CN113754705A - Method for separating and preparing two petunidin compounds from lycium ruthenicum murr - Google Patents
Method for separating and preparing two petunidin compounds from lycium ruthenicum murr Download PDFInfo
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- 241000169546 Lycium ruthenicum Species 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 28
- 150000002981 petunidin Chemical class 0.000 title claims abstract 4
- 239000002253 acid Substances 0.000 claims abstract description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 44
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 18
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 11
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- 239000003960 organic solvent Substances 0.000 claims description 10
- 239000011347 resin Substances 0.000 claims description 9
- 229920005989 resin Polymers 0.000 claims description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
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- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
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- 238000000926 separation method Methods 0.000 claims description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 229940125904 compound 1 Drugs 0.000 claims description 4
- 229940125782 compound 2 Drugs 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 238000004237 preparative chromatography Methods 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 235000002639 sodium chloride Nutrition 0.000 claims description 2
- 235000010208 anthocyanin Nutrition 0.000 abstract description 22
- 229930002877 anthocyanin Natural products 0.000 abstract description 22
- 239000004410 anthocyanin Substances 0.000 abstract description 22
- 150000004636 anthocyanins Chemical class 0.000 abstract description 22
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- 238000000605 extraction Methods 0.000 abstract description 6
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 3
- 238000002390 rotary evaporation Methods 0.000 description 8
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- -1 petunidin compound Chemical class 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- RJRZRMYKFWYPNX-UHFFFAOYSA-N [NH4+].[NH4+].CCO.[O-]S([O-])(=O)=O Chemical compound [NH4+].[NH4+].CCO.[O-]S([O-])(=O)=O RJRZRMYKFWYPNX-UHFFFAOYSA-N 0.000 description 4
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- 238000007254 oxidation reaction Methods 0.000 description 2
- QULMBDNPZCFSPR-UHFFFAOYSA-N petunidin chloride Chemical compound [Cl-].OC1=C(O)C(OC)=CC(C=2C(=CC=3C(O)=CC(O)=CC=3[O+]=2)O)=C1 QULMBDNPZCFSPR-UHFFFAOYSA-N 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- JPFCOVZKLAXXOE-XBNSMERZSA-N (3r)-2-(3,5-dihydroxy-4-methoxyphenyl)-8-[(2r,3r,4r)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2h-chromen-4-yl]-3,4-dihydro-2h-chromene-3,5,7-triol Chemical compound C1=C(O)C(OC)=C(O)C=C1C1[C@H](O)CC(C(O)=CC(O)=C2[C@H]3C4=C(O)C=C(O)C=C4O[C@@H]([C@@H]3O)C=3C=CC(O)=CC=3)=C2O1 JPFCOVZKLAXXOE-XBNSMERZSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
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- 229940124600 folk medicine Drugs 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- UHNSRFWQBVXBSK-UHFFFAOYSA-N methanol;2,2,2-trifluoroacetic acid Chemical compound OC.OC(=O)C(F)(F)F UHNSRFWQBVXBSK-UHFFFAOYSA-N 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
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- 150000007965 phenolic acids Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000004262 preparative liquid chromatography Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 150000008265 rhamnosides Chemical class 0.000 description 1
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- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention provides a method for simultaneously separating two petunidin compounds from lycium ruthenicum, which comprises the steps of obtaining a lycium ruthenicum anthocyanin extract by utilizing double-aqueous-phase extraction, optimizing process parameters of a high performance liquid chromatography mixed acid mobile phase system, and separating and preparing 2 high-purity petunidin compounds from the lycium ruthenicum anthocyanin extract.
Description
Technical Field
The invention relates to the technical field of plant extraction, in particular to a method for separating a petunidin compound from lycium ruthenicum.
Background
Lycium ruthenicum (Lycium ruthenicum Murr) belongs to Lycium of Solanaceae, is mainly distributed in middle Asia regions, is distributed in Qinghai, Ningxia, Xinjiang, Gansu, Shaanxi and other places in China, and is a special perennial shrub wild plant in arid regions of China. According to records in the four medical classics, the Tibetan medicine uses the lycium ruthenicum mill to treat diseases such as heart heat disease, heart disease, irregular menstruation, menopause and the like; folk medicines are used as tonics, strengthening bodies, improving eyesight, lowering blood pressure and the like. Researches show that the lycium ruthenicum contains various active ingredients such as polysaccharide, flavone, proanthocyanidin, anthocyanin and phenolic acid, and has the effects of resisting fatigue, reducing blood sugar, resisting oxidation, inhibiting tumors, nourishing, protecting health and the like.
Anthocyanins (Anthocyanins) are water-soluble natural pigments widely present in plants in nature, and belong to flavonoid compounds. Anthocyanins mainly exist in the forms of glucoside, rhamnoside, etc. in fruits, epidermis and flowers of plants. The content of anthocyanin in the lycium ruthenicum is extremely high, wherein more than 90% of anthocyanin is a petunidin derivative, has the functions of resisting oxidation, protecting eyesight, inhibiting tumors and the like, and has important biological activity and extraction value. With the increasing demand for functional components of natural origin and no toxic or side effects, anthocyanins are the most representative functional components of natural origin. Because of good coloring function and excellent antioxidant activity, the pigment is popular among consumers and the market.
For example, publication numbers CN108517000A, CN108409806A, and CN108516999A all refer to the separation of petunidin compounds from blueberries, but the method comprises four steps: the steps of alcohol extraction and concentration, resin adsorption, preparation liquid chromatography purification and high-speed countercurrent chromatography separation are simple in CN104513218A, but the resolution of the high-speed countercurrent chromatography used in the method is low, and only one high-purity petunidin compound can be obtained; CN103626814B, the steps are simple, but the separation efficiency is low, and a plurality of lycium ruthenicum anthocyanin monomers cannot be prepared at one time.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for simultaneously separating a plurality of petunidin compounds from lycium ruthenicum.
Specifically, the invention provides a method for simultaneously separating the following two petunidin compounds from lycium ruthenicum mill: the compound is:
the method comprises the following steps:
(1) adding lycium ruthenicum superfine powder into an inorganic salt water solution and a hydrophilic low-molecular organic solvent, extracting, standing and phase splitting;
(2) taking the upper phase, removing the solvent, adding absolute ethyl alcohol to precipitate inorganic salt, filtering, and removing the absolute ethyl alcohol from the liquid part to obtain a solid matter;
(3) the solid was dissolved in acidified methanol and separated by preparative chromatography under the following conditions:
detection wavelength: 520 +/-2 nm;
a chromatographic column: c18;
mobile phase: a phase is acetonitrile containing 0-5% v/v acid, B phase is water containing 0-5% v/v acid, and the gradient elution procedure is as follows:
| time (min) | |
| 0 | 10 |
| 20 | 17 |
| 25 | 17 |
| 55 | 25 |
| 60 | 90 |
；
(4) Respectively collecting target eluates, removing solvent, dissolving in water, adsorbing with deacidification resin, and eluting with 50-95% v/v ethanol to obtain compound 1 and compound 2.
The invention utilizes double-aqueous phase extraction to obtain the lycium ruthenicum anthocyanin extract, optimizes the process parameters of a high performance liquid chromatography mixed acid mobile phase system, and separates and prepares 2 high-purity petunidin compounds from the lycium ruthenicum anthocyanin extract
Wherein the inorganic salt is selected from: one or more of sodium chloride, ammonium sulfate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate. Further, the inorganic salt is selected from ammonium sulfate.
Wherein the hydrophilic low molecular organic solvent is one or more of methanol, ethanol, acetone, isopropanol, and n-butanol. Further, the hydrophilic low-molecular organic solvent is ethanol.
Wherein the hydrophilic low molecular organic solvent accounts for 20-40% of the total mass of the inorganic salt A aqueous solution and the hydrophilic low molecular organic solvent.
Wherein the inorganic salt accounts for 15-25% of the total mass of the inorganic salt water solution and the hydrophilic low-molecular organic solvent.
Wherein the total mass ratio of the lycium ruthenicum superfine powder to the inorganic salt A water solution and the hydrophilic low-molecular organic solvent is 1: 5-1: 20.
wherein the acid is selected from one or more mixed acids of phosphoric acid, trifluoroacetic acid, citric acid and dilute nitric acid; further selected from phosphoric acid: trifluoroacetic acid: citric acid: the mol ratio of the dilute nitric acid is 2: 6: 4: 1.
further, collecting the eluent for 18-20min at the flow rate of 100mL/min and the column temperature of 30 ℃, and separating to obtain a compound 1; collecting eluate for 34-35min, and separating to obtain compound 2.
Further, the column model is 50mm × 500 mm.
Further, the concentration of ethanol used for eluting the deacidification resin in the step (4) is 80-95%.
The purpose of the acid-removing resin is to exchange and remove acid to prepare an acid-free product, and all the existing resin chips capable of meeting the requirement can be used in the invention. For example selected from TulsionT CH-32 or A-722MP or A654.
In the early experiments, the mixed acid mobile phase system can effectively improve the resolution of the anthocyanin on the reverse chromatographic column. As the radius of the chromatography column increases, the column efficiency is affected by an increase in the molecular diffusion terms of the chromatography material. By using a mixed acid mobile phase system, the column effect reduction caused by the influence of molecular diffusion when the inner diameter of the column is increased is counteracted, so that the resolution is improved to achieve the purpose of separating two types of petunidin at one time, and the method is more suitable for large-scale production.
The method adopts a mixed acid system to be matched with a dynamic axial compression column to prepare the petunidin compound in the lycium ruthenicum, and the purity of the petunidin compound is as high as 95%. Compared with the common semi-preparative column, the separation method improves the sample loading amount by more than 4 times under the condition of not reducing the separation rate, has simple method, can prepare 2 types of petunidin compounds at one time, and can safely apply the petunidin to various industries after acidification treatment.
Drawings
FIG. 1 is a high performance liquid chromatogram of the anthocyanin extract of Lycium ruthenicum Murr of example 1;
FIG. 2 is a liquid chromatogram of the anthocyanin extract of Lycium ruthenicum Murr of example 1;
FIG. 3 is a high performance liquid chromatogram of product 1 of example 1;
FIG. 4 is a high performance liquid chromatogram of product 2 of example 1;
FIG. 5 is a nuclear magnetic H spectrum of product 1 of example 1;
FIG. 6 is a nuclear magnetic H spectrum of product 2 from example 1.
Detailed Description
In order that the present invention may be more readily understood, the present invention is further described without limiting in any way the invention by reference to the following specific examples which are intended only to illustrate the invention and are not intended to limit the scope of the invention, which are intended to be within the scope of the claims of the invention without departing from the technical solution of the invention.
Example 1
1. Anthocyanin extract preparation
Crushing the dried lycium ruthenicum mill by using an ultrafine crusher, and sieving by using a 100-mesh sieve to obtain lycium ruthenicum submicron powder; weighing 170g of ammonium sulfate, adding 590g of water to prepare an aqueous solution, then adding 240g of absolute ethyl alcohol to prepare an ethanol-ammonium sulfate two-aqueous-phase system, adding 100g of lycium ruthenicum superfine powder into the two-aqueous-phase system, carrying out ultrasonic assisted extraction for 30min, and standing for 1h at room temperature for layering; after the upper phase is subjected to rotary evaporation of the solvent, absolute ethyl alcohol is used for dissolving, filtering and recovering inorganic salt, and the absolute ethyl alcohol is subjected to rotary evaporation and recovery again to obtain 5.0 g of a product, wherein the content of anthocyanin is 34% and the extraction rate reaches 79%;
2. preparation of mixed acid
Taking analytically pure phosphoric acid, trifluoroacetic acid, citric acid and dilute nitric acid according to a molar mass ratio of 2: 6: 4: 1, configuring; 3. high performance liquid chromatography preparation
Dissolving lycium ruthenicum anthocyanin powder with 1% trifluoroacetic acid methanol by using a dynamic axial compression column C18-ODS (50mm multiplied by 500mm), an air pressure of 3.5Mpa and an oil pressure of 5Mpa to ensure that the final concentration is 100mg/mL, injecting the solution into a preparative liquid chromatography, wherein the sample injection amount is 20mL, the effective anthocyanin loading amount is about 680mg at a flow rate of 100mL/min, and the mobile phase A: acetonitrile, phase B: mixed acid (5%): water (95%); the column temperature is 30 ℃; the mobile phase gradient elution procedure was:
4. detection and characterization of prepared fractions
Detecting wavelength at 520nm, collecting 18-20min and 34-35min fractions, removing acetonitrile under reduced pressure, adsorbing with deacidification resin A654, and eluting with 90% ethanol to obtain 173.5mg of product 1(18-20min) with purity of 95% and 421.8mg of product 2(34-35min) with purity of 99%; nuclear magnetic hydrogen spectrum data of components 18-20min and 34-35min are shown in fig. 5 and fig. 6, and the structure is identified, the product 1 is petronidin-3-O- (glucosyl-trans-p-coenzyme rho-xylopyranoside) -5-O-glucoside, the product 2 is petronidin 3-O- [6-O- (4-O-E-p-coenzyme- α -L-rhodopyranosyl) - β -D-glucopyranoside ] -5-O- β -D-glucopyranoside:
example 2
The anthocyanin extract was prepared as follows, with other steps seen in example 1.
Crushing the dried lycium ruthenicum mill by using an ultrafine crusher, and sieving by using a 150-mesh sieve to obtain lycium ruthenicum submicron powder; weighing 300g of ammonium sulfate, adding 1300g of water to prepare an aqueous solution, then adding 400g of absolute ethyl alcohol to prepare an ethanol-ammonium sulfate two-aqueous-phase system, adding 100g of lycium ruthenicum superfine powder into the two-aqueous-phase system, carrying out ultrasonic assisted extraction for 60min, and standing for 3h at room temperature for layering; and (3) after the solvent is subjected to rotary evaporation on the upper phase, dissolving the solvent by using absolute ethyl alcohol, filtering and recovering inorganic salt, and performing rotary evaporation again to recover the absolute ethyl alcohol to obtain the product.
Example 3
The anthocyanin extract was prepared as follows, with other steps seen in example 1.
Crushing the dried lycium ruthenicum mill by using an ultrafine crusher, and sieving by using a 80-mesh sieve to obtain lycium ruthenicum submicron powder; weighing 125g of ammonium sulfate, adding 200g of water to prepare an aqueous solution, then adding 200g of absolute ethyl alcohol to prepare an ethanol-ammonium sulfate two-aqueous-phase system, adding 100g of lycium ruthenicum superfine powder into the two-aqueous-phase system, carrying out ultrasonic assisted extraction for 20min, and standing at room temperature for 2h for layering; and (3) after the solvent is subjected to rotary evaporation on the upper phase, dissolving the solvent by using absolute ethyl alcohol, filtering and recovering inorganic salt, and performing rotary evaporation again to recover the absolute ethyl alcohol to obtain the product.
Example 4
The anthocyanin extract was prepared as follows, with other steps seen in example 1.
Crushing the dried lycium ruthenicum mill by using an ultrafine crusher, and sieving by using a 100-mesh sieve to obtain lycium ruthenicum submicron powder; weighing 200g of ammonium sulfate, adding 500g of water to prepare an aqueous solution, then adding 300g of absolute ethyl alcohol to prepare an ethanol-ammonium sulfate two-aqueous-phase system, adding 100g of lycium ruthenicum superfine powder into the two-aqueous-phase system, carrying out ultrasonic assisted extraction for 40min, and standing for 2h at room temperature for layering; and (3) after the solvent is subjected to rotary evaporation on the upper phase, dissolving the solvent by using absolute ethyl alcohol, filtering and recovering inorganic salt, and performing rotary evaporation again to recover the absolute ethyl alcohol to obtain the product.
Claims (10)
1. The method for simultaneously separating and preparing two petunidin compounds from lycium ruthenicum is characterized by comprising the following steps of: the compound is:
the method comprises the following steps:
(1) adding lycium ruthenicum superfine powder into an inorganic salt water solution and a hydrophilic low-molecular organic solvent, extracting, standing and phase splitting;
(2) taking the upper phase, removing the solvent, adding absolute ethyl alcohol to precipitate inorganic salt, filtering, removing the absolute ethyl alcohol from the liquid part to obtain a solid, and dissolving the solid with methanol or acetonitrile containing 0.1-5% v/v acid;
(3) the separation was carried out by preparative chromatography under the following chromatographic conditions:
detection wavelength: 520 +/-2 nm;
a chromatographic column: c18;
mobile phase: a phase is acetonitrile containing 0-5% v/v acid, B phase is water containing 0-5% v/v acid, and the gradient elution procedure is as follows:
；
(4) Respectively collecting target eluates, removing solvent, dissolving in water, adsorbing with deacidification resin, and eluting with 50-95% v/v ethanol to obtain compound 1 and compound 2.
2. The method of claim 1, wherein: the inorganic salt is selected from: one or more of sodium chloride, ammonium sulfate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate; further, the inorganic salt is selected from ammonium sulfate.
3. The method of claim 1, wherein: the hydrophilic low molecular organic solvent is one or more of methanol, ethanol, acetone, isopropanol, and n-butanol.
4. The method of claim 1, wherein: the hydrophilic low molecular weight organic solvent is ethanol.
5. The method of claim 1, wherein: in the methanol or acetonitrile containing 0.1-5% v/v of acid in the step (2), the acid is one or more mixed acid selected from phosphoric acid, trifluoroacetic acid, citric acid and dilute nitric acid; further selected from trifluoroacetic acid or an acid for the mobile phase.
6. The method according to claim 1 or 5, characterized in that: the acid used in the mobile phase is one or more mixed acid selected from phosphoric acid, trifluoroacetic acid, citric acid and dilute nitric acid; further selected from phosphoric acid: trifluoroacetic acid: citric acid: the mol ratio of the dilute nitric acid is 2: 6: 4: 1.
7. the method of claim 1, wherein: collecting eluate at the flow rate of 100mL/min and the column temperature of 30 deg.C for 18-20min, and separating to obtain compound 1; collecting eluate for 34-35min, and separating to obtain compound 2.
8. The method according to claim 1 or 5, characterized in that: the type of the chromatographic column is 50mm multiplied by 500 mm.
9. The method of claim 1, wherein: and (4) the concentration of the ethanol used for eluting the deacidification resin in the step (4) is 80-95%.
10. The method according to claim 1 or 7, characterized in that: the acid-removing resin is TulsionT CH-32 or A-722MP or A654.
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