CN114832440B - Preparation method and application of antioxidant component in paeonia lactiflora - Google Patents
Preparation method and application of antioxidant component in paeonia lactiflora Download PDFInfo
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- 244000236658 Paeonia lactiflora Species 0.000 title claims abstract description 23
- 235000008598 Paeonia lactiflora Nutrition 0.000 title claims abstract description 23
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 18
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000004185 countercurrent chromatography Methods 0.000 claims abstract description 97
- YKRGDOXKVOZESV-WRJNSLSBSA-N Paeoniflorin Chemical compound C([C@]12[C@H]3O[C@]4(O)C[C@](O3)([C@]1(C[C@@H]42)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C)OC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-WRJNSLSBSA-N 0.000 claims abstract description 77
- 238000000926 separation method Methods 0.000 claims abstract description 72
- YKRGDOXKVOZESV-UHFFFAOYSA-N paeoniflorin Natural products O1C(C)(C2(CC34)OC5C(C(O)C(O)C(CO)O5)O)CC3(O)OC1C24COC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-UHFFFAOYSA-N 0.000 claims abstract description 70
- 239000002904 solvent Substances 0.000 claims abstract description 41
- 239000000287 crude extract Substances 0.000 claims abstract description 33
- -1 monoterpene glycoside compounds Chemical class 0.000 claims abstract description 31
- 229930182470 glycoside Natural products 0.000 claims abstract description 24
- 229930003658 monoterpene Natural products 0.000 claims abstract description 24
- 235000002577 monoterpenes Nutrition 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 21
- 239000004480 active ingredient Substances 0.000 claims abstract description 16
- 238000002347 injection Methods 0.000 claims abstract description 15
- 239000007924 injection Substances 0.000 claims abstract description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 90
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 33
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- 238000007254 oxidation reaction Methods 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 235000013824 polyphenols Nutrition 0.000 abstract description 14
- 238000004925 denaturation Methods 0.000 abstract description 2
- 230000036425 denaturation Effects 0.000 abstract description 2
- 239000004615 ingredient Substances 0.000 abstract description 2
- 230000002427 irreversible effect Effects 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- 229940126680 traditional chinese medicines Drugs 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 description 40
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 28
- VFPFQHQNJCMNBZ-UHFFFAOYSA-N ethyl gallate Chemical compound CCOC(=O)C1=CC(O)=C(O)C(O)=C1 VFPFQHQNJCMNBZ-UHFFFAOYSA-N 0.000 description 23
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 20
- 239000012071 phase Substances 0.000 description 19
- FBSFWRHWHYMIOG-UHFFFAOYSA-N methyl 3,4,5-trihydroxybenzoate Chemical compound COC(=O)C1=CC(O)=C(O)C(O)=C1 FBSFWRHWHYMIOG-UHFFFAOYSA-N 0.000 description 17
- 230000000694 effects Effects 0.000 description 11
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- 239000004262 Ethyl gallate Substances 0.000 description 8
- 235000006708 antioxidants Nutrition 0.000 description 8
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- 239000003446 ligand Substances 0.000 description 7
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 6
- QQUHMASGPODSIW-ICECTASOSA-N albiflorin Chemical compound O([C@@]12C[C@H]3[C@H](O)C[C@@]1(OC(=O)[C@]32COC(=O)C=1C=CC=CC=1)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QQUHMASGPODSIW-ICECTASOSA-N 0.000 description 6
- 229910052739 hydrogen Inorganic materials 0.000 description 6
- 239000001257 hydrogen Substances 0.000 description 6
- IBKQQKPQRYUGBJ-UHFFFAOYSA-N methyl gallate Natural products CC(=O)C1=CC(O)=C(O)C(O)=C1 IBKQQKPQRYUGBJ-UHFFFAOYSA-N 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 5
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 102000004034 Kelch-Like ECH-Associated Protein 1 Human genes 0.000 description 5
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- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241000736199 Paeonia Species 0.000 description 3
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 3
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 3
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- 238000002474 experimental method Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 229960001285 quercetin Drugs 0.000 description 3
- 235000005875 quercetin Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000003064 anti-oxidating effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229940125898 compound 5 Drugs 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
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- 230000000670 limiting effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 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 2
- 230000004048 modification Effects 0.000 description 2
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- 230000004792 oxidative damage Effects 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 101100536885 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) THI5 gene Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- 238000003570 cell viability assay Methods 0.000 description 1
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- 238000004737 colorimetric analysis Methods 0.000 description 1
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- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- JYVHOGDBFNJNMR-UHFFFAOYSA-N hexane;hydrate Chemical compound O.CCCCCC JYVHOGDBFNJNMR-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
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- 201000007270 liver cancer Diseases 0.000 description 1
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- 230000001404 mediated effect Effects 0.000 description 1
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- 239000000178 monomer Substances 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000010587 phase diagram Methods 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- 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
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
-
- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention belongs to the technical field of separation and extraction of active ingredients of traditional Chinese medicines, and relates to a preparation method and application of antioxidant ingredients in paeonia lactiflora. And (3) performing linear countercurrent chromatography on the paeoniflorin crude extract to obtain monoterpene glycoside compounds and a stripping sample, separating the monoterpene glycoside compounds by adopting internal circulation countercurrent chromatography, and separating the stripping sample by adopting an off-line linear countercurrent chromatography continuous sample injection mode. The extraction method can separate various active ingredients including monoterpene glycoside compounds, polyphenol compounds and the like from the paeonia lactiflora crude extract, and has the advantages of no irreversible adsorption, high sample injection recovery rate, large capacity, low sample denaturation risk, low solvent consumption and the like.
Description
Technical Field
The invention belongs to the technical field of separation and extraction of active ingredients of traditional Chinese medicines, and relates to a preparation method and application of antioxidant ingredients in paeonia lactiflora.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
According to research of the inventor, the current extraction method of the active ingredients of the paeonia lactiflora comprises silica gel column chromatography, sephadex LH-20 column chromatography, a silica gel and Sephadex LH-20 combined technology, simulated moving bed chromatography and the like. However, the inventors have found that since paeoniflorin and paeoniflorin, which are major compounds in paeonia, are isomers, have similar polarities (compounds 3 and 4 of fig. 1). When the sample injection amount is high, peaks overlap, further limiting large-scale separation, so that the separation of monoterpene glycoside compounds from paeonia lactiflora is challenging.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a preparation method of antioxidant components in paeonia lactiflora and application thereof, various active components including monoterpene glycoside compounds, polyphenol compounds and the like can be separated from a paeonia lactiflora crude extract, and meanwhile, the extraction method has the advantages of no irreversible adsorption, high sample recovery rate, large capacity, low sample denaturation risk, low solvent consumption and the like.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
Firstly, because the paeoniflorin and the paeoniflorin have similar polarity, the separation of the paeoniflorin and the paeoniflorin is difficult to realize by optimizing a solvent system, the invention adopts an internal circulation countercurrent chromatography mode to separate, no matter the paeoniflorin or the paeoniflorin crude extract components are complex, other components are easy to mix in by directly adopting the internal circulation countercurrent chromatography mode, and the separation of the paeoniflorin and the paeoniflorin is difficult to carry out.
The first separation mode (linear countercurrent chromatography mode) is: the two countercurrent chromatographic devices are connected in parallel for separation and extraction;
the second separation mode is: the two countercurrent chromatographic devices are connected in series for separation and extraction;
The third separation mode (internal circulation countercurrent chromatography mode) is: the back counter-current chromatographic device after the series connection of the second separation mode circularly separates and extracts.
The target mixture with similar polarity is separated and extracted through the first separation mode, then the target mixture with similar polarity extracted through the first separation mode can be conveyed to a subsequent countercurrent chromatography device through the second separation mode, and then the target mixture with similar polarity is subjected to circulating countercurrent chromatography separation through the third separation mode. Greatly simplifies the extraction operation of polar similar active compounds in the traditional Chinese medicine.
Preferably, the connection mode is as follows: the countercurrent chromatography device comprises a pump, an injector, a countercurrent chromatography device and a collector which are sequentially connected, wherein a detector is arranged at an outlet of the countercurrent chromatography device, a first six-way valve is arranged between the countercurrent chromatography device and the collector of the first countercurrent chromatography device, a second six-way valve is arranged at a pump inlet of the second countercurrent chromatography device, a third six-way valve is arranged between the countercurrent chromatography device and the collector of the second countercurrent chromatography device, the first six-way valve is directly connected with the second six-way valve, and the first six-way valve is directly connected with the third six-way valve.
On the other hand, the extraction method of the active ingredients in the paeoniflorin crude extract is characterized in that the paeoniflorin crude extract is subjected to linear countercurrent chromatography to obtain monoterpene glycoside compounds and blow-off samples, the monoterpene glycoside compounds are separated by adopting internal circulation countercurrent chromatography, and the blow-off samples are separated by adopting an off-line linear countercurrent chromatography continuous sample injection mode.
Preferably, the paeoniflorin crude extract is separated and extracted by adopting the online switching 2D-CCC system.
In a third aspect, an extraction method of active ingredients in paeoniflorin crude extract is applied to extraction of active ingredients in paeoniflorin.
In a fourth aspect, a method for preparing an antioxidant component in paeonia lactiflora is provided, wherein paeoniflorin crude extract is obtained by extracting paeoniflorin from paeonia lactiflora, and the obtained paeoniflorin crude extract is treated by adopting the method for extracting active components in the paeoniflorin crude extract.
The activity research of the paeoniflorin shows that the paeoniflorin has higher antioxidant activity than quercetin and has potential hydrogen bond effect with VAL418, VAL467, VAL465, VAL561, thr-560 and VAL608, so that oxidation-induced diseases can be prevented, and in a fifth aspect, the paeoniflorin is applied to the preparation of drugs for preventing the oxidation-induced diseases.
The beneficial effects of the invention are as follows:
1. The invention provides a line switching 2D-CCC system, which connects two countercurrent chromatographic devices through three six-way valves, so that three modes of conversion can be formed, the combination of linear countercurrent chromatographic separation and extraction and internal circulation countercurrent chromatographic separation and extraction is realized, the extraction of a target mixture with similar polarity in traditional Chinese medicine or crude extract of traditional Chinese medicine and the separation of the target mixture with similar polarity are realized, and the operation is more convenient.
2. According to the method disclosed by the invention, gallic acid, methyl gallate and ethyl gallate can be extracted, and simultaneously, the paeoniflorin and the paeoniflorin can be completely separated, and researches show that the compounds have excellent antioxidant activity. Meanwhile, the purity of the extracted gallic acid, gallic acid methyl ester, gallic acid ethyl ester, paeoniflorin and paeoniflorin is higher than 98%.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a diagram of the structural formula of an isolated compound in an embodiment of the present invention;
FIG. 2 is a schematic diagram of separation of an on-line switching two-dimensional internal circulation countercurrent chromatography (on-line switching 2D-CCC system) in an embodiment of the invention, wherein a is parallel connection, b is series connection, and c is internal circulation;
FIG. 3 is a chromatogram of stationary phase retention and inner circulation CCC separation of compounds 3 and 4 at different flow rates in the examples of the present invention, a is a histogram of S f at different flow rates, b is a chromatogram of compounds 3 and 4 at a loading of 500mg, c is a chromatogram of compounds 3 and 4 at a loading of 1.0g, d is a chromatogram of compounds 3 and 4 at a loading of 1.5g, e is a chromatogram of a mixed sample of compounds 3 and 4 in d for 3 cycles using inner circulation countercurrent chromatography;
FIG. 4 is a chromatogram of a continuous sample injection CCC separating compounds 1, 2 and 5 in an example of the invention;
FIG. 5 is a liquid phase diagram of a white peony extract and separated components in an embodiment of the present invention, a is a total sample, b is gallic acid, c is methyl gallate, d is paeoniflorin, e is paeoniflorin, f is ethyl gallate, and g is a blown sample);
FIG. 6 is a graph showing the cell viability (a) and antioxidant effect (b) of five compounds on HepG2 cells and the 3D interaction of five compound ligands with KEAP1 protein (c) in the examples of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the problem that in the prior art, monoterpene glycoside compounds are difficult to separate in a large scale from paeonia lactiflora, the invention provides a preparation method and application of an antioxidant component in paeonia lactiflora.
In an exemplary embodiment of the present invention, an on-line switching 2D-CCC system is provided, comprising two countercurrent chromatography devices and three six-way valves, three separation modes being formed by the control of the three six-way valves:
The first separation mode (linear countercurrent chromatography mode) is: the two countercurrent chromatographic devices are connected in parallel for separation and extraction;
the second separation mode is: the two countercurrent chromatographic devices are connected in series for separation and extraction;
The third separation mode (internal circulation countercurrent chromatography mode) is: the back counter-current chromatographic device after the series connection of the second separation mode circularly separates and extracts.
The target mixture with similar polarity is separated and extracted through the first separation mode, then the target mixture with similar polarity extracted through the first separation mode can be conveyed to a subsequent countercurrent chromatography device through the second separation mode, and then the target mixture with similar polarity is subjected to circulating countercurrent chromatography separation through the third separation mode. Greatly simplifies the extraction operation of polar similar active compounds in the traditional Chinese medicine.
In some examples of this embodiment, the connection means are: the countercurrent chromatography device comprises a pump, an injector, a countercurrent chromatography device and a collector which are sequentially connected, wherein a detector is arranged at an outlet of the countercurrent chromatography device, a first six-way valve is arranged between the countercurrent chromatography device and the collector of the first countercurrent chromatography device, a second six-way valve is arranged at a pump inlet of the second countercurrent chromatography device, a third six-way valve is arranged between the countercurrent chromatography device and the collector of the second countercurrent chromatography device, the first six-way valve is directly connected with the second six-way valve, and the first six-way valve is directly connected with the third six-way valve.
In another embodiment of the invention, an extraction method of active ingredients in paeoniflorin crude extract is provided, wherein the paeoniflorin crude extract is subjected to linear countercurrent chromatography to obtain monoterpene glycoside compounds and blow-off samples, the monoterpene glycoside compounds are separated by adopting internal circulation countercurrent chromatography, and the blow-off samples are separated by adopting an off-line linear countercurrent chromatography continuous sample injection mode.
In some examples of this embodiment, paeoniflorin crude extract is isolated and extracted using the on-line switching 2D-CCC system described above.
In some examples of this embodiment, the solvent system for the linear countercurrent chromatographic separation is n-butanol, ethyl acetate and water in a volume ratio of 1.6 to 2.4:2.6 to 3.4:4.6 to 5.4. Research shows that the monoterpene glycoside isomer and the polyphenol compound can be separated by adopting linear countercurrent chromatography under the solvent system.
In some examples of this embodiment, the solvent system for the internal circulation countercurrent chromatography separation is n-butanol, ethyl acetate, and water, the volume ratio of n-butanol, ethyl acetate, and water being 1.6-2.4:2.6-3.4:4.6-5.4. Research shows that the paeoniflorin and paeoniflorin in the monoterpene glycoside isomer can be separated by using internal circulation countercurrent chromatography under the solvent system.
In some examples of this embodiment, the flow rate of the internal circulation countercurrent chromatography separation is 2 to 7mL/min. Studies have shown that peak resolution is better when the flow rate is 2-4 mL/min. Within this range, the faster the flow rate, the higher the separation efficiency. Thus, 4mL/min was chosen as the more optimal flow rate, in combination with peak resolution and separation efficiency.
In some examples of this embodiment, the solvent system for continuous sample injection separation for offline linear countercurrent chromatography is n-hexane/ethyl acetate/methanol/water, and the volume ratio of n-hexane, ethyl acetate, methanol, and water is 1.6-2.4:7.6-8.4:3.6-4.4:5.6-6.4. Researches show that the polyphenol compounds can be separated by using an off-line linear countercurrent chromatography continuous sample injection mode under the solvent system.
In some examples of this embodiment, the separation using countercurrent chromatography is performed by: adding the stationary phase into a countercurrent chromatograph, adding the mobile phase, and adding the sample solution to be separated after the equilibrium is reached.
In one or more embodiments, the ratio of the addition flow rate of the stationary phase to the addition flow rate of the mobile phase is 10:1-2.
In a third embodiment of the present invention, an application of the method for extracting active ingredients from paeoniflorin crude extract in extracting active ingredients from paeoniflorin is provided.
In a fourth embodiment of the present invention, a method for preparing an antioxidant component in paeonia is provided, wherein paeoniflorin crude extract is obtained by extracting paeoniflorin from paeonia, and the obtained paeoniflorin crude extract is treated by adopting the method for extracting active components in the paeoniflorin crude extract.
In a fifth embodiment of the present invention, an application of albiflorin in preparing a medicament for preventing oxidation-induced diseases is provided.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Examples
Instrument and materials
N-butanol, ethyl acetate, methanol and n-hexane were all analytical grade (Shanghai national pharmaceutical chemical Co., ltd.). High performance liquid chromatography grade acetonitrile was purchased from fischer company (fei laugh, new jersey, usa). Reverse osmosis water is produced from the Milli-Q system (millbo, bedford, ma, usa). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum, streptomycin, phosphate buffered saline and penicillin were purchased from Jinan Hui technology (Jinan, china). HepG2 cell lines were purchased from China center for type culture Collection (national cancer institute, beijing, china).
The paeoniflorin crude extract is provided by Shaanxi herbal Biotechnology Co.Ltd (western An, china) and has a paeoniflorin content of about 10%.
Measuring distribution coefficient
Based on the structural characteristics of Paeonia lactiflora, partition coefficients (K D values) were determined using n-butanol/ethyl acetate/water and n-hexane/ethyl acetate/methanol/water solvent systems.
First, a series of different proportions of solvent system were added to 10mL tubes and shaken vigorously. After separation of the upper and lower phases, 1mL of each phase was transferred to a centrifuge tube and the crude extract (5 mg) was completely dissolved under sonication. After the upper phase and the lower phase are completely separated, the two phases are taken out respectively and dried by a centrifugal concentrator. The dried sample was then redissolved in methanol and filtered with a 0.22 μm membrane. The K D value of the target compound was calculated using equation K D＝AU/AL, where a U and a L are the peak areas of the target compound in the upper and lower phases, respectively.
Solvent system and preparation of sample solutions
Separating polyphenol compounds from the stripping part by using an off-line 2D-CCC mode and an n-hexane/ethyl acetate/methanol/water (2:8:4:6, v/v) solvent system, and simultaneously separating main monoterpene glycoside components in paeonia lactiflora by using an on-line 2D-CCC mode and an n-butanol/ethyl acetate/water (2:3:5, v/v) solvent system. First, the solvent system was added to the separation funnel in proportion and the two-phase solvent system was vigorously shaken until phase equilibrium was reached. Separating the upper phase and the lower phase to respectively serve as a stationary phase and a mobile phase.
An on-line switch 2D-CCC sample solution was prepared by dissolving 500mg of the crude extract in 10mL of n-butanol/ethyl acetate/water (2:3:5, v/v) solution. 100mg of crude polyphenol (blow-off sample) was dissolved in 10mL of n-hexane/ethyl acetate/methanol/water (2:8:4:6, v/v) solvent system to prepare an off-line 2D-CCC separation sample solution.
High-speed countercurrent chromatographic separation procedure
On-line switching 2D-CCC combined internal circulation mode
The on-line switching 2D-CCC system consists of two independent CCC devices (fig. 2), including independent pumps, syringes, CCC instruments, detectors and automatic collectors. The two separate CCC devices are connected by three six-way valves.
First, the six-way valve is set to the mode shown in fig. 2 a. The stationary phase was pumped into the CCC instrument at a flow rate of 20mL/min in a head-to-tail direction. The mobile phase was pumped into the instrument at a flow rate of 4mL/min with a rotational speed set at 800rpm. After equilibrium is reached, a sample solution containing 500mg of the crude sample is injected into CCC-1. The effluent was continuously monitored at 230nm and collected in an automatic collector. When the target component is eluted, the six-way valves 1 and 2 are switched. The effluent from the CCC-1 outlet flows into CCC-2 (FIG. 2 b). When the target composition is completely transferred into CCC-2, the six-way valve is set to the mode in fig. 2 c. During this time, CCC-2 was used as an internal circulation means to isolate two monoterpene glycosides. And simultaneously, blowing off a solvent system in the CCC-1 to obtain crude polyphenol. And then pumping a new solvent system to reach equilibrium, and loading the sample solution to realize large-scale separation. After complete separation of the two monoterpene glycosides in CCC-2, the six-way valve 3 was opened and the effluent was collected. The retention of the stationary phase after separation (S f value) was defined as the stationary phase relative to the total column capacity.
Separating crude polyphenol by combining offline 2D-CCC with continuous sample injection
The upper phase (stationary phase) of the n-hexane/ethyl acetate/methanol/water (2:8:4:6, v/v) solvent system was first pumped into the CCC column at a flow rate of 20mL/min. The rotational speed was set at 800rpm and the mobile phase was pumped at a flow rate of 2 mL/min. After equilibration, 100mg of crude polyphenol was injected through a sample loop, the detection wavelength was set at 230nm, and the measurement was continued. During a certain period of time, sample solution was again injected into CCC through the sample loop, and continuous sample separation was performed.
High performance liquid chromatography and structural identification
The crude and CCC fractions were subjected to high performance liquid chromatography using Wooking K2025 high performance liquid chromatography system, WATERS SYMMETRY C 18 column (25 x 4.6mm,5 μm). The mobile phase was acetonitrile/0.1% phosphoric acid solution (14:86, v/v), the flow rate was 1.0mL/min, and the detection wavelength was 230nm.
NMR spectra were recorded on a Bruker AV-400 spectrometer (Bruker 125biospin, rheinstetten, germany) using dimethyl sulfoxide (DMSO). With tetramethylsilane as an internal standard, the chemical shift (δ) value is expressed in parts per million (ppm) and the coupling constant (J) is expressed in Hz.
Cell culture and cell viability assay
HepG2 cell lines were purchased from China center for type culture Collection (national cancer institute, beijing, china). These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100ng/mL streptomycin and 100U/mL penicillin. The cells were cultured in a humidified incubator at 37℃with 5% CO 2. To determine cell viability, hepG2 cells were seeded into 96-well plates at a density of 2 x 10 4 cells/well, then cells were treated with the compounds at concentrations of 0, 50, 100 and 200 μm and cell viability was determined by MTT method.
Role of five components in oxidative damage caused by ABAP
The sample stock was prepared with dimethyl sulfoxide, and HepG2 cells were cultured in 96-well plates at a density of 5×10 4 cells/well, and cultured at 37 ℃ under 5% co 2 for 24 hours. HepG2 cells were incubated with DMEM medium. ABAP solution containing the sample was added to the cells for 24h. About 50. Mu.L of 1mg/mL MTT solution was added to each well, incubated at 37℃for 4 hours, and after removal of the supernatant, 500. Mu.L of dimethyl sulfoxide was added to lyse the cells on a rotary shaker. Absorbance was recorded at 450nm using a microplate reader and the results were expressed as the average of the cell viability of the standardized controls.
Molecular docking
In order to confirm the interaction between the core antioxidant target and the component, a key oxidation stress protein KEAP1 with a high median value is selected as a receptor, and 5 compounds obtained by separation are used as ligands for molecular docking.
The crystal structure of the KEAP1 protein was downloaded from the protein database (http:// www.rcsb.org) and saved in PDB format. And drawing a three-dimensional chemical structure diagram of the candidate compound, and storing the three-dimensional chemical structure diagram in a MOL2 format. All files are converted to PDB format for subsequent molecular docking. In AutoDock software, the water molecules in the ligand were removed, nonpolar hydrogen was added, and the Gasteiger charge was calculated and stored in PDBQT format. And (3) performing energy minimization treatment on the potential core ligands, calculating to obtain the atom types of the ligands, and finally storing the atom types in PDBQT format. And adopting AutoDock software to perform butt joint calculation on the semi-soft molecules.
Statistical analysis
The data obtained was analyzed on SPSS software using one-way analysis of variance followed by LSD testing. Data are expressed as mean ± Standard Deviation (SD), P <0.05 being statistically significant.
Results and discussion
Selection of HSCCC solvent systems
To determine the appropriate two-phase solvent system, the K D value for the target compound was calculated. When the separation coefficient (α value) (α=k 1/K2,K1>K2) is greater than 1.5, the K D value is suitably between 0.5 and 2.0. The K D value is more than 2.0, the retention time is long, the K D value is less than 0.5, and the separation effect is poor.
According to the structural characteristics, solubility and polarity of the target compound, a series of n-butanol/ethyl acetate/water two-phase solvent systems are selected to separate main monoterpene glycoside compounds. As shown in Table 1, the K D values of target compounds 3 and 4 were smaller in the solvent system n-butanol/ethyl acetate/water (0:5:5 and 1:4:5, v/v), indicating poor isolation. In contrast, the K D values for target compounds 3 and 4 were higher in n-butanol/ethyl acetate/water (3:2:5; 4:1:5;5:0:5, v/v) solvent systems. The ratio of n-butanol/ethyl acetate/water was 2:3: in a solvent system of 5 (v/v), compounds 3 and 4 have K D values of 0.98 and 1.15, respectively, and the corresponding alpha value is 1.19. The K D value for compound 1 was 2.31, while the K D values for both compounds 4 and 5 were greater than 5. In all solvent systems, the alpha values of compounds 3 and 4 were less than 1.5, indicating that compounds 3 and 4 did not separate well in one step of isolation. Therefore, in order to improve the separation effect of the main monoterpene glycoside in paeonia lactiflora, it is necessary to introduce on-line conversion 2D-CCC combined with an internal circulation mode.
Partition coefficient of the compounds of Table 1
A series of n-hexane/ethyl acetate/methanol/water solvent systems were also tested for separation of the blow-off samples. As shown in Table 2, when a solvent system of n-hexane/ethyl acetate/methanol/water (1:9:1:9, v/v) was used, the K D values of both compounds 2 and 5 were greater than 4, indicating that the solvent consumption was large, and further increasing the ratio of methanol resulted in a decrease in the K D value. In a solvent system of n-hexane/ethyl acetate/methanol/water (3:7:3:7, v/v), the K D value of compound 5 was in the appropriate range, while the K D value of compound 1 was too small, indicating insufficient resolution. Suitable K D values were measured in an n-hexane/ethyl acetate/methanol/water (2:8:3:7; 2:8:4:6, v/v) solvent system. Considering the K D value and the separation coefficient, the ratio of n-hexane/ethyl acetate/methanol/water is selected to be 2:8:4:6 (v/v) solvent system further experiments were performed.
TABLE 2K D values for off-line 2D-CCC mode isolated polyphenols
Optimization of HSCCC conditions
According to the countercurrent chromatographic separation theory, the higher the S f value, the higher the theoretical plate number, and the better the peak resolution. FIG. 3a shows the effect of flow rate of n-butanol/ethyl acetate/water (2:3:5, v/v) solvent system on the S f value. When the flow rate is 2mL/min, the S f value reaches about 70%. Further increases in flow rate resulted in a decrease in the value of S f, with a value of S f below 60% at flow rates of 5-7 mL/min. According to the separation efficiency and the S f value, 4mL/min is selected as the optimal flow rate.
FIGS. 3b-D show the results (0.5, 1.0 and 1.5 g) of separation from the crude extract of Paeonia lactiflora, using on-line switching of 2D-CCC and internal circulation modes. As summarized in FIG. 3b, compounds 3 and 4 were well isolated at a loading of 500mg in yields of 19.9 and 36.9mg, respectively. The purity can reach more than 98 percent by High Performance Liquid Chromatography (HPLC) determination. The loading was further increased to 1.0g, peak resolution was reduced, and yields were 19.9mg (3) and 47.8mg (4), respectively (FIG. 3 c). At the highest loading of 1.5g, the peaks of compounds 3 and 4 overlap, indicating poor separation. The countercurrent chromatography technique is preparative chromatography, the larger the loading, the less solvent is consumed. Therefore, the sample loading amount is increased while the separation effect is maintained, and the solvent consumption is reduced by adopting a mode of combining on-line switching of the 2D-CCC and an internal circulation mode. Table 3 shows the results of a comparison of conventional and on-line switching of 2D-CCC-inner loop CCC separation of monoterpene glycosides. At the highest loading of 1.5g, the yields of compounds 3 and 4 were 31.2mg/L and 60.3mg/L, respectively, which were increased by 26.3% and 24.6% compared to 1.0g loading, respectively (FIG. 3 d). After the second and third sample injections, the yield of the compound 3 is further improved by 35.6% and 38.9%, respectively, which indicates that the developed online switching 2D-CCC internal circulation separation method can improve the separation efficiency and reduce the solvent consumption. FIG. 3e is an internal circulation CCC process for preparing monoterpene glycosides. After 3 cycles, the compounds 3 and 4 were successfully separated, the yields were 54.5 and 105.4mg, respectively, and the purities were above 98% as determined by high performance liquid chromatography. After concentration in vacuo, a blow-off sample (49.7 mg) was obtained from the CCC column, which was rich in polyphenols (blow-off sample) and could be further separated by off-line 2D-CCC.
TABLE 3 comparison of yields of isolated monoterpene glycosides for conventional and on-line switching of 2D-CCC-inner circulation CCC
a The conventional CCC mode b switches 2D-CCC-inner loop CCC on-line.
FIG. 4 shows the separation effect of the blown-off sample by combining the offline 2D-CCC with the continuous sample injection mode, so that the separation time is shortened and the separation efficiency is improved. The total sample injection amount was 300mg, and compound 1 (26.8 mg), compound 2 (40.0 mg) and compound 5 (6.7 mg) were successfully separated, and the purity was more than 98% by HPLC measurement, as shown in FIG. 5.
Structural identification
In summary, 5 monomers were successfully isolated from 10% of the crude paeonia lactiflora extract. Their structure was determined by spectroscopic data, including electrospray mass spectrometry (ESI-MS) and Nuclear Magnetic Resonance (NMR) data.
Gallic acid C 7H6O5, ESI-MS (positive ion mode) )m/z 170.15.1H-NMR(DMSO,400MHz):δH6.91(2H,s,H-2,6).13C-NMR(DMSO,100MHz):δC 121.0(C-1),109.2(C-2),145.9(C-3),138.4(C-4),145.9(C-5),109.2(C-6),168.0(C-7).
Methyl gallate C 8H8O5, ESI-MS (positive ion mode) )m/z 184.27.1H-NMR(DMSO,400MHz):δH6.94(2H,s,H-2,6),3.74(3H,s,CH3);13C-NMR(DMSO,100MHz):δC 119.7(C-1),109.0(C-2),146.1(C-3),139.0(C-4),146.1(C-5),109.0(C-6),166.8(C-7),52.0(CH3).
Paeoniflorin lactone C 23H28O11, ESI-MS (positive ion mode) )m/z 480.63.1H-NMR(DMSO,400MHz):δH1.85(1H,d,J＝15.1Hz,H-3a),2.30(1H,dd,J＝15.1,6.5Hz,H-3b),4.12(1H,t,J＝5.6Hz,H-4),2.68(1H,m,H-5),1.90(1H,d,J＝10.7Hz,H-7a),2.77(1H,m,H-7b),4.64(1H,d,J＝12.1Hz,H-8a),4.55(1H,d,J＝12.1Hz,H-8b),1.39(3H,s,H-10),4.40(1H,d,J＝7.5Hz,H-1'),3.65(1H,m,H-6'a),3.38(1H,m,H-6'b),2.96-3.11(4H,m,H-2'-5'),8.02(2H,d,J＝7.5Hz,H-2",6"),7.55(2H,t,J＝7.5Hz,H-3",5"),7.68(1H,t,J＝7.5Hz,H-4");13C-NMR(DMSO,100MHz):δC 91.5(C-1),85.4(C-2),41.0(C-3),66.5(C-4),41.0(C-5),55.1(C-6),27.4(C-7),61.6(C-8),175.2(C-9),20.5(C-10),99.0(C-1'),73.8(C-2'),77.5(C-3'),70.6(C-4'),77.3(C-5'),61.2(C-6'),130.1(C-1"),129.8(C-2",6"),129.2(C-3",C-5"),133.8(C-4"),166.2(C-7").
Paeoniflorin C 23H28O11, ESI-MS (positive ion mode) )m/z 480.65.1H-NMR(DMSO,400MHz):δH2.05(1H,d,J＝12.4Hz,H-3a),1.65(1H,d,J＝12.4Hz,H-3b),2.44(1H,d,J＝6.4Hz,H-5),2.38(1H,dd,J＝10.4,6.4Hz,H-7a),1.81(1H,d,J＝10.4Hz,H-7b),4.64(2H,d,J＝9.3Hz,H-8),5.33(1H,s,H-9),1.24(3H,s,H-10),4.39(1H,d,J＝7.6Hz,H-1'),3.65(1H,m,H-6'a),3.18-3.45(5H,m,H-2'-6'b),7.99(2H,d,J＝7.5Hz,H-2",6"),7.56(2H,t,J＝7.4Hz,H-3",5"),7.67(1H,t,J＝7.2Hz,H-4");13C-NMR(DMSO,100MHz):δC 88.0(C-1),85.4(C-2),44.1(C-3),105.2(C-4),42.8(C-5),70.7(C-6),22.5(C-7),60.9(C-8),100.5(C-9),19.6(C-10),99.1(C-1'),73.9(C-2'),77.4(C-3'),70.4(C-4'),77.4(C-5'),61.7(C-6'),130.1(C-1"),129.7(C-2",6"),129.2(C-3",C-5"),133.9(C-4"),166.2(C-7").
Ethyl gallate, C 9H10O5, ESI-MS (positive ion mode) )m/z 198.26.1H-NMR(DMSO,400MHz):δH 6.94(2H,s,H-2,6),4.20(2H,q,J＝7.1Hz,CH2),1.27(3H,t,J＝7.1Hz,CH3);13C-NMR(DMSO,100MHz):δC 120.0(C-1),109.0(C-2),146.1(C-3),139.0(C-4),146.1(C-5),109.0(C-6),166.3(C-7),60.4(CH2),14.7(CH3).
Cytotoxicity assay and antioxidant effect
The cytotoxic activities of the compounds gallic acid (1), gallic acid methyl ester (2), paeoniflorin (3), paeoniflorin (4) and gallic acid ethyl ester (5) obtained by separation are measured by adopting an MTT colorimetric method of a human liver cancer cell line HepG2 cell. In this example, four different concentrations (12.5, 25, 50 and 100. Mu.g/mL) were studied and the results are shown in FIG. 6 a. The results show that all compounds showed better cell viability at the concentrations tested. At the highest concentration of 100. Mu.M, the activity of HepG2 cells treated by paeoniflorin (4) was only slightly decreased (87.03.+ -. 0.97%). The experimental result shows that the maximum concentration is 100 mu M, and the method can be used for further antioxidation experiments.
Figure 6b summarizes the results of antioxidant experiments for five compounds at different concentrations (25, 50 and 100 μm). The results show that all tested compounds showed significant protection against ABAP-induced oxidative damage even at the lowest concentration compared to the positive control quercetin. Wherein, the albiflorin (3) has the strongest antioxidation effect, and is secondarily ethyl gallate (5), paeoniflorin (4), gallic acid (1) and methyl gallate (2). Similar to the cytotoxicity test results, gallic acid (1) and paeoniflorin (4) showed slight cytotoxicity at high concentration. The antioxidative activity of the albiflorin (3) and the ethyl gallate (5) is strongest at the concentration of 100 mu M, and the cell activities are 90.78+/-7.34% and 84.88 +/-5.00% (P < 0.05) respectively. This example demonstrates that albiflorin has protective effect on oxidative stress-mediated toxicity, further demonstrating that albiflorin can be used to treat diseases caused by oxidation.
Molecular docking results
The crystalline ligand derived from PDB is docked to the active site to determine binding energy and intermolecular interactions. 5 compounds with excellent oxidation resistance were docked using Autodock. The hydrogen bonding of VAL418 is critical to KEAP1 inhibition properties, indicating hinge region interactions. Table 4 shows the binding energy results and FIG. 6c shows the 3D interaction pattern of the isolated compounds. As shown, all 5 compounds have very high binding affinity. The docking fraction of the paeoniflorin (3) is highest and is-8.22 kcal/mol, and the docking fraction of the ethyl gallate (5) is lowest and is-3.79 kcal/mol. The polyphenols obtained by separation, including gallic acid (1), methyl gallate (2) and ethyl gallate (5), have lower binding energy and weaker hydrogen bonding with VAL606, VAL465, GLY367 and GLY 462. The monoterpene glycoside including paeoniflorin (3) and paeoniflorin (4) has highest negative binding energy and has strong hydrogen bond action with VAL418, VAL467, VAL465, VAL561, thr-560 and VAL 608.
TABLE 4 investigation of the binding energy values of KEAP1 proteins to ligands using Autodock technology
Conclusion(s)
The invention utilizes the efficient 2D-HSCCC combined internal circulation mode to successfully separate main monoterpene glycoside isomers and polyphenol compounds from paeonia lactiflora. For separation of HSCCC, 1D-CCC separation was performed using an n-butanol/ethyl acetate/water (2:3:5, v/v) solvent system. Then, paeoniflorin (54.5 mg, 3), paeoniflorin (105.4 mg, 4) and a mixture (49.7 mg) were isolated from 1.5g of the crude sample using IRCCC mode. The mixture with polyphenols as the main component was further separated using the continuous injection CCC mode. The purity of gallic acid (26.8 mg, 1), methyl gallate (40.0 mg, 2) and ethyl gallate (6.7 mg, 5) was measured by high performance liquid chromatography and was higher than 98%. The result of the antioxidant activity shows that the antioxidant activity of the paeoniflorin is stronger than that of quercetin (positive control). Molecular docking results show that the affinity energy of the paeoniflorin is at least-8.22 kcal/mol, and the paeoniflorin has potential hydrogen bonding effect with VAL418, VAL467, VAL465, VAL561, thr-560 and VAL 608. These results indicate that albiflorin can prevent oxidation-induced diseases.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A method for extracting active ingredients from paeoniflorin crude extract is characterized in that the paeoniflorin crude extract is subjected to linear countercurrent chromatography to obtain monoterpene glycoside compounds and blow-off samples, the monoterpene glycoside compounds are separated by adopting internal circulation countercurrent chromatography, and the blow-off samples are separated by adopting an off-line linear countercurrent chromatography continuous sample injection mode;
The paeoniflorin crude extract is separated and extracted by adopting an on-line switching 2D-CCC system, wherein the on-line switching 2D-CCC system comprises two countercurrent chromatographic devices and three six-way valves, and three separation modes are formed by controlling the three six-way valves:
The first split mode is: the two countercurrent chromatographic devices are connected in parallel for separation and extraction;
the second separation mode is: the two countercurrent chromatographic devices are connected in series for separation and extraction;
the third split mode is: the back counter-current chromatographic device after the series connection of the second separation mode is used for circularly separating and extracting;
The solvent system for the linear countercurrent chromatographic separation is n-butanol, ethyl acetate and water, and the volume ratio of the n-butanol to the ethyl acetate to the water is 1.6-2.4:2.6-3.4:4.6-5.4;
the solvent system for the internal circulation countercurrent chromatographic separation is n-butanol, ethyl acetate and water, and the volume ratio of the n-butanol to the ethyl acetate to the water is 1.6-2.4:2.6-3.4:4.6-5.4;
the flow rate of the internal circulation countercurrent chromatographic separation is 2-7 mL/min;
The solvent system for continuous sample injection separation of the offline linear countercurrent chromatography is n-hexane/ethyl acetate/methanol/water, and the volume ratio of n-hexane to ethyl acetate to methanol to water is 1.6-2.4:7.6-8.4:3.6-4.4:5.6-6.4.
2. The method for extracting active ingredients from paeoniflorin crude extract as claimed in claim 1, wherein the countercurrent chromatography device comprises a pump, an injector, a countercurrent chromatography device and a collector which are sequentially connected, wherein a detector is arranged at an outlet of the countercurrent chromatography device, a first six-way valve is arranged between the countercurrent chromatography device and the collector of the first countercurrent chromatography device, a second six-way valve is arranged at a pump inlet of the second countercurrent chromatography device, a third six-way valve is arranged between the countercurrent chromatography device and the collector of the second countercurrent chromatography device, the first six-way valve is directly connected with the second six-way valve, and the first six-way valve is directly connected with the third six-way valve.
3. The method for extracting active ingredients from paeoniflorin crude extract as claimed in claim 1, wherein the separation process using countercurrent chromatography is as follows: adding the stationary phase into a countercurrent chromatograph, adding the mobile phase, and adding the sample solution to be separated after the equilibrium is reached.
4. The method for extracting active ingredients from paeoniflorin crude extract as claimed in claim 3, wherein the ratio of the addition flow rate of the stationary phase to the addition flow rate of the mobile phase is 10:1-2.
5. Use of the method for extracting active ingredients from paeoniflorin crude extract according to any one of claims 1 to 4 in extracting active ingredients from paeonia lactiflora.
6. A method for preparing antioxidant components in paeonia lactiflora, which is characterized in that paeoniflorin crude extract is obtained by extracting paeoniflorin from paeonia lactiflora, and the obtained paeoniflorin crude extract is treated by adopting the extraction method of active components in the paeoniflorin crude extract according to any one of claims 1-4.
7. Use of paeoniflorin in the preparation of a medicament for preventing oxidation-induced disorders, wherein said paeoniflorin is obtained by the preparation method of claim 6.
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