CN117339239A - Method for preparing epigallocatechin gallate by dynamic axial compression column - Google Patents
Method for preparing epigallocatechin gallate by dynamic axial compression column Download PDFInfo
- Publication number
- CN117339239A CN117339239A CN202311130968.1A CN202311130968A CN117339239A CN 117339239 A CN117339239 A CN 117339239A CN 202311130968 A CN202311130968 A CN 202311130968A CN 117339239 A CN117339239 A CN 117339239A
- Authority
- CN
- China
- Prior art keywords
- axial compression
- dynamic axial
- compression column
- egcg
- column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 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 132
- 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 title claims abstract description 132
- 230000006835 compression Effects 0.000 title claims abstract description 95
- 238000007906 compression Methods 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 42
- 229940030275 epigallocatechin gallate Drugs 0.000 title claims abstract description 18
- 150000008442 polyphenolic compounds Chemical class 0.000 claims abstract description 32
- 235000013824 polyphenols Nutrition 0.000 claims abstract description 32
- 239000002994 raw material Substances 0.000 claims abstract description 4
- 241001122767 Theaceae Species 0.000 claims abstract 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 54
- 238000000926 separation method Methods 0.000 claims description 51
- 239000000178 monomer Substances 0.000 claims description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 38
- 238000010828 elution Methods 0.000 claims description 32
- 238000000746 purification Methods 0.000 claims description 26
- 238000012856 packing Methods 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 15
- 238000002386 leaching Methods 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 14
- 238000010829 isocratic elution Methods 0.000 claims description 11
- 238000007670 refining Methods 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 244000269722 Thea sinensis Species 0.000 description 45
- 235000013616 tea Nutrition 0.000 description 42
- 238000001514 detection method Methods 0.000 description 36
- 239000000523 sample Substances 0.000 description 21
- 230000000052 comparative effect Effects 0.000 description 18
- 239000000945 filler Substances 0.000 description 17
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 15
- 238000011049 filling Methods 0.000 description 13
- 239000007788 liquid Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 12
- 238000009210 therapy by ultrasound Methods 0.000 description 12
- 239000007791 liquid phase Substances 0.000 description 11
- 230000014759 maintenance of location Effects 0.000 description 11
- 238000011010 flushing procedure Methods 0.000 description 10
- 239000012535 impurity Substances 0.000 description 10
- 239000012071 phase Substances 0.000 description 10
- 238000003756 stirring Methods 0.000 description 9
- 238000004811 liquid chromatography Methods 0.000 description 7
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 6
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 6
- 229960001948 caffeine Drugs 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 235000009569 green tea Nutrition 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 description 2
- 235000005487 catechin Nutrition 0.000 description 2
- -1 catechin compounds) Chemical class 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229950001002 cianidanol Drugs 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000002031 ethanolic fraction Substances 0.000 description 2
- 238000002761 liquid phase assay Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 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 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- XMWRBQBLMFGWIX-UHFFFAOYSA-N C60 fullerene Chemical compound C12=C3C(C4=C56)=C7C8=C5C5=C9C%10=C6C6=C4C1=C1C4=C6C6=C%10C%10=C9C9=C%11C5=C8C5=C8C7=C3C3=C7C2=C1C1=C2C4=C6C4=C%10C6=C9C9=C%11C5=C5C8=C3C3=C7C1=C1C2=C4C6=C2C9=C5C3=C12 XMWRBQBLMFGWIX-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000002790 anti-mutagenic effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000012539 chromatography resin Substances 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000002781 deodorant agent Substances 0.000 description 1
- 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
- 230000002708 enhancing effect Effects 0.000 description 1
- DQYBDCGIPTYXML-UHFFFAOYSA-N ethoxyethane;hydrate Chemical compound O.CCOCC DQYBDCGIPTYXML-UHFFFAOYSA-N 0.000 description 1
- 229930003949 flavanone Natural products 0.000 description 1
- 150000002208 flavanones Chemical class 0.000 description 1
- 235000011981 flavanones Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229910003472 fullerene Inorganic materials 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides a method for preparing epigallocatechin gallate by a dynamic axial compression column. The method for preparing epigallocatechin gallate by utilizing the dynamic axial compression column provided by the invention comprises the following steps: the tea polyphenol extract is used as a raw material, and the EGCG is separated and purified by adopting a dynamic axial compression column. The method provided by the invention can safely and efficiently prepare the high-purity EGCG.
Description
Technical Field
The invention relates to a production method of natural products, in particular to a method for preparing epigallocatechin gallate by a dynamic axial compression column.
Background
Along with the change of the life style of the current society, the consumption demand of people on tea leaves gradually develops from the traditional single primary product form to the high-quality, variety and high-value utilization direction of the product. Therefore, there is a trend in developing tea deep processing into world tea processing.
The tea polyphenol is the sum of polyphenols in tea, and the content of tea polyphenol in green tea is higher and accounts for 15% -30% of the mass of the tea. The tea polyphenol is most important by flavanones (mainly catechin compounds), and accounts for 60% -80% of the total amount of the tea polyphenol. EGCG (epigallocatechin gallate) is the most effective active component in tea polyphenol, is one of catechin monomers, and has antioxidant, antimutagenic, anticancer, cardiovascular disease resisting, ultraviolet radiation resisting, antidiabetic, antibacterial and antiinflammatory effects. EGCG has the functions of preventing and treating various diseases such as cancers, enhancing immunity and the like in medicine and health care; can be used as an antioxidant, antibacterial, fresh-keeping and deodorant agent in the food industry; the product can be used as special functional quality-keeping agent and skin care agent.
EGCG is white to yellow powder or irregular crystal, has no special smell, is slightly soluble in cold water and diethyl ether, and can be dissolved in hot water, ethanol, methanol, glacial acetic acid and acetone. The existing EGCG preparation technology mainly comprises methanol liquid phase separation, supercritical extraction, column chromatography and the like.
Disclosure of Invention
In the prior art, the EGCG is purified by adopting a simulated moving bed chromatography method, a column chromatography resin adsorption method and other methods, and the methods have the advantages of high toxicity, potential safety hazard, long time consumption, low yield and low final purity of the obtained EGCG product by taking methanol as a mobile phase.
Aiming at the problems in the prior art, the invention provides a safe and efficient method for preparing high-purity EGCG.
The invention provides a method for preparing epigallocatechin gallate by utilizing a dynamic axial compression column, which comprises the following steps: the tea polyphenol extract is used as a raw material, and the EGCG is separated and purified by adopting a dynamic axial compression column.
Preferably, the method comprises the steps of:
(1) And (3) carrying out primary separation and purification on the epigallocatechin gallate monomer by using a dynamic axial compression column: injecting the tea polyphenol extract into a balanced dynamic axial compression column for gradient elution, detecting sample separation condition by an ultraviolet detector, concentrating the collected EGCG fraction, and removing the solvent;
(2) Refining epigallocatechin gallate monomer by dynamic axial compression column: and (3) injecting the EGCG fraction obtained in the step (1) into a balanced dynamic axial compression column for isocratic elution, detecting the sample separation condition by using an ultraviolet detector, concentrating the collected EGCG fraction, and removing the solvent to obtain the refined epigallocatechin gallate monomer.
Preferably, the time t is collected in step (1) R Fraction of =50-58 min,
preferably, the time t is collected in step (2) R Fraction =25-33 min.
Preferably, the packing of the dynamic axial compression column in the step (1) is C18 packing with the particle size of 20-40 mu m;
preferably, the dynamic axial compression column has a column inner diameter of 200mm.
Preferably, the gradient elution method in the step (1) is as follows: eluting with pure water for 0-20 min; eluting with 10% ethanol and 90% pure water for 20-40 min; eluting with 20% ethanol and 80% pure water for 40-60 min;
preferably, the elution flow rate is 600-800ml/min and the pressure is 1.0-1.2MPa.
Preferably, the packing of the dynamic axial compression column in the step (2) is C18 packing with the particle size of 10-20 mu m;
preferably, the particle size of the packing of the dynamic axial compression column in step (1) is 20um when different from the particle size of the packing of the dynamic axial compression column in step (2);
preferably, the dynamic axial compression column has a column inner diameter of 100mm.
Preferably, the elution method in the step (2) is as follows: eluting with 15% ethanol and 85% pure water for 0-35 min;
preferably, the elution flow rate is 100-200ml/min and the pressure is 4.0-4.5MPa.
Preferably, the ultraviolet detector wavelength in the step (1) and the step (2) is 280nm.
Preferably, the concentration mode in the step (1) and the step (2) is reduced pressure concentration.
Preferably, the method further comprises the steps of: mixing fresh tea with water, leaching, separating, concentrating, and filtering to obtain tea polyphenols extract.
Preferably, the leaching liquor ratio is 1:20-30 (m/v), the leaching temperature is 70-90 ℃, and the leaching time is 40-60min.
Preferably, the water content of the fresh tea leaves is 70% -80%.
The invention also provides an application of the method for preparing epigallocatechin gallate by utilizing the dynamic axial compression column in preparation of epigallocatechin gallate.
The invention has the beneficial effects that:
(1) The invention adopts sectional extraction and refining. Firstly, crude extraction of tea polyphenol in fresh tea leaves is carried out by a water extraction method, and then EGCG monomer in the fresh tea leaves is separated and purified.
(2) The mobile phases adopted by chromatographic separation are ethanol and water, both embody the green and safe production concepts, are environment-friendly and pollution-free, can be recycled, reduce the environmental-friendly treatment cost of wastewater discharge, and have no toxic residual solution in the finished EGCG.
(3) The dynamic axial compression technology is the only technology which can keep good separation effect in various scale column chromatography, and is the best column loading technology in the field of the current preparation chromatography. The dynamic axial compression column purification preparation system has the advantages of simple operation, large treatment capacity, good separation effect, reusable filler and the like, and is suitable for industrial production of EGCG.
(4) According to the invention, a step-by-step separation mode is adopted to separate and purify EGCG, firstly, a large-particle-size C18 packing dynamic axial compression column 200 column (column inner diameter of 200 mm) is adopted to perform preliminary purification to remove most impurities, and a small-particle-size C18 packing dynamic axial compression column 100 column (column inner diameter of 100 mm) is adopted to perform refining purification in the second step, so that two-stage separation is only needed to be completed for 95min, the purity of the finished EGCG can reach more than 99%, the steps are simple and convenient to operate, and the production efficiency is high.
(5) The invention combines the optimization of gradient elution, mobile phase concentration and proportion, flow speed, sample injection quantity, peak time and the like, and combines the automatic sample injection, continuous sample injection and fraction sectional collection, thereby having high intelligent degree, high target fraction and strong repeatability and being capable of realizing intelligent continuous production under the condition of unmanned control.
Drawings
FIG. 1 shows a chromatogram of a primary separation purification EGCG monomer in example 1;
FIG. 2 a chromatogram of refined EGCG monomer in example 1;
FIG. 3 is a liquid chromatogram of the tea polyphenol extract of example 1;
FIG. 4 shows a liquid chromatogram of a primary separation purification EGCG monomer in example 1;
FIG. 5 shows a liquid chromatogram of refined EGCG monomer in example 1;
FIG. 6 comparative example 1 primary isolation and purification EGCG monomer chromatogram;
FIG. 7 is a chromatogram of a first separation purification EGCG monomer liquid phase in comparative example 1;
FIG. 8 comparative example 2 refined EGCG monomer chromatogram;
FIG. 9 comparative example 2 refined EGCG monomer liquid chromatogram;
FIG. 10 comparative example 3 refined EGCG monomer chromatogram;
FIG. 11 comparative example 3 refined EGCG monomer liquid chromatogram.
Detailed Description
The invention takes tea polyphenol extract as raw material, adopts dynamic axial compression column to separate and purify EGCG, and provides a safe and efficient method for preparing high-purity EGCG.
According to the invention, pollution-free and friendly reagent ethanol is selected as a mobile phase, and EGCG in green tea is separated and purified, so that high-purity EGCG can be prepared in a green and efficient manner.
In one specific embodiment of the present invention, the preparation method of epigallocatechin gallate comprises the following steps:
(1) Fresh tea leaves and water are mixed according to the following ratio of 1: mixing at 20-30 (m/v) ratio, leaching at 70-90deg.C for 40-60min, separating solid and liquid, concentrating, and filtering with 0.45 μm filter membrane to obtain tea polyphenols extract.
(2) And (3) carrying out primary separation and purification on EGCG monomer by using a dynamic axial compression column: filling a dynamic axial compression column 200 (column inner diameter of 200 mm) with 40 μmC18 filler, continuously flushing the dynamic axial compression column with pure water until the chromatograph baseline is stable, injecting proper amount of tea polyphenol extract into the balanced dynamic axial compression column for gradient elution, wherein the elution flow rate is 600-800ml/min, the pressure is 1.0-1.2Mpa, detecting sample separation condition by an ultraviolet detector, setting the detection wavelength to 280nm, and determining the start-stop collection time t of EGCG according to the retention time and the chromatographic peak height identification R =50-58 min, concentrating the collected EGCG fraction under reduced pressure, removing the solvent: the gradient elution method is as follows: eluting with pure water for 0-20 min; eluting with 10% ethanol and 90% pure water for 20-40 min; eluting with 20% ethanol and 80% pure water for 40-60min.
(3) Refining EGCG monomer by dynamic axial compression column: filling a dynamic axial compression column 100 (column inner diameter is 100 mm) with a 20 mu mC18 packing, continuously flushing the dynamic axial compression column with pure water until a chromatograph baseline is stable, injecting EGCG fraction obtained in the step (2) into the balanced dynamic axial compression column for isocratic elution, detecting sample separation condition by an ultraviolet detector at an elution flow rate of 100-200ml/min and a pressure of 4.0-4.5Mpa, setting a detection wavelength to 280nm, and determining a start-stop collection time t of EGCG according to retention time and chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 25-33min, the solvent was removed, and the gradient elution method was as follows: eluting with 15% ethanol and 85% pure water for 0-35min
(4) Detecting purity of EGCG refined fraction by high performance liquid chromatograph, and drying sample in low temperature drying equipment after detection result reaches standard (more than 99%), to obtain EGCG pure powder, and storing at-80deg.C in dark place.
The source information of the instrument used in the present invention is as follows:
a horizontal spiral centrifuge, WZ20, ancient cooking vessel;
a membrane concentration device, CFE-lab, rayleigh;
dynamic axial compression column used in the preliminary purification step, 200dac200, ymc;
dynamic axial compression column used for refining and purifying, 100DAC100, YMC;
high performance liquid chromatograph, chromater 5000, hitachi;
vacuum freeze dryer, FD50S, fullerene;
rotary evaporator, R1005, bai xiang.
The source information of the reagents and instruments used in the present invention are as follows:
isopropyl alcohol, 32064, westernized;
ethanol, 2023031401, colone;
formic acid, F112032, aladine;
methanol, 015323, rich in space;
fresh green tea leaves, maojian tea leaves and Hubei tea leaves are collected.
The present invention will be described in detail with reference to the following embodiments.
Example 1
Mixing 5kg of fresh tea leaves with 100L of pure water (1:20), stirring and leaching in an extraction tank at 70 ℃ for 60min, and performing solid-liquid separation, film concentration and 0.45 mu m organic film filtration by a horizontal spiral centrifuge after leaching to obtain tea polyphenol extract.
Adding 4.6kg of C18 filler with the particle size of 40 mu m into 11L of homogenizing reagent isopropanol, setting the pressure of a DAC chromatographic column system, performing ultrasonic treatment at room temperature for 30min, performing ultrasonic treatment and stirring for 20min, then sending the homogenate into a dynamic axial compression column with the inner diameter of 200mm, compacting the chromatographic column filler after removing a mobile phase by means of compressed air, and completing the filling of a dynamic axial compression column 200 column with the pressure of 90 Bar.
Adding 1.2kg of C18 filler with the particle size of 20 mu m into 2.5L of homogenizing agent isopropanol, setting the pressure of a DAC chromatographic column system, carrying out ultrasonic treatment at room temperature for 30min, carrying out ultrasonic treatment and stirring for 20min, then sending the homogenate into a dynamic axial compression column with the inner diameter of 100mm, compacting the chromatographic column filler after removing a mobile phase by means of compressed air, and completing the filling of the dynamic axial compression column 100 column under the pressure of 150 Bar.
And (3) carrying out primary separation and purification on EGCG monomer by using a dynamic axial compression column: continuously washing 200 columns with pure water until the chromatograph baseline is stable, injecting concentrated tea polyphenols extract into balanced dynamic axial compression column, gradient eluting (shown in table 1 below) with flow rate of 700ml/min and pressure of 1.0-1.2Mpa, detecting sample separation with ultraviolet detector, setting detection wavelength to 280nm, and determining EGCG start-stop collecting time t according to retention time and chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 50-58min, and the solvent was removed. The separating chromatogram is shown in figure 1, t R Fractions containing EGCG were collected for 50-58min, other chromatographic peaks were all impurities.
TABLE 1 gradient elution
| Time/min | Ethanol/% | Water/% |
| 0-20 | 0 | 100 |
| 20-40 | 10 | 90 |
| 40-60 | 20 | 80 |
Refining EGCG monomer by dynamic axial compression column: continuously flushing 100 columns of dynamic axial compression column with pure water until the chromatograph baseline is stable, injecting the monomer fraction of EGCG as the primary separation and purification product into the balanced dynamic axial compression column for isocratic elution (as shown in the following table 2), detecting sample separation condition with ultraviolet detector at flow rate of 150ml/min and pressure of 4.0-4.5Mpa, setting detection wavelength to 280nm, and determining start-stop collection time t of EGCG according to retention time and chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 25-33min, and the solvent was removed. The separating chromatogram is shown in figure 2, t R Fraction containing EGCG is collected for 25-33min, t R =17-24 min is impurity caffeine.
TABLE 2 isocratic elution
| Time/min | Ethanol/% | Water/% |
| 0-35 | 15 | 85 |
Detecting EGCG content in tea polyphenol extract, primary separation and purification EGCG monomer fraction and refined EGCG monomer fraction by high performance liquid chromatograph, and setting the instrument parameters as follows:
instrument: HITACHI Chromaster high performance liquid chromatograph, chromatographic column: waters XBridge C18 (4.6x250 mm,5 μm), flow rate: 1mL/min, column temperature: 30 ℃, sample injection amount: 10 μl, detection wavelength: 278nm, elution conditions are shown in Table 3 below.
TABLE 3 elution conditions
The liquid phase detection results of the tea polyphenol extract are shown in fig. 3 and table 4.
TABLE 4 liquid phase detection data for tea polyphenol extract of examples
As shown in the liquid chromatography detection results of the tea polyphenol extract shown in FIG. 3 and Table 4, wherein t is R Chromatographic peak 13 at 10.38min was EGCG with a detection purity of 14.5%.
The results of the liquid phase detection of the first separation and purification EGCG monomer fraction are shown in FIG. 4 and Table 5.
TABLE 5 initial separation and purification of EGCG monomer liquid phase detection data
As shown in the liquid chromatography detection results of the primary separation and purification of EGCG monomer shown in FIG. 4 and Table 5, wherein t is R At 10.30min chromatographic peak 12 is EGCG, the detection purity is 39.6%, and right chromatographic peak 13 is caffeine.
The results of liquid chromatography detection of the refined EGCG monomer fraction are shown in FIG. 5 and Table 6.
Table 6 example refined EGCG monomer liquid phase detection data
Liquid chromatography detection results of refined EGCG monomer as shown in FIG. 5 and Table 6, wherein t R Chromatographic peak 1 at 10.27min was EGCG and the assay purity was 100%.
Concentrating the refined EGCG pure product solution under reduced pressure, and freeze-drying to obtain EGCG lyophilized powder.
Example 2
Mixing 5kg of fresh tea leaves with 150L of pure water (1:30), stirring and leaching in an extraction tank at 90 ℃ for 40min, and performing solid-liquid separation, film concentration and 0.45 mu m organic film filtration by a horizontal spiral centrifuge after leaching to obtain tea polyphenol extract.
Adding 4.6kg of C18 filler with the particle size of 20 mu m into 11L of homogenizing reagent isopropanol, setting the pressure of a DAC chromatographic column system, performing ultrasonic treatment at room temperature for 30min, performing ultrasonic treatment and stirring for 20min, then sending the homogenate into a dynamic axial compression column with the inner diameter of 200mm, compacting the chromatographic column filler after removing a mobile phase by means of compressed air, and completing the filling of a dynamic axial compression column 200 column with the pressure of 90 Bar.
Adding 1.2kg of C18 filler with the particle size of 10 mu m into 2.5L of homogenizing agent isopropanol, setting the pressure of a DAC chromatographic column system, carrying out ultrasonic treatment at room temperature for 30min, carrying out ultrasonic treatment and stirring for 20min, then sending the homogenate into a dynamic axial compression column with the inner diameter of 100mm, compacting the chromatographic column filler after removing a mobile phase by means of compressed air, and completing the filling of the dynamic axial compression column 100 column under the pressure of 150 Bar.
And (3) carrying out primary separation and purification on EGCG monomer by using a dynamic axial compression column: continuously flushing the dynamic axial compression column 200 with pure water until the chromatograph baseline is stable, injecting the concentrated tea polyphenol extract into the balanced dynamic axial compression column for gradient elution (elution conditions are the same as those of example 1), wherein the elution flow rate is 600ml/min, the pressure is 1.0-1.2Mpa, detecting the sample separation condition by an ultraviolet detector, setting the detection wavelength to 280nm, and determining the start-stop collection time t of EGCG according to the retention time and the chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 50-58min, and the solvent was removed. t is t R Fractions containing EGCG were collected for 50-58min, other chromatographic peaks were all impurities.
Refining EGCG monomer by dynamic axial compression column: continuously flushing the dynamic axial compression column 100 with pure water until the chromatograph baseline is stable, and injecting the EGCG monomer fraction as the primary separation and purification product into the dynamic axial compression column after balancing for isocratic elution (eluting condition and reality)The same as in example 1), the elution flow rate was 200ml/min, the pressure was 4.0-4.5MPa, the sample separation was detected by an ultraviolet detector, the detection wavelength was set to 280nm, and the start-stop collection time t of EGCG was determined based on the retention time and the chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 25-33min, and the solvent was removed. t is t R Fraction containing EGCG is collected for 25-33min, t R =17-24 min is impurity caffeine.
Example 3
Mixing 5kg of fresh tea leaves with 150L of pure water (1:30), stirring and leaching in an extraction tank at 80 ℃ for 50min, and performing solid-liquid separation, film concentration and 0.45 mu m organic film filtration by a horizontal spiral centrifuge after leaching to obtain tea polyphenol extract.
Adding 4.6kg of C18 filler with the particle size of 20 mu m into 11L of homogenizing reagent isopropanol, setting the pressure of a DAC chromatographic column system, performing ultrasonic treatment at room temperature for 30min, performing ultrasonic treatment and stirring for 20min, then sending the homogenate into a dynamic axial compression column with the inner diameter of 200mm, compacting the chromatographic column filler after removing a mobile phase by means of compressed air, and completing the filling of a dynamic axial compression column 200 column with the pressure of 90 Bar.
Adding 1.2kg of C18 filler with the particle size of 10 mu m into 2.5L of homogenizing agent isopropanol, setting the pressure of a DAC chromatographic column system, carrying out ultrasonic treatment at room temperature for 30min, carrying out ultrasonic treatment and stirring for 20min, then sending the homogenate into a dynamic axial compression column with the inner diameter of 100mm, compacting the chromatographic column filler after removing a mobile phase by means of compressed air, and completing the filling of the dynamic axial compression column 100 column under the pressure of 150 Bar.
And (3) carrying out primary separation and purification on EGCG monomer by using a dynamic axial compression column: continuously flushing the dynamic axial compression column 200 with pure water until the chromatograph baseline is stable, injecting the concentrated tea polyphenol extract into the balanced dynamic axial compression column for gradient elution (elution conditions are the same as those of example 1), wherein the elution flow rate is 800ml/min, the pressure is 1.0-1.2Mpa, detecting the sample separation condition by an ultraviolet detector, setting the detection wavelength to 280nm, and determining the start-stop collection time t of EGCG according to the retention time and the chromatographic peak height identification R Concentrating the collected EGCG fraction under reduced pressure for 50-58min, and removing solvent。t R Fractions containing EGCG were collected for 50-58min, other chromatographic peaks were all impurities.
Refining EGCG monomer by dynamic axial compression column: continuously flushing the dynamic axial compression column 100 with pure water until the chromatograph baseline is stable, injecting the EGCG monomer fraction as the primary separation and purification product into the balanced dynamic axial compression column for isocratic elution (the elution condition is the same as that of example 1), the elution flow rate is 100ml/min, the pressure is 4.0-4.5Mpa, detecting the sample separation condition by an ultraviolet detector, the detection wavelength is set to 280nm, and determining the start-stop collection time t of EGCG according to the retention time and the chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 25-33min, and the solvent was removed. t is t R Fraction containing EGCG is collected for 25-33min, t R =17-24 min is impurity caffeine.
Comparative example 1
The preparation method of the tea polyphenol extract is the same as that of the embodiment 1, and the packing methods of the dynamic axial compression column 200 column and the dynamic axial compression column 100 column are the same as those of the embodiment.
And (3) carrying out primary separation and purification on EGCG monomer by using a dynamic axial compression column: filling a dynamic axial compression column with 40 μmC18 filler, continuously flushing the dynamic axial compression column with pure water until the chromatograph baseline is stable, injecting concentrated tea polyphenol extract into the balanced dynamic axial compression column for gradient elution (as shown in table 7 below), eluting at a flow rate of 600ml/min and a pressure of 0.8-1.0Mpa, detecting sample separation condition with an ultraviolet detector, setting detection wavelength to 280nm, and determining start-stop collection time t of EGCG according to retention time and chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 45-54min, and the solvent was removed. The current separation chromatogram is shown in FIG. 6, t R Fractions containing EGCG were collected for 45-54min, other chromatographic peaks were impurities.
TABLE 7 comparative example 1 gradient elution
| Time/min | Ethanol/% | Water/% |
| 0-20 | 0 | 100 |
| 20-40 | 10 | 90 |
| 40-60 | 25 | 75 |
The liquid phase detection method and instrument parameter settings were the same as in example 1, and the primary separation and purification EGCG monomer sample solution obtained above was detected, and the results are shown in fig. 7 and table 8.
TABLE 8 comparative example 1 Primary separation purification EGCG monomer liquid phase detection data
As shown in FIG. 7 and Table 8, the results of liquid chromatography detection of the EGCG monomer first separated and purified in comparative example 1, wherein t R Chromatographic peak 9 at 10.34min was EGCG with a detection purity of 21.3%. In this example, the ethanol ratio in the first separation was increased and the flow rate was decreased, and the second separation was not performed, resulting in a sample having a lower purity than the first separation in example 1.
Comparative example 2
The preparation method of the tea polyphenol extract is the same as that of the embodiment 1, the filling method of the dynamic axial compression column 200 column and the dynamic axial compression column 100 column is the same as that of the embodiment, and the method for primarily separating and purifying EGCG monomer by the dynamic axial compression column is the same as that of the embodiment 1.
Refining EGCG monomer by dynamic axial compression column: filling a dynamic axial compression column with 20 mu mC18 filler, continuously flushing the dynamic axial compression column with pure water until the chromatograph baseline is stable, injecting the EGCG fraction as a primary separation and purification product into the balanced dynamic axial compression column for isocratic elution (as shown in the following table 9), wherein the elution flow rate is 100ml/min, the pressure is 4.0-4.2Mpa, detecting the sample separation condition by an ultraviolet detector, setting the detection wavelength to 280nm, and determining the start-stop collection time t of EGCG according to the retention time and the chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 23-30min, and the solvent was removed. The current separation chromatogram is shown in FIG. 8, t R Fraction containing EGCG is collected for 23-30min, t R =15-22 min is impurity caffeine.
TABLE 9 comparative example 2 isocratic elution
| Time/min | Ethanol/% | Water/% |
| 0-35 | 20 | 80 |
The liquid phase detection method and instrument parameter settings were the same as in the examples, and the sample solutions obtained above were subjected to detection, and the results are shown in fig. 9 and table 10.
Table 10 comparative example 2 refined EGCG monomer liquid phase assay data
The results of liquid chromatography detection of the purified EGCG monomer in comparative example 2 are shown in FIG. 9 and Table 10, wherein t R Chromatographic peak 1 at 10.26min was EGCG with a detection purity of 97.7%, increasing the ethanol fraction in the secondary separation and decreasing the flow rate, resulting in a sample with a purity lower than that of EGCG prepared in the examples.
Comparative example 3
The preparation method of the tea polyphenol extract is the same as that of the embodiment 1, the filling method of the dynamic axial compression column 200 and the dynamic axial compression column 100 is the same as that of the embodiment, and the method for primarily separating and purifying EGCG monomer by the dynamic axial compression column is the same as that of the embodiment.
Refining EGCG monomer by dynamic axial compression column: filling a dynamic axial compression column with 20 mu mC18 filler, continuously flushing the dynamic axial compression column with pure water until the chromatograph baseline is stable, injecting the EGCG fraction as a primary separation and purification product into the balanced dynamic axial compression column for isocratic elution (as shown in the following table 11), wherein the elution flow rate is 200ml/min, the pressure is 4.3-4.8Mpa, detecting the sample separation condition by an ultraviolet detector, setting the detection wavelength to 280nm, and determining the start-stop collection time t of EGCG according to the retention time and the chromatographic peak height identification R The collected EGCG fraction was concentrated under reduced pressure for 15-22min, and the solvent was removed. The current separation chromatogram is shown in FIG. 10, t R Collecting fraction containing EGCG for 15-22min, t R =11-14 min is impurity caffeine.
TABLE 11 comparative example 3 isocratic elution
| Time/min | Ethanol/% | Water/% |
| 0-35 | 10 | 90 |
The liquid phase detection method and instrument parameter settings were the same as in the examples, and the sample solutions obtained above were subjected to detection, and the results are shown in fig. 11 and table 12.
Table 12 comparative example 3 refined EGCG monomer liquid phase assay data
The results of liquid chromatography detection of the purified EGCG monomer in comparative example 3 are shown in FIG. 11 and Table 12, wherein t R Chromatographic peak 5 at 10.25min was EGCG with a detection purity of 89.3%. This example reduced the ethanol fraction in the secondary separation and increased the flow rate, resulting in a sample of lower purity than the EGCG prepared in the examples.
Claims (13)
1. A method for preparing epigallocatechin gallate by using a dynamic axial compression column, which is characterized by comprising the following steps: the tea polyphenol extract is used as a raw material, and the EGCG is separated and purified by adopting a dynamic axial compression column.
2. The method according to claim 1, characterized in that it comprises the steps of:
(1) And (3) carrying out primary separation and purification on the epigallocatechin gallate monomer by using a dynamic axial compression column: injecting the tea polyphenol extract into a balanced dynamic axial compression column for gradient elution, detecting sample separation condition by an ultraviolet detector, concentrating the collected EGCG fraction, and removing the solvent;
(2) Refining epigallocatechin gallate monomer by dynamic axial compression column: and (3) injecting the EGCG fraction obtained in the step (1) into a balanced dynamic axial compression column for isocratic elution, detecting the sample separation condition by using an ultraviolet detector, concentrating the collected EGCG fraction, and removing the solvent to obtain the refined epigallocatechin gallate monomer.
3. The process according to claim 1 or 2, wherein the packing of the dynamic axial compression column in step (1) is a C18 packing having a particle size of 20-40 μm;
preferably, the dynamic axial compression column has a column inner diameter of 200mm.
4. A method according to any one of claims 1 to 3, wherein the gradient elution method in step (1) is: eluting with pure water for 0-20 min; eluting with 10% ethanol and 90% pure water for 20-40 min; eluting with 20% ethanol and 80% pure water for 40-60 min;
preferably, the elution flow rate is 600-800ml/min and the pressure is 1.0-1.2MPa.
5. The method according to any one of claims 1 to 4, wherein the packing of the dynamic axial compression column in step (2) is a C18 packing having a particle size of 10 to 20 μm;
preferably, the particle size of the packing of the dynamic axial compression column in step (1) is 20um when different from the particle size of the packing of the dynamic axial compression column in step (2);
preferably, the dynamic axial compression column has a column inner diameter of 100mm.
6. The method according to any one of claims 1 to 5, wherein the elution method in step (2) is: eluting with 15% ethanol and 85% pure water for 0-35 min;
preferably, the elution flow rate is 100-200ml/min and the pressure is 4.0-4.5MPa.
7. The method according to any one of claims 1 to 6, wherein the ultraviolet detector wavelength in step (1) and step (2) is 280nm.
8. The method according to any one of claims 1 to 7, wherein the concentration in step (1) and step (2) is performed by vacuum concentration.
9. The method according to any one of claims 1-8, characterized in that the method further comprises the steps of: mixing fresh tea with water, leaching, separating, concentrating, and filtering to obtain tea polyphenols extract.
10. The method of claim 9, wherein the leach liquor ratio is 1:20-30 (m/v), the leaching temperature is 70-90 ℃, and the leaching time is 40-60min.
11. A method according to claim 9 or claim 10 wherein the fresh tea leaves have a moisture content of from 70% to 80%.
12. The method according to any one of claims 1 to 11, wherein the time t is collected in step (1) R Fraction of =50-58 min,
preferably, the time t is collected in step (2) R Fraction =25-33 min.
13. Use of the method of any one of claims 1-12 in the preparation of epigallocatechin gallate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311130968.1A CN117339239A (en) | 2023-08-31 | 2023-08-31 | Method for preparing epigallocatechin gallate by dynamic axial compression column |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311130968.1A CN117339239A (en) | 2023-08-31 | 2023-08-31 | Method for preparing epigallocatechin gallate by dynamic axial compression column |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117339239A true CN117339239A (en) | 2024-01-05 |
Family
ID=89354731
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311130968.1A Pending CN117339239A (en) | 2023-08-31 | 2023-08-31 | Method for preparing epigallocatechin gallate by dynamic axial compression column |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117339239A (en) |
-
2023
- 2023-08-31 CN CN202311130968.1A patent/CN117339239A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11795130B2 (en) | Process for separating a constituent/cannabinoid using a chromatographic resin | |
| CN100364985C (en) | Method of preparing tea polyphenol by resin adsorption method | |
| CN110642679A (en) | Method for preparing cannabidiol and coproducing cannabis full spectrum oil | |
| CN102276679A (en) | Method for extracting high-purity tea saponin from oil-tea-cake by decompression boiling | |
| CN110724589B (en) | Method for preparing cannabidiol and coproducing cannabis full spectrum oil | |
| CN111960930A (en) | Method for separating and purifying cannabidiol from industrial cannabis sativa leaves | |
| CN109134560A (en) | A method of extracting anthocyanin from Roselle calyx | |
| CN112010738B (en) | Industrial method for producing cannabinoid compound by utilizing chromatographic separation | |
| CN103772339B (en) | A kind of method extracting NVP-XAA 723 from tealeaves tankage | |
| CN107304213B (en) | Method and equipment for processing poppy extract | |
| CN103058871B (en) | Separation and purification method of tobacco chlorogenic acid | |
| CN103910705B (en) | The method of rapid extraction separation and purification NVP-XAA 723 from the tankage of green tea | |
| CN111978156B (en) | Method for preparing cannabidiol | |
| CN105287690A (en) | Bilberry extract and preparation method thereof | |
| CN115260005B (en) | Method for purifying and extracting cannabidiol and application thereof | |
| CN117339239A (en) | Method for preparing epigallocatechin gallate by dynamic axial compression column | |
| CN103570647A (en) | Method for preparing high-purity paclitaxel from taxus chinensis cell culture fluid | |
| CN107382943B (en) | Method for subcritical water extraction of dihydroquercetin in sorghum bran | |
| CN107245046B (en) | A method of extracting separating pepper total alkaloids and capsicum red pigment from capsicum | |
| CN111253221B (en) | Method for separating and purifying cannabidiol | |
| CN112939744A (en) | Efficient extraction method of cannabidiol | |
| CN102276570A (en) | Method for purifying epigallo catechin gallate (EGCG) | |
| CN109180485A (en) | A kind of method of separating chlorogenic acid | |
| CN107739306A (en) | A kind of processing method for preparing refined Rosmarinus officinalis extract | |
| CN107298688A (en) | A kind of purification process for extracting concentration qinghaosu |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |