyphenols the measurement of catechin and
Column chromatography extracts the comparison chemical industry progress of EGCG, 2008,27 (5):749-752.||Bis- chromatography systems of Zhu Bin, Chen Xiaoguang
The technical study food of standby high-purity Epigallo-catechin gallate (EGCG) (EGCG) and machinery, 2009,25 (4): 83-
85.).The method that inventor is provided with document is that column chromatography filler is pure to tea catechin progress in commercially available tea polyphenols using silica gel
Change confirmatory experiment.As described in document, glue silicon using anhydrous ethyl acetate as dress column and elution solvent, tea polyphenols can be divided into compared with
For apparent visible component up and down, but contain tea catechin in component up and down, separation is not thorough；And the polarity adsorbed compared with
Strong pigment constituents, which are difficult to elute, can not be ignored.On the other hand, it could be used after the silica gel after use need to be dried so that silica gel
Special separating property is difficult to industrialization application and preparation.If being purified from tea polyphenols using gel Sephadex LH-20 as column packing
Refined tea catechin also has that pigment pollution is difficult to elute, and regenerative elution need to use the highly basic and salt of high concentration, increase and divide
From period and experiment difficulty, to reduce the application prospect of its industrialization.Therefore using existing technique filler as absorption and layer
Analysis, obtains the tea catechin of high-purity, and effectively removing is seemed particularly by the pigment of filling adsorption and the improvement of the Combinatorial Optimization of technique
It is necessary and urgent.
Invention content
The purpose of the present invention is to provide a kind of mode with silica gel and low degree of substitution hydroxypropyl cellulose tandem compound is pure
Change prepares the technology of tea catechin, and particular technique route is shown in Fig. 1.Compared with the existing technology using only silica gel column chromatography, the technique
It can make the significantly more efficient separation of tea catechin and enrichment.
The present invention also aims to provide it is a kind of effectively remove by the complete solidity pigment after silica gel absorption, after adsorption chromatography
Silica gel activating it is simpler feasible, recycled for a long time so as to realize.One kind provided by the invention is pure from tea polyphenols
Change the technology of tea catechin, including：
(1) by water dissolution of the tea polyphenols powder containing acetic acid, tea polyphenols solution is obtained；
(2) the prepared tea polyphenols solution of centrifugation step (1) obtains supernatant；
(3) supernatant of step (2) is sufficiently mixed with water saturated ethyl acetate, after being layered, obtains upper layer ester phase
Extract liquor；
(4) the upper layer ester phase extract liquor of concentration step (3) obtains concentrate；
(5) concentrate of step (4) is splined on silica gel column chromatography；
(6) it is eluted with water saturation ethyl acetate, and collects eluent；
(7) eluent of concentration step (6) obtains tea catechin purified product (I) to remove ethyl acetate；
(8) silica gel in aqueous sodium potassium tartrate zeolite regeneration step (5) is utilized；
(9) the tea catechin purified product (I) in step (7) is diluted with water, obtains aqueous solution；
(10) by the aqueous solution loading of step (9) and it is adsorbed in low degree of substitution hydroxypropyl cellulose；
(11) it is eluted with water；
(12) ethanol elution of 15-45 % (v/v) is used, and collects eluent；
(13) eluent of concentration step (12) obtains concentrate；
(14) low degree of substitution hydroxypropyl cellulose in the water elution regeneration step (10) containing ethyl acetate is utilized；
(15) ethyl acetate is added into the concentrate of step (13) to be sufficiently mixed, after being layered, obtains upper layer ester and mutually extract
Take liquid；
(16) the upper layer ester phase extract liquor of concentration step (15) obtains tea catechin purified product to remove ethyl acetate
(II)；
(17) the tea catechin purified product (II) of further vacuum drying step (16), obtains the tea catechu of polishing purification
Element.
Preferentially, the wherein water containing acetic acid described in step (1), acetate concentration is in 1-4 mol/L.
Preferentially, wherein the water containing acetic acid described in step (1), acetate concentration are 2 mol/L.
Preferentially, the tea polyphenols solution wherein described in step (1), tea polyphenols concentration are higher than 5 % (w/v).
Preferentially, the tea polyphenols solution wherein described in step (1), a concentration of 10 % (w/v) of tea polyphenols.
Preferentially, the r/min of the centrifuge speed wherein described in step (2) >=3000.
Preferentially, the min of the centrifugation time wherein described in step (2) >=10.
Preferentially, the water saturation ethyl acetate wherein described in step (3) is 0.5-1.5 times of volume of supernatant.
Preferentially, wherein step (3) the water saturated ethyl acetate is 1 times of supernatant.
Preferentially, wherein step (3) the water saturated ethyl acetate is 1 times of supernatant, is extracted 2 times.
Preferentially, wherein step (4), (7), (13), (16) and (17) is in -0.07 MPa ~ -0.1 of vacuum degree
MPa is carried out under the conditions of 35 DEG C ~ 50 DEG C of temperature.
Preferentially, the concentration of solution is controlled in the % of 0.5 % ~ 5 (w/v) wherein in step (9).
Preferentially, wherein step (11) elutes remaining acetic acid.
Preferentially, the wherein a concentration of mmol/L of 35 mmol/L ~ 50 of the sodium potassium tartrate tetrahydrate of step (8).
Main advantages of the present invention are：
(1) not purified tea polyphenols (polyphenol content is generally below 80 %, w/w) have a part with room temperature water dissolution
Adhesive component is difficult to dissolve, insoluble that precipitation is collectively formed at branch's a certain amount of tea catechin of absorption；Such as with a certain concentration
Acetate dissolution tea polyphenols, then can promote precipitation in tea catechin be redissolved in water, to reduce the damage of tea catechin
It loses.
(2) it is extracted by water saturation ethyl acetate before silicagel column adsorption chromatography, is on the one hand enriched tea catechin, another party
Face reduces water colo(u)r ingredient and excessive suction-operated occurs with silica gel column chromatography filler.
(3) water loads silica gel, fully after activation, then with water saturation ethyl acetate balance, and is eluted in this, as column chromatography
Agent can then enable caffeine and water-soluble strong pigment retain by force and adsorb, and tea catechin and fat-soluble stronger yellowish red color
Pigment elutes simultaneously with eluant, eluent, to realize more sufficient first step chromatographic purifying.
(4) activating and regenerating of silica gel column chromatography directly uses the aqueous solution of sodium potassium tartrate tetrahydrate, is convenient for water saturation ethyl acetate
Balance recycle, to simple realization column chromatography filler the repeatable purpose recycled.
Description of the drawings
Fig. 1 is the purifying tea catechin technology path schematic diagram from tea polyphenols.
Fig. 2 is tea polyphenol raw materials, silica gel column chromatography water saturation ethyl acetate eluent, low-substituted hydroxypropyl cellulose washing
(gallic acid, epigallocatechin, caffeine, epicatechin, epigallocatechin do not have each ingredient in de- liquid and desorbed solution
Infanticide acid esters, L-Epicatechin gallate) HPLC collection of illustrative plates.
Specific implementation mode
With reference to embodiment, the purport of the present invention is further illustrated.
It is following that examples are only for illustrating the present invention and not for limiting the scope of the present invention.
The analysis of tea catechin is all made of national standard method (GB/T 8313-2008, Tea Polyphenols in Tea in following embodiments
With the detection method of catechin content).High performance liquid chromatography Waters (1525 pumps, column oven, 2487 UV, visible light bilaterals
Road detector, 717-plus autosamplers,BreezeControl software packet), chromatographic column is 5 μm of Phenomenex Luna
Phenyl-Hexyl, 250 mm × 4.6 mm.Using caffeine as each ingredient of internal standard coefficient standard measure.Coffee is taken respectively
50 mg/L of alkali standard solution, 100 mg/L, 150 mg/L sample introductions, 10 μ L, after high performance liquid chromatography separation, in 278 nm items
Corresponding product peak area is respectively 1311154 (μ V × Sec), 2671194 (μ V × Sec), 4058875 (μ V under part
× Sec)；The standard curve for being forced across coordinate axis origin is ψ (peak area)=26900 × χ (concentration mg/L), R2 =
0.999。
Tea polyphenols described in following embodiments are same batch, tea catechu prepared by tea polyphenol raw materials, silica gel column chromatography
The HPLC figures of each ingredient are as shown in Figure 2 in plain crude product, low-substituted hydroxypropyl cellulose water elution and desorbed solution.The GA,
EGC, CAF, EC, EGCG, ECG are respectively gallic acid, epigallocatechin, caffeine, epicatechin, epi-nutgall catechu
Plain gallate, L-Epicatechin gallate.The L-HPC is low degree of substitution hydroxypropyl cellulose.
Material, reagent etc., are commercially available unless otherwise specified used in following experimental examples.
The acquisition of experimental data is completed in October, 2014 in December, 2014 in following embodiments.
1 pure water of embodiment dissolves tea polyphenols powder
It takes 0.5 g of tea polyphenols powder to be placed in 10mL plastics graded tubes, and adds the pure water of 5 mL, be sufficiently stirred and through 3500
After r/min centrifuges 10 min, constant volume in supernatant to 10 mL volumetric flasks is shifted.After 50 times of dilution, efficient liquid phase is carried out into 10 μ L
Chromatography.Precipitation in plastics graded tube takes out precision weighing after fully dry 5 h of 80 DEG C of hot winds.The result shows that through fixed
A concentration of 164.2 mg/L of EGCG in the supernatant of appearance, a concentration of 22.2 mg/L of ECG, precipitation weigh 0.1088 g.
Water dissolution tea polyphenols powder of the embodiment 2 containing acetic acid
It takes 0.5 g of tea polyphenols powder to set in 10 mL plastics graded tubes, and adds the aqueous acetic acid of 5 mL (acetate concentration is
0.2 mol/L), it is sufficiently stirred and after 3500 r/min centrifuge 10 min, shifts constant volume in supernatant to 10 mL volumetric flasks.It is dilute
After releasing 50 times, efficient liquid phase chromatographic analysis is carried out into 10 μ L.Precipitation in plastics graded tube is through fully dry 5 h of 80 DEG C of hot winds
After take out precision weighing.The result shows that in the supernatant through constant volume EGCG a concentration of 189.8 mg/L, ECG's is a concentration of
22.9 mg/L, precipitation weigh 0.0901 g.
Water dissolution tea polyphenols powder of the embodiment 3 containing acetic acid
It takes 0.5 g of tea polyphenols powder to set in 10 mL plastics graded tubes, and adds the aqueous acetic acid of 5mL (acetate concentration is
0.5 mol/L), it is sufficiently stirred and after 3500 r/min centrifuge 10 min, shifts constant volume in supernatant to 10 mL volumetric flasks.It is dilute
After releasing 50 times, efficient liquid phase chromatographic analysis is carried out into 10 μ L.Precipitation in plastics graded tube is through fully dry 5 h of 80 DEG C of hot winds
After take out precision weighing.The result shows that in the supernatant through constant volume EGCG a concentration of 206.2 mg/L, ECG's is a concentration of
29.5 mg/L, precipitation weigh 0.0735 g.
Water dissolution tea polyphenols powder of the embodiment 4 containing acetic acid
It takes 0.5 g of tea polyphenols powder to set in 10 mL plastics graded tubes, and adds the aqueous acetic acid (acetate concentration 1 of 5mL
Mol/L), it is sufficiently stirred and after 3500 r/min centrifuge 10 min, shifts constant volume in supernatant to 10 mL volumetric flasks.Dilution 50
After times, efficient liquid phase chromatographic analysis is carried out into 10 μ L.Precipitation in plastics graded tube takes after the fully dry 5h of 80 DEG C of hot winds
Go out precision weighing.The result shows that in the supernatant through constant volume EGCG a concentration of 253.4 mg/L, a concentration of the 37.1 of ECG
Mg/L, precipitation weigh 0.0427 g.
Water dissolution tea polyphenols powder of the embodiment 5 containing acetic acid
It takes 0.5 g of tea polyphenols powder to set in 10 mL plastics graded tubes, and adds the aqueous acetic acid (acetate concentration 2 of 5 mL
Mol/L), it is sufficiently stirred and after 3500 r/min centrifuge 10 min, shifts constant volume in supernatant to 10 mL volumetric flasks.Dilution 50
After times, efficient liquid phase chromatographic analysis is carried out into 10 μ L.Precipitation in plastics graded tube takes after fully dry 5 h of 80 DEG C of hot winds
Go out precision weighing.The result shows that in the supernatant through constant volume EGCG a concentration of 258.8 mg/L, a concentration of the 37.6 of ECG
Mg/L, precipitation weigh 0.025 g.
Water dissolution tea polyphenols powder of the embodiment 6 containing acetic acid
It takes 0.5 g of tea polyphenols powder to set in 10 mL plastics graded tubes, and adds the aqueous acetic acid (acetate concentration 4 of 5 mL
Mol/L), it is sufficiently stirred and after 3500 r/min centrifuge 10 min, shifts constant volume in supernatant to 10 mL volumetric flasks.Dilution 50
After times, efficient liquid phase chromatographic analysis is carried out into 10 μ L.Precipitation in plastics graded tube takes after fully dry 5 h of 80 DEG C of hot winds
Go out precision weighing.The result shows that in the supernatant through constant volume EGCG a concentration of 267.5 mg/L, a concentration of the 40.8 of ECG
Mg/L, precipitation weigh 0.012 g.
In above-described embodiment, control takes 20 mg tea polyphenols to be completely dissolved with 50 % (v/v) ethyl alcohol to be dissolved in 10 mL surely, into
10 μ L carry out efficient liquid phase chromatographic analysis.The result shows that in the solution through constant volume GA, EGC, CAF, EC, EGCG and ECG it is dense
Degree is respectively 11.9 mg/L, 68.6 mg/L, 97.1 mg/L, 17.8 mg/L, 100.2 mg/L, 23.7 mg/L.Tea polyphenols are former
The mass fraction of each ingredient GA, EGC, CAF, EC, EGCG and ECG are respectively 0.6 %, 3.4 %, 4.8 %, 0.9 %, 5.0 in material
%、1.2 %。
7 silica gel column chromatography purification enrichment tea catechin of embodiment
With commercially available 5 g of tea polyphenols powder for a purification enrichment unit, used per unit steps are as follows：
1. accurately weighing 5 g of tea polyphenols powder, 50 mL of acetic acid water of 2 mol/L is added, after being sufficiently stirred, is put into centrifugation
After 3500 r/min centrifuge 10 min in machine, takes supernatant and 25 mL water saturation ethyl acetate are added, fully shake simultaneously stratification
Afterwards, upper layer ester is taken mutually to be splined on silica gel column chromatography (100 g of about 200 mesh silica gel, the column diameter 3.5 of water saturation ethyl acetate balance
Cm, 26 cm of pillar height, 80 mL of interstitial volume in column bed), and eluted with water saturation ethyl acetate, collection starts from eluent and contains
Yellowish red color pigment is until eluent is colourless.Concentrate eluant is dried in vacuo to obtain 0.42 g of tea catechin crude product to after doing.
2. accurately weighing 5 g of tea polyphenols powder, 50 mL of acetic acid water of 2 mol/L is added, after being sufficiently stirred, is put into centrifugation
After 3500 r/min centrifuge 10 min in machine, takes supernatant and 50 mL water saturation ethyl acetate are added, fully shake simultaneously stratification
Afterwards, upper layer ester is taken mutually to be splined on silica gel column chromatography (100 g of about 200 mesh silica gel, the column diameter 3.5 of water saturation ethyl acetate balance
Cm, 26 cm of pillar height, 80 mL of interstitial volume in column bed), and eluted with water saturation ethyl acetate, collection starts from eluent and contains
Yellowish red color pigment is until eluent is colourless.Concentrate eluant is dried in vacuo to obtain tea catechin crude product 0.53g to after doing.
3. accurately weighing 5 g of tea polyphenols powder, 50 mL of acetic acid water of 2 mol/L is added, after being sufficiently stirred, is put into centrifugation
After 3500 r/min centrifuge 10 min in machine, takes supernatant and 150 mL water saturation ethyl acetate are added, fully shake and stand point
After layer, takes upper layer ester phase and be concentrated into its volume less than 100 mL；Concentrate is taken to be splined on the silicon of water saturation ethyl acetate balance
Glue chromatographic column (100 g of about 200 mesh silica gel, 3.5 cm of column diameter, 26 cm of pillar height, 80 mL of interstitial volume in column bed), and with water
It is saturated ethyl acetate elution, collection starts from eluent and contains yellowish red color pigment until eluent is colourless.Concentrate eluant is to dry
Afterwards, it is dried in vacuo to obtain 0.57 g of tea catechin crude product.
It is 1.52 to merge the above tea catechin crude product (tea catechin purified product (I)) 1. 2. 3. obtained by unit of mixing
g.The mass fraction (%, w/w) that GA, EGC, EC, EGCG and ECG contained by it are detected through HPLC be respectively 2.3 %, 4.1 %,
4.9 %,34.0 %,9.1 %.Using the aqueous solution zeolite regeneration silica gel of sodium potassium tartrate tetrahydrate.
8 L-HPC adsorption columns of embodiment are enriched with tea catechin
7 gained tea catechin crude product of Example, 0.5 g is dissolved in and is settled to 100 mL, with the flow velocity of 1 cm/min
It is splined on the L-HPC (5 g L-HPC, 1.6 cm of column diameter, 13.5 cm of pillar height, 10 mL of interstitial volume in column bed) of water balance,
And eluted with 30 % (v/v) ethanol water, it is collected simultaneously eluent.It has been observed that eluent is slight yellowish, and
The peony pigment of L-HPC absorption is not eluted.By eluent vacuum carry out concentration fling to ethyl alcohol after, carried out using ethyl acetate
It is enriched with dry that refined 0.25 g of tea catechin product, the wherein mass fraction (%, w/w) of GA, EGC, EC, EGCG and ECG are distinguished
For 3.2 %, 4.5 %, 7.7 %, 59.5 %, 13.0 %.
9 L-HPC adsorption columns of embodiment are enriched with tea catechin
7 gained tea catechin crude product of Example, 0.5 g is dissolved in and is settled to 100 mL, with the flow velocity of 1 cm/min
It is splined on the L-HPC (5 g L-HPC, 1.6 cm of column diameter, 13.5 cm of pillar height, 10 mL of interstitial volume in column bed) of water balance,
And eluted with 40 % (v/v) ethanol water, it is collected simultaneously eluent.It has been observed that eluent is faint yellow, and L-
The peony pigment of HPC absorption is not eluted.Eluent vacuum is carried out after concentration flings to ethyl alcohol, to utilize ethyl acetate to carry out rich
Collect dry that refined 0.27 g of tea catechin product, the wherein mass fraction (%, w/w) of GA, EGC, EC, EGCG and ECG are respectively
3.7 %、6.5 %、7.9 %、56.8 %、16.0 %。
10 L-HPC adsorption columns of embodiment are enriched with tea catechin
7 gained tea catechin crude product of Example, 0.5 g is dissolved in and is settled to 100 mL, with the flow velocity of 1 cm/min
It is splined on the L-HPC (5 g L-HPC, 1.6 cm of column diameter, 13.5 cm of pillar height, 10 mL of interstitial volume in column bed) of water balance,
And eluted with 50 % (v/v) ethanol water, it is collected simultaneously eluent.It has been observed that eluent is faint yellow, and L-
The peony pigment of HPC absorption is not eluted.Eluent vacuum is carried out after concentration flings to ethyl alcohol, to utilize ethyl acetate to carry out rich
Collect dry that refined 0.31 g of tea catechin product, the wherein mass fraction (%, w/w) of GA, EGC, EC, EGCG and ECG are respectively
3.8 %、6.6 %、7.6 %、52.8 %、16.5 %。
Claims (15)
1. a kind of method isolating and purifying tea catechin class compound from tea polyphenols, including：
(1) by water dissolution of the tea polyphenols powder containing acetic acid, tea polyphenols solution is obtained；
(2) the prepared tea polyphenols solution of centrifugation step (1) obtains supernatant；
(3) supernatant of step (2) is sufficiently mixed with water saturated ethyl acetate, after being layered, obtains upper layer ester and mutually extract
Liquid；
(4) the upper layer ester phase extract liquor of concentration step (3) obtains concentrate；
(5) concentrate of step (4) is splined on silica gel column chromatography；
(6) it is eluted with water saturation ethyl acetate, and collects eluent；
(7) eluent of concentration step (6) obtains tea catechin crude product to remove ethyl acetate；
(8) silica gel in aqueous sodium potassium tartrate zeolite regeneration step (5) is utilized；
(9) the tea catechin crude product in step (7) is diluted with water, obtains aqueous solution；
(10) by the aqueous solution loading of step (9) and it is adsorbed in low degree of substitution hydroxypropyl cellulose；
(11) it is eluted with water；
(12) ethanol elution of 15-45v/v% is used, and collects eluent；
(13) eluent of concentration step (12) obtains concentrate；
(14) low degree of substitution hydroxypropyl cellulose in the water elution regeneration step (10) containing ethyl acetate is utilized；
(15) ethyl acetate is added into the concentrate of step (13) to be sufficiently mixed, after being layered, obtains upper layer ester phase extract liquor；
(16) the upper layer ester phase extract liquor of concentration step (15), to remove ethyl acetate, rear further vacuum drying obtains refining pure
The tea catechin class compound of change, the tea catechin class compound are epigallocatechin, epicatechin, epigallocatechin gallate
Catechin gallate, L-Epicatechin gallate.
2. the water containing acetic acid described in the method as described in claim 1, wherein step (1), acetate concentration is in 1-4mol/L.
3. the water containing acetic acid described in method as claimed in claim 2, wherein step (1), acetate concentration 2mol/L.
4. the tea polyphenols solution described in the method as described in claim 1, wherein step (1), tea polyphenols concentration is higher than 5w/v%.
5. the tea polyphenols solution described in method as claimed in claim 4, wherein step (1), a concentration of 10w/v% of tea polyphenols.
6. centrifuge speed >=3000r/min described in the method as described in claim 1, wherein step (2).
7. centrifugation time >=10min described in the method as described in claim 1, wherein step (2).
8. the water saturation ethyl acetate described in the method as described in claim 1, wherein step (3) is the 0.5-1.5 of supernatant
Times volume.
9. method as claimed in claim 8, wherein step (3) the water saturated ethyl acetate are 1 times of supernatant.
10. the method as described in claim 1, wherein step (3) water saturated ethyl acetate are 1 times of supernatant, extraction
It takes 2 times.
11. the method as described in claim 1, wherein step (4), (7), (13), (16) vacuum degree -0.07MPa~-
0.1MPa is carried out under the conditions of 35 DEG C~50 DEG C of temperature.
12. the concentration of solution is controlled in 0.5%~5w/v% in the method as described in claim 1, wherein step (9).
13. the method as described in claim 1, wherein step (11) elute remaining acetic acid.
14. a concentration of 35mmol/L~50mmol/ of the sodium potassium tartrate tetrahydrate of the method as described in claim 1, wherein step (8)
L。
15. the tea polyphenols used in the method as described in claim 1, wherein step (1) derive from green tea or oolong tea.
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