CN109232253B - Preparation method of ferulic acid saturated fat diester - Google Patents
Preparation method of ferulic acid saturated fat diester Download PDFInfo
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- CN109232253B CN109232253B CN201811238591.0A CN201811238591A CN109232253B CN 109232253 B CN109232253 B CN 109232253B CN 201811238591 A CN201811238591 A CN 201811238591A CN 109232253 B CN109232253 B CN 109232253B
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- ethyl acetate
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 title abstract description 9
- 229940114124 ferulic acid Drugs 0.000 title abstract description 9
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 title abstract description 9
- 235000001785 ferulic acid Nutrition 0.000 title abstract description 9
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 title abstract description 9
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 title abstract description 7
- 150000005690 diesters Chemical class 0.000 title abstract description 5
- 235000021003 saturated fats Nutrition 0.000 title description 2
- 150000001875 compounds Chemical class 0.000 claims abstract description 49
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000000284 extract Substances 0.000 claims abstract description 17
- 239000003960 organic solvent Substances 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 238000004440 column chromatography Methods 0.000 claims abstract description 9
- 229920005654 Sephadex Polymers 0.000 claims abstract description 7
- 239000012507 Sephadex™ Substances 0.000 claims abstract description 7
- 239000012043 crude product Substances 0.000 claims abstract description 6
- 239000003480 eluent Substances 0.000 claims abstract description 6
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 57
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 18
- 238000000605 extraction Methods 0.000 claims description 17
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- 241000209140 Triticum Species 0.000 claims description 13
- 235000021307 Triticum Nutrition 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 12
- 239000003208 petroleum Substances 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 9
- 239000000796 flavoring agent Substances 0.000 claims description 6
- 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 claims description 6
- 230000008569 process Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 125000003158 alcohol group Chemical group 0.000 claims description 2
- 239000002994 raw material Substances 0.000 abstract description 12
- -1 ferulic acid ester compounds Chemical class 0.000 abstract description 9
- 229920006395 saturated elastomer Polymers 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 150000003254 radicals Chemical class 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000003078 antioxidant effect Effects 0.000 description 5
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000004809 thin layer chromatography Methods 0.000 description 4
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 3
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 230000002000 scavenging effect Effects 0.000 description 3
- MUUHXGOJWVMBDY-UHFFFAOYSA-L tetrazolium blue Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC=CC=2)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 MUUHXGOJWVMBDY-UHFFFAOYSA-L 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000013124 brewing process Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 150000001793 charged compounds Chemical class 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N deuterated chloroform Substances [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 240000006053 Garcinia mangostana Species 0.000 description 1
- 235000017048 Garcinia mangostana Nutrition 0.000 description 1
- 241001620225 Garcinia multiflora Species 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- VPUSOBLQICITSB-UHFFFAOYSA-N [NH4+].[NH4+].C(C)N1CSC2=C1C=CC(=C2)S(=O)(=O)[O-].C(C)N2CSC1=C2C=CC(=C1)S(=O)(=O)[O-].[N] Chemical compound [NH4+].[NH4+].C(C)N1CSC2=C1C=CC(=C2)S(=O)(=O)[O-].C(C)N2CSC1=C2C=CC(=C1)S(=O)(=O)[O-].[N] VPUSOBLQICITSB-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004898 kneading Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
- C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
- C07C69/734—Ethers
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a preparation method of ferulic acid saturated fatty diester, belonging to the technical field of preparation of ferulic acid ester compounds. The invention provides a preparation method of a compound shown as a formula I or a mixture of more than two compounds, which is obtained by separating the compound from Daqu. Specifically, the method comprises the following steps: a. crushing Daqu, extracting with an organic solvent, and concentrating to obtain Daqu extract; b. carrying out normal phase silica gel column chromatography on the Daqu extract, collecting eluent containing the compound shown in the formula I, and concentrating to obtain a crude product; c. and (3) performing sephadex column chromatography on the crude product, collecting eluent containing the compound shown in the formula I, and concentrating to obtain the compound. The invention directly uses the yeast for brewing wine as a preparation raw material, is cheap and easy to obtain, and greatly improves the availability of the compound in the formula I.
Description
Technical Field
The invention relates to a preparation method of ferulic acid saturated fatty diester, belonging to the technical field of preparation of ferulic acid ester compounds.
Background
Ferulic acid saturated fatty diester compound (1E,22Z) -1, 22-dieruloyloxydocosine (1), molecular weight: 694.44, respectively; (1E,24Z)1, 24-dieruloyloxyyter-acoside (2), molecular weight: 722.48, respectively; (1E,26Z) -1, 26-dierulyloxyhexacosane (3), molecular weight: 750.51 are partially symmetrical compounds containing two feruloyl groups, and have the following chemical structure:
in 2003, (1E,22Z) -1, 22-diphenoxydocosane and (1E,24Z) -1, 24-diphenoxyxydocosane were isolated from the stems of Garcinia multiflora by Yi-Ming Chiang et al. At present, only one of the methods for producing (1E,22Z) -1, 22-difluorooxydocosine and (1E,24Z) -1, 24-difluorooxybenzosine is reported. The structure of (1E,26Z) -1, 26-dierulyloxyhexacosane has not been reported.
Daqu is a raw material for brewing wine, which is prepared by using wheat and the like as raw materials, crushing, adding water, kneading, pressing into fermented grains and allowing various microorganisms in the nature to grow on the fermented grains. The Daqu is an indispensable part in the brewing process of the white spirit, accounts for about 20 percent of brewing raw materials, has the functions of saccharification, fermentation, aroma generation and flavor development, and also directly influences the quality and the yield of the white spirit. During the distillation of the white spirit, part of beneficial components from the Daqu can be brought into the spirit body under the actions of azeotropy, entrainment and the like, so that the white spirit product has a certain health-care effect. Separating the compound with physiological activity in the Daqu can lay a foundation for improving and innovating the white spirit brewing process and producing white spirit products rich in healthy active ingredients.
At present, reports related to the presence of the compounds (1E,22Z) -1, 22-dioloxyocane, (1E,24Z) -1, 24-dioloxyocane and (1E,22Z) -1, 26-dioloxyocane in the Daqu are not found, and reports of the separation and preparation of the compounds from the Daqu are not found.
Disclosure of Invention
The invention aims to provide a preparation method of ferulic acid saturated fatty diester.
The invention provides a method for preparing a compound shown as a formula I or a mixture of more than two compounds: separating from the yeast for making hard liquor:
wherein n is 22, 24 or 26.
Further, the preparation method comprises the following steps:
a. crushing Daqu, extracting with an organic solvent, and concentrating to obtain Daqu extract;
b. carrying out normal phase silica gel column chromatography on the Daqu extract, collecting eluent containing the compound shown in the formula I, and concentrating to obtain a crude product;
c. and (3) performing sephadex column chromatography on the crude product, collecting eluent containing the compound shown in the formula I, and concentrating to obtain the compound.
The invention firstly selects an organic solvent to extract trace target substances in the yeast and simultaneously avoids the main components of the yeast such as starch, sugar, protein and the like from entering the extracting solution. Then, normal phase silica gel column chromatography is adopted, and the principle is that separation is carried out according to different polarities of the compounds, so that the purposes of separation and purification are achieved. The column chromatography is carried out by adopting sephadex column, the principle is that the separation is carried out according to the difference of the molecular weight of the compounds, the compounds with large molecular weight flow out firstly, and the compounds with small molecular weight flow out later. The method can obviously reduce the loss of the target compound in the column chromatography process. In order to separate (1E,22Z) -1, 22-diepoxyldocosane, (1E,24Z) -1, 24-diepoxyldocosane and (1E,26Z) -1, 26-diepoxylhexosane from a yeast with a complex composition, the above separation means must be combined, and the above three steps a, b and c must be omitted.
Further, the organic solvent is an alcohol solvent or ethyl acetate.
Preferably, the alcohol solvent is methanol.
The invention adopts alcohol solvent or ethyl acetate as extraction solvent, can fully extract (1E,22Z) -1, 22-diferroxydocosane, (1E,24Z) -1, 24-diferroxydocosane and (1E,26Z) -1, 26-diferroxyhexocosane in the yeast, and can simultaneously prevent components such as starch, sugar, protein and the like from entering into the extracting solution. In a preferred embodiment, the boiling points of the methanol and the ethyl acetate are low, and the temperature does not need to be set too high during the decompression concentration, so that the target compound can be prevented from being damaged by high temperature, the time can be saved, and the solvent can be conveniently recovered.
Further, when the organic solvent is methanol, at least one of the following is satisfied:
the using amount of the methanol is 2-4 times of the weight of the Daqu;
the extraction temperature is 15-40 ℃;
the extraction times are 2-4 times;
the extraction time is 5-15 days each time.
Further, when the organic solvent is methanol, the step a further comprises the following steps: concentrating the extractive solution, adding water, dispersing, adding ethyl acetate, extracting, and concentrating ethyl acetate phase to obtain Daqu extract.
Preferably, the dispersion is homogeneous at 60 ℃.
Through investigation, the methanol dosage is 2-4 times of the weight of the yeast, so that the (1E,22Z)1, 22-dipheyloxycocosane, (1E,24Z) -1, 24-dipheyloxytocosane and (1E,26Z) -1, 26-dipheyloxycoane in the yeast can be fully extracted, and the waste of the solvent is avoided.
The extraction temperature is the best between 15 and 40 ℃, so that the (1E,22Z) -1, 22-diolyoxydocosane, (1E,24Z) -1, 24-diolyoxydocosane and (1E,26Z) -1, 26-diolyoxyhexocosane in the yeast can be fully extracted, and the serious volatilization and excessive loss of a solvent can not be caused.
The extraction frequency is 2-4 times, each extraction time is 5-15 days, not only can (1E,22Z) -1, 22-diphenoxydocosane, (1E,24Z) -1, 24-diphenoxydocosane and (1E,26Z) -1, 26-diphenoxyhexocosane in the yeast be fully extracted, but also the impurities can be prevented from entering the extracting solution too much, and the difficulty is brought to the subsequent separation and purification.
When methanol is used as an extraction solvent, a plurality of impurities with different polarities in the yeast can be dissolved in the yeast, so that the invention adopts ethyl acetate with medium polarity to extract the concentrated extracting solution, and extracts compounds with medium polarity to reduce the subsequent separation difficulty.
Further, when the organic solvent is ethyl acetate, at least one of the following is satisfied:
the using amount of the ethyl acetate is 2-4 times of the weight of the Daqu;
the extraction temperature is 15-40 ℃;
the extraction times are 2-4 times;
the extraction time is 5-15 days each time.
Further, step b satisfies at least one of the following:
the silica gel is 100-300 mesh normal phase silica gel;
uniformly stirring the Daqu extract and normal phase silica gel in a weight ratio of 1: 1;
chloroform-methanol or petroleum ether-ethyl acetate is used as an elution reagent;
gradient elution is carried out according to the proportion of chloroform/methanol, v/v, 1:0,50:1,30:1 and 10:1 in sequence, or gradient elution is carried out according to the proportion of petroleum ether/ethyl acetate, v/v, 30:1,10:1,5:1,2:1 and 1:1 in sequence.
The invention selects 100-300 mesh normal phase silica gel preferentially, which not only can achieve better purification effect, but also can ensure the separation speed.
Chloroform-methanol or petroleum ether-ethyl acetate is used as an elution reagent, and different compounds in the Daqu extract can be gradually eluted from weak polarity to strong polarity by adjusting the gradient of the eluent.
Further, step c satisfies at least one of the following:
the sephadex is sephadex LH-20;
chloroform-methanol is used as an elution reagent;
preferably, the chloroform-methanol elution reagent has a chloroform to methanol volume ratio of 1: 1.
The sephadex LH-20 column is adopted for chromatography, and chloroform-methanol is used as an elution reagent for elution, so that sephadex LH-20 has gel filtration and reverse distribution effects, compounds with large molecular weight and large polarity are weakly retained and are eluted firstly, and compounds with small molecular weight and small polarity are strongly retained and are eluted later. Meanwhile, the solvent amount used by the scheme is small, the separation efficiency is high, and the cost is low.
Further, the Daqu is medium-temperature wheat starter. The medium-temperature yeast for wheat is prepared from wheat-containing raw materials, and the temperature of a yeast blank is controlled to be 35-60 ℃.
Preferably, the Daqu is a strong-flavor medium-temperature pure wheat starter. The medium-temperature yeast for the strong-flavor pure wheat is prepared by completely taking wheat as a raw material, and the temperature of a yeast blank is controlled to be not more than 35-60 ℃.
The invention provides an application of a compound shown as a formula I or a mixture of more than two compounds in the formula I in preparing antioxidant medicines, health-care products or additives.
Further, the medicine, health product or additive has effects of scavenging DPPH free radical, ABTS free radical or O2 -The efficacy of free radicals.
The present invention provides compounds of formula ii:
wherein n is 26.
The invention provides a preparation method of ferulic acid saturated fatty diester compounds (1E,22Z) -1, 22-diferroyloxydocosane, (1E,24Z) -1, 24-diferroyloxyeicosane and (1E,22Z) -1, 26-diferroyloxycyclohexacoane, and the compounds are prepared by separating from Daqu for the first time by combining a plurality of chromatographic techniques, and mainly have the following advantages:
1. the raw materials are easy to obtain: in the prior art, two compounds of (1E,22Z) -1, 22-diphenoxydocosane and (1E,24Z) -1, 24-diphenoxyoxycosane are separated from stems of mangosteen, and the preparation raw materials are not easy to obtain.
2. The invention discovers the compound (1E,26Z) -1, 26-dierulyloxyhexacosane for the first time: in the existing literature, no structural report of (1E,26Z) -1, 26-dieruloyloxyhexocosane is found, and no report of the compound is isolated. The invention discovers (1E,26Z) -1, 26-diphenoxyhexocosane which is a substance with antioxidant activity for the first time, and separates the (1E,26Z) -1, 26-diphenoxyhexocosane from the Daqu.
Drawings
FIG. 1 is an ESI mass spectrum of (1E,22Z) -1, 22-dieruloyloxydocoane;
FIG. 2 is an ESI mass spectrum of (1E,24Z) -1, 24-dieruloyloxyteracosane;
FIG. 3 is an ESI mass spectrum of (1E,22Z) -1, 26-dierulyloxyhexacosane.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
The invention provides a method for preparing a compound shown as a formula I or a mixture of more than two compounds: separating from the yeast for making hard liquor:
wherein n is 22, 24 or 26.
In the process of exploring health functional factors in white spirit, the inventor discovers that (1E,22Z) -1, 22-dioleyloxydocosane, (1E,24Z) -1, 24-dioleyloxydocosane and (1E,22Z) -1, 26-dioleyloxyhexacosane exist in the brewing raw material Daqu for the first time, provides possibility for separating and preparing the compounds from the Daqu, and also provides a new method for obtaining the compounds in large quantity.
Furthermore, the invention creatively combines a plurality of separation technologies such as silica gel normal phase column chromatography, sephadex column chromatography and the like, and separates the yeast from the yeast with complex components for the first time to obtain the yeast
The target compound is qualitatively analyzed by detection means such as mass spectrum and nuclear magnetism, and the detection means is adopted for (1E,22Z) -1, 22-diphenoxydocosane, (1E,24Z) -1, 22-diphenoxyyteracosane and (1E,22Z) -1, 26-diphenoxyhexocosane.
EXAMPLE 1 preparation of (1E,22Z) -1,22-Diferuloyloxydocoane, (1E,24Z) -1, 24-Diferuloyloxyeicosane and (1E,22Z) -1, 26-Diferuloyloxycyclohexa-cane by the Process of the invention
(1) Taking 30kg of strong-flavor pure wheat medium-temperature yeast (prepared by completely taking wheat as a raw material and controlling the temperature of a yeast blank to be 35-60 ℃), crushing, adding 90L of methanol, extracting for 2 times at 20 ℃ for 5 days each time, filtering an extracting solution, and concentrating under reduced pressure to obtain a yeast extract. Adding 5L of distilled water into the crude extract of Daqu, dispersing at 60 deg.C, adding 5L of ethyl acetate into a separating funnel, extracting for 3 times, collecting ethyl acetate phase, and concentrating under reduced pressure to obtain 420g of Daqu extract.
(2) Uniformly stirring the Daqu extract and 100-mesh normal phase silica gel according to the weight ratio of 1:1, performing normal phase silica gel column chromatography, performing gradient elution by using chloroform-methanol as an elution reagent and using the ratios of 1:0,50:1,30:1 and 10:1 (chloroform/methanol, v/v), and eluting 4 liters in each ratio; collecting the elution parts at a ratio of 30:1-10:1 in sections, carrying out thin layer chromatography (petroleum ether/acetone, 3:1, v/v), combining the samples with the Rf value of 0.5, and drying under reduced pressure to obtain the component EA-5.
(3) Subjecting the component EA-5 obtained in step (2) to sephadex LH-20 column chromatography, eluting with chloroform/methanol as an eluting reagent at a ratio of 1:1 (chloroform/methanol, v/v), subjecting the eluate to thin layer chromatography (petroleum ether/acetone, 3:1, v/v), combining samples with Rf value of 0.50, and drying under reduced pressure.
Subjecting the separated sample to nuclear magnetic resonance test, 1H-NMR (400MHz, CDCl)3) The data are as follows: 1.25(38H, brs),4.14,4.17(2H, t, J ═ 6.6Hz),3.92(6H, s, OMe-3 '), 5.84(2H, s, OH-4 and OH-4'), 5.81,6.78(1H, d, J ═ 13.0Hz, H-8 ', H-7'), 6.29,7.61(1H, d, J ═ 15.7Hz, H-8, H-7),6.88,6.90(1H, d, J ═ 8.3Hz, H-5 '), 7.03,7.72(1H, d, J ═ 1.8Hz, H-2'), 7.07,7.11(1H, dd, J ═ 8.3,1.8, H-6, 6-6 Hz);13C NMR(CDCl3100MHz) δ: 29.9,29.5(n-CH2),65.1(O-CH2),56.62,56.10(OMe-3, OMe-3 '), 109.5(C-2),113.0 (C-2'), 113.9(C-5),114.9(C-5 '), 116.5(C-8),117.8 (C-8'), 123.18(C-6),125.68(C-6 '), 128.3(C-1),128.1 (C-1'), 143.2(C-7),144.5(C-7 '), 146.1(C-3),146.9 (C-3'), 147.1(C-4),148.0(C-4 '), 166.2(C-9),166.9 (C-9'). Analyzing the mixture obtained in the step (3) by using a mass spectrum (HPLC-Qtof-MS), wherein the detected result shows 3 molecular ion peaks which are respectively: m/z 547.3996[ M + H ]]+Characteristic daughter ion 177.0549, see fig. 1; m/z 575.4348[ M + H ]]+Characteristic daughter ion 177.0543, see fig. 2; m/z603.4657[ M + H ]]+Its characteristic daughter ion is 177.0540, see FIG. 3. The above spectral data demonstrate that the present invention provides a mixture of three compounds, i.e., (1E,22Z) -1, 22-dierulyloxydocosane, (1E,24Z) -1, 24-dierulyloxyphytacosane and (1E,22Z) -1, 26-dierulyloxyhexacosane, isolated from the yeast.
Example 2 preparation of (1E,22Z) -1, 22-Diferroxydocoane, (1E,24Z) -1, 24-Diferroxynico and (1E,22Z) -1, 26-Diferroxyhexaco-cane by the Process of the invention
(1) Taking 30kg of Luzhou-flavor pure wheat medium-temperature yeast (prepared by completely taking wheat as a raw material and controlling the temperature of a yeast blank to be 35-60 ℃), crushing, adding 90L of ethyl acetate, extracting for 2 times at 30 ℃ for 7 days each time, filtering an extracting solution, and concentrating under reduced pressure to obtain a Daqu extract.
(2) Uniformly stirring the Daqu extract and 300-mesh normal-phase silica gel in a weight ratio of 1:1, performing normal-phase silica gel column chromatography, performing gradient elution by using petroleum ether-ethyl acetate as an elution reagent and in a ratio of 30:1,10:1,5:1,2:1,1:1 (petroleum ether/ethyl acetate, v/v), and eluting 5 liters in each ratio; collecting the eluted part at ratio of 2:1, performing thin layer chromatography (petroleum ether/acetone, 3:1, v/v), combining the samples with Rf value of 0.50, and drying under reduced pressure to obtain component EA-5.
(3) Subjecting the component EA-5 obtained in step (2) to sephadex LH-20 column chromatography, eluting with chloroform/methanol as an eluting reagent at a ratio of 1:1 (chloroform/methanol, v/v), subjecting the eluate to thin layer chromatography (petroleum ether/acetone, 3:1, v/v), combining samples with Rf value of 0.50, and drying under reduced pressure.
Subjecting the separated sample to nuclear magnetic resonance test, 1H-NMR (400MHz, CDCl)3) The data are as follows: 1.25(38H, brs),4.14,4.17(2H, t, J ═ 6.6Hz),3.92(6H, s, OMe-3 '), 5.84(2H, s, OH-4 and OH-4'), 5.81,6.78(1H, d, J ═ 13.0Hz, H-8 ', H-7'), 6.29,7.61(1H, d, J ═ 15.7Hz, H-8, H-7),6.88,6.90(1H, d, J ═ 8.3Hz, H-5 '), 7.03,7.72(1H, d, J ═ 1.8Hz, H-2'), 7.07,7.11(1H, dd, J ═ 8.3,1.8, H-6, 6-6 Hz);13C NMR(CDCl3100MHz) δ: 29.9,29.5(n-CH2),65.1(O-CH2),56.62,56.10(OMe-3, OMe-3 '), 109.5(C-2),113.0 (C-2'), 113.9(C-5),114.9(C-5 '), 116.5(C-8),117.8 (C-8'), 123.18(C-6),125.68(C-6 '), 128.3(C-1),128.1 (C-1'), 143.2(C-7),144.5(C-7 '), 146.1(C-3),146.9 (C-3'), 147.1(C-4),148.0(C-4 '), 166.2(C-9),166.9 (C-9'). Analyzing the mixture obtained in the step (3) by using a mass spectrum (HPLC-Qtof-MS), wherein the detected result shows 3 molecular ion peaks which are respectively: m/z 547.3996[ M + H ]]+Characteristic daughter ion 177.0549, see fig. 1; m/z 575.4348[ M + H ]]+Characteristic daughter ion 177.0543, see fig. 2; m/z603.4657[ M + H ]]+Its characteristic daughter ion is 177.0540, see FIG. 3. The above spectral data demonstrate that the present invention provides three types of (1E,22Z) -1, 22-dioloxyoctane, (1E,24Z) -1, 24-dioloxyocane and (1E,22Z) -1, 26-dioloxyocane separated from the yeastMixtures of compounds.
EXAMPLE 3 antioxidant Activity test of (1E,22Z) -1, 22-Difuloyloxydocosane, (1E,24Z) -1, 24-Difuloyloxyphytocosane and (1E,22Z) -1, 26-Difuloyloxyhexo-san
The invention separates (1E,22Z) -1, 22-dieruloyloxydocosine, (1E,24Z)1, 24-dieruloyloxyster-aconane and (1E,26Z) -1, 26-dieruloyloxydaxacosine from the Daqu, the structures and properties of the three compounds are very similar, and the further separation and purification are more complicated, so the following activity test is directly carried out in the form of mixture (yellow oil).
DPPH free radical, ABTS free radical and O of the present experiment on the Compound2 -The scavenging ability of free radicals was measured to evaluate their antioxidant activity.
DPPH experiment
Accurately weighing 58.9mg of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) powder, dissolving with ethanol, diluting to 100mL, and storing at-20 ℃ for later use. When in use, the solution is diluted by 20 times with ethanol and is ready to use. mu.L of DPPH solution is added into 10. mu.L of 0.6mg/mL sample solution (the final concentration of the sample in the reaction system is 20. mu.g/mL) which is placed in a 96-well plate, and after the reaction is carried out for 30min in the absence of light at room temperature, the absorption value of the reaction solution is measured at 517nm by using a multifunctional reading instrument.
ABTS experiment
ABTS (2, 2-linked nitrogen-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) powder 96.2mg and potassium persulfate 16.5mg are precisely weighed, dissolved in water and added to 25mL in constant volume, and the mixture is transposed and stored at 4 ℃ for later use after being reacted for 16 hours in a dark place. When in use, 350 mu L of the solution is diluted to 25mL by 80% ethanol and is ready to use. 250 mu L of ABTS solution is added into 10 mu L of 0.52mg/mL sample solution which is placed in a 96-well plate (the final concentration of the sample in the reaction system is 20 mu g/mL), and after the reaction is carried out for 5min in a dark place at room temperature, the absorption value of the reaction solution is measured by a multifunctional reading instrument at 734 nm.
3. Superoxide anion radical scavenging experiment
150 μ M NBT (blue tetrazolium), 234 μ M NADH (reduced coenzyme I) and 30 μ M PMS (phenazine methosulfate) solutions were prepared with 16mM Tris-HCl buffer (pH 8.0), and stored in a refrigerator at 4 ℃ for further use. When in use, 100. mu.L of NBT solution, 100. mu.L of NADH solution and 100. mu.L of PMS solution (the final concentration of the sample in the reaction system is 20. mu.g/mL) are sequentially added to 10. mu.L of 0.62mg/mL sample solution which has been placed in a 96-well plate. After 5min reaction at room temperature, the absorbance of the reaction solution was measured at 560nm using a multifunctional reader.
Results of the experiment
The positive control is vitamin VcWhen the concentration is 5.0 mu M, the clearance rate of the compound on DPPH free radicals is 48.6 +/-0.5 percent, and the clearance rate of the compound on ABTS free radicals is 18.3 +/-0.6 percent; to O2 -The clearance rate of free radicals is 87.6 +/-0.3%. . At a concentration of 20. mu.g/mL, the compound has a DPPH radical clearance of 62.3% + -0.5%; the scavenging capacity for ABTS free radicals is 18.1 +/-0.2%; to O2 -The clearance rate of free radical clearance is 54.2 +/-0.6%. . The experimental results show that the compound has obvious antioxidant activity.
Claims (10)
1. A process for the preparation of a compound of formula I or a mixture of two or more compounds thereof, characterized in that: the yeast is obtained by separating yeast from yeast for making hard liquor, wherein the yeast for making hard liquor is wheat medium temperature yeast:
wherein n is 22, 24 or 26;
the preparation method comprises the following steps:
a. crushing Daqu, extracting with an organic solvent, and concentrating to obtain Daqu extract; the organic solvent is an alcohol solvent or ethyl acetate;
b. carrying out normal phase silica gel column chromatography on the Daqu extract, collecting eluent containing the compound shown in the formula I, and concentrating to obtain a crude product; wherein, chloroform-methanol or petroleum ether-ethyl acetate is used as an elution reagent in the step b;
c. performing Sephadex column chromatography on the crude product, collecting eluate containing compound of formula I, and concentrating; wherein, chloroform-methanol is used as an elution reagent in the step c.
2. The method of claim 1, wherein: the alcohol solvent is methanol.
3. The method of claim 1, wherein: when the organic solvent is methanol, at least one of the following conditions is satisfied:
the using amount of the methanol is 2-4 times of the weight of the Daqu;
the extraction temperature is 15-40 ℃;
the extraction times are 2-4 times;
the extraction time is 5-15 days each time.
4. The method of claim 1, wherein: when the organic solvent is methanol, the step a further comprises the following steps: concentrating the extractive solution, adding water, dispersing, adding ethyl acetate, extracting, and concentrating ethyl acetate phase to obtain Daqu extract.
5. The method of claim 4, wherein: dispersing uniformly at 60 ℃.
6. The method of claim 1, wherein: when the organic solvent is ethyl acetate, at least one of the following conditions is satisfied:
the using amount of the ethyl acetate is 2-4 times of the weight of the Daqu;
the extraction temperature is 15-40 ℃;
the extraction times are 2-4 times;
the extraction time is 5-15 days each time.
7. The method of claim 1, wherein: step b satisfies at least one of the following:
the silica gel is 100-300 mesh normal phase silica gel;
uniformly stirring the Daqu extract and normal phase silica gel in a weight ratio of 1: 1;
gradient elution is carried out according to the proportion of chloroform/methanol, v/v, 1:0,50:1,30:1 and 10:1 in sequence, or gradient elution is carried out according to the proportion of petroleum ether/ethyl acetate, v/v, 30:1,10:1,5:1,2:1 and 1:1 in sequence.
8. The method of claim 1, wherein: the sephadex in the step c is sephadex LH-20.
9. The method of claim 1, wherein: the volume ratio of chloroform to methanol in the chloroform-methanol elution reagent is 1: 1.
10. The method of claim 1, wherein: the Daqu is a strong-flavor medium-temperature yeast of pure wheat.
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