CN114573474A - Method for preparing hydroxy sanshool by deep eutectic solvent extraction and dynamic axial chromatography - Google Patents
Method for preparing hydroxy sanshool by deep eutectic solvent extraction and dynamic axial chromatography Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 30
- BHHBIFKHVGSQFJ-UHFFFAOYSA-N Hydroxy-Sanshool Natural products CC=CC=CC=CCCC=CC=CC(=O)NC(C)(C)O BHHBIFKHVGSQFJ-UHFFFAOYSA-N 0.000 title claims abstract description 24
- 230000005496 eutectics Effects 0.000 title claims abstract description 19
- 238000000638 solvent extraction Methods 0.000 title abstract description 7
- 238000004587 chromatography analysis Methods 0.000 title abstract description 5
- 238000000605 extraction Methods 0.000 claims abstract description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000002904 solvent Substances 0.000 claims abstract description 29
- 239000012071 phase Substances 0.000 claims abstract description 19
- 150000001875 compounds Chemical class 0.000 claims abstract description 16
- 230000006835 compression Effects 0.000 claims abstract description 12
- 238000007906 compression Methods 0.000 claims abstract description 12
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 241000949456 Zanthoxylum Species 0.000 claims abstract description 8
- 239000002994 raw material Substances 0.000 claims abstract description 8
- 239000003960 organic solvent Substances 0.000 claims abstract description 6
- 238000000746 purification Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000004237 preparative chromatography Methods 0.000 claims abstract description 5
- 239000007791 liquid phase Substances 0.000 claims abstract description 3
- 238000005191 phase separation Methods 0.000 claims abstract description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 17
- 239000001257 hydrogen Substances 0.000 claims description 17
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 239000000178 monomer Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- SBXYHCVXUCYYJT-UEOYEZOQSA-N alpha-Sanshool Chemical class C\C=C\C=C\C=C/CC\C=C\C(=O)NCC(C)C SBXYHCVXUCYYJT-UEOYEZOQSA-N 0.000 claims description 8
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims description 7
- 235000019743 Choline chloride Nutrition 0.000 claims description 7
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical group [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims description 7
- 229960003178 choline chloride Drugs 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 7
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 235000011054 acetic acid Nutrition 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000002390 rotary evaporation Methods 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 2
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 1
- 238000005352 clarification Methods 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
- 229920001144 Hydroxy alpha sanshool Polymers 0.000 abstract description 22
- -1 hydroxyl sanshool Chemical compound 0.000 abstract description 9
- PSKIOIDCXFHNJA-UHFFFAOYSA-N Sanshool Natural products CC=CC=CC=CCCC=CC=CC(=O)NC(C)C PSKIOIDCXFHNJA-UHFFFAOYSA-N 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 6
- 238000009776 industrial production Methods 0.000 abstract description 4
- 229930014626 natural product Natural products 0.000 abstract description 2
- LHFKHAVGGJJQFF-UHFFFAOYSA-N hydroxyl-alpha-sanshool Natural products CC=CC=CC=CCCC=CC(=O)NCC(C)(C)O LHFKHAVGGJJQFF-UHFFFAOYSA-N 0.000 description 18
- LHFKHAVGGJJQFF-JRNWQWJGSA-N hydroxy-α-sanshool Chemical compound C\C=C/C=C/C=C\CC\C=C\C(\O)=N\CC(C)(C)O LHFKHAVGGJJQFF-JRNWQWJGSA-N 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 11
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 235000002566 Capsicum Nutrition 0.000 description 7
- 239000006002 Pepper Substances 0.000 description 7
- 241000722363 Piper Species 0.000 description 7
- 235000016761 Piper aduncum Nutrition 0.000 description 7
- 235000017804 Piper guineense Nutrition 0.000 description 7
- 235000008184 Piper nigrum Nutrition 0.000 description 7
- LHFKHAVGGJJQFF-UMYNZBAMSA-N (2e,6e,8e,10e)-n-(2-hydroxy-2-methylpropyl)dodeca-2,6,8,10-tetraenamide Chemical compound C\C=C\C=C\C=C\CC\C=C\C(=O)NCC(C)(C)O LHFKHAVGGJJQFF-UMYNZBAMSA-N 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- LHFKHAVGGJJQFF-GUXLVXIJSA-N hydroxy epsilon-sanshool Natural products CC=C/C=C/C=CCCC=CC(=O)NCC(C)(C)O LHFKHAVGGJJQFF-GUXLVXIJSA-N 0.000 description 6
- BHHBIFKHVGSQFJ-JDXPBYPHSA-N (2e,4e,8z,10e,12e)-n-(2-hydroxypropan-2-yl)tetradeca-2,4,8,10,12-pentaenamide Chemical compound C\C=C\C=C\C=C/CC\C=C\C=C\C(=O)NC(C)(C)O BHHBIFKHVGSQFJ-JDXPBYPHSA-N 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- CRPPMKFSMRODIQ-UHFFFAOYSA-N hydroxy gamma-sanshooel Natural products CC=CC=CC=CCCC=CC=CC(=O)NCC(C)(C)O CRPPMKFSMRODIQ-UHFFFAOYSA-N 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000002137 ultrasound extraction Methods 0.000 description 5
- 241000345998 Calamus manan Species 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 244000131415 Zanthoxylum piperitum Species 0.000 description 4
- 235000008853 Zanthoxylum piperitum Nutrition 0.000 description 4
- 241001079064 Zanthoxylum schinifolium Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 150000001408 amides Chemical class 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 235000012950 rattan cane Nutrition 0.000 description 4
- 244000089698 Zanthoxylum simulans Species 0.000 description 3
- 239000000370 acceptor Substances 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 235000019654 spicy taste Nutrition 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000208341 Hedera Species 0.000 description 1
- 101000764872 Homo sapiens Transient receptor potential cation channel subfamily A member 1 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 108090000862 Ion Channels Proteins 0.000 description 1
- 102100026186 Transient receptor potential cation channel subfamily A member 1 Human genes 0.000 description 1
- 235000004417 Zanthoxylum alatum Nutrition 0.000 description 1
- 241000949457 Zanthoxylum armatum Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- KVUKDCFEXVWYBN-UHFFFAOYSA-N gamma-sanshooel Natural products CC=CC=CC=CCCC=CC=CC(=O)NCC(C)C KVUKDCFEXVWYBN-UHFFFAOYSA-N 0.000 description 1
- KVUKDCFEXVWYBN-JDXPBYPHSA-N gamma-sanshool Chemical compound C\C=C\C=C\C=C/CC\C=C\C=C\C(=O)NCC(C)C KVUKDCFEXVWYBN-JDXPBYPHSA-N 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011894 semi-preparative HPLC Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- 230000001052 transient effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/22—Separation; Purification; Stabilisation; Use of additives
- C07C231/24—Separation; Purification
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention relates to a method for preparing hydroxyl sanshool by deep eutectic solvent extraction and dynamic axial chromatography, belonging to the field of natural product extraction. The method comprises the following steps: s1, supercritical CO2Extracting the zanthoxylum oil as a raw material, adding a eutectic solvent for ultrasonic auxiliary extraction, centrifuging after extraction is finished, and collecting a lower DES layer to obtain a DES extracting solution of the hydroxysanshool compound; recovering hydroxy sanshool components from the obtained extracting solution by an organic solvent reverse extraction method to obtain a hydroxy sanshool component enrichment product; s2, dynamic axial preparative chromatography separation and purification: the hydroxy sanshool component enrichment product is completely dissolved by using a mobile phase with the mass volume of 1-3 times, and the dynamic axial compression is adopted to prepare liquid phase separation, wherein the mobile phase comprises methanol, water and the like. The process is simple, does not need a large number of purification steps, is convenient to recover the solvent, has high purity, is suitable for large-scale industrial production, and has strong use value.
Description
Technical Field
The invention relates to a method for preparing hydroxy sanshool by eutectic solvent extraction and dynamic axial chromatography, belonging to the field of natural product extraction.
Background
Zanthoxylum armatum DC is a traditional Chinese medicine and food dual-purpose resource, and has faint scent, softness, no stimulation and bitter taste. Researches show that sanshool and amide homologues thereof with structural changes caused by alkyl unsaturation difference or carbon chain oxidation are regarded as the basis of zanthoxylum schinifolium substances and are also important quality guarantee, and hydroxyl-alpha-sanshool, hydroxyl-beta-sanshool, hydroxyl-gamma-sanshool and hydroxyl-epsilon-sanshool are taken as main materials, so that the zanthoxylum schinifolium can cause the pungent sense by activating Transient Receptor Potential (TRP) V1 and TRPA1 or blocking an ion channel, and simultaneously has the functions of anesthesia, analgesia, inflammation diminishing, intestinal protection and the like. However, the compounds are unstable in structure and are very easy to isomerize, hydrolyze and oxidize in the air, so that technical bottlenecks exist in large-scale preparation of high-purity sanshools. At present, a standard method for evaluating the spicy taste of the zanthoxylum/zanthoxylum piperitum and extracting the spicy taste substances applicable to industrial production is not established at home and abroad, so that the quality of the zanthoxylum bungeanum, the zanthoxylum piperitum raw materials and the products thereof is uneven, and the benefit of consumers and the effective supervision of the market are seriously influenced.
The preparation method of sanshool compounds disclosed in Chinese patent CN102771747B, CN102690208A and CN105237430A comprises using organic solvent or supercritical CO2Extracting pepper/rattan pepper samples, purifying by silica gel and gel chromatography, collecting fractions under different elution conditions, obtaining higher-purity monomers by utilizing instruments such as preparative thin-layer chromatography, preparative/semi-preparative HPLC and the like, and having the advantages of complex and complicated preparation process, small preparation amount and low efficiency, so that the research on the rapid and macro extraction of pepper/rattan pepper samplesThe preparation process for preparing various high-purity monomers has great scientific significance and engineering application value.
Disclosure of Invention
The invention aims to provide a method for preparing hydroxy sanshool by deep eutectic solvent extraction and dynamic axial chromatography, which is a method for rapidly preparing high-purity hydroxy sanshool compounds in an industrial grade based on green novel deep eutectic solvent extraction and provides a reference substance for analyzing spicy components of zanthoxylum piperitum and evaluating quality.
The purpose of the invention is realized by the following technical scheme:
a method for rapidly preparing high-purity sanshool compounds at an industrial level comprises the following steps:
s1, by supercritical CO2Extracting the zanthoxylum oil as a raw material, adding a eutectic solvent (DES), performing ultrasonic-assisted extraction, centrifuging after extraction is finished, and collecting a lower DES layer to obtain a DES extracting solution of hydroxysanshool components; and (3) recovering the hydroxy sanshool components from the obtained extracting solution by an organic solvent reverse extraction method to obtain a hydroxy sanshool component enriched product.
Preferably, the eutectic solvent is prepared by a method that a hydrogen bond donor and a hydrogen bond acceptor are mixed according to a certain molar ratio, sealed and then heated at 80 ℃ to be co-dissolved until the mixture is clear, so as to form a DES solvent, and the DES solvent is kept stand at normal temperature for later use.
Preferably, the hydrogen bond donor is choline chloride, the hydrogen bond acceptor is one of formic acid, acetic acid and lactic acid, and the molar ratio is 1: 2.
Preferably, the centrifugal speed is 4000r/mi, and the centrifugal time is 10 min.
Preferably, the ultrasonic-assisted treatment conditions comprise that the material-liquid ratio is 1: 5-1: 10(w/v), the extraction power is 150-200 w, and the extraction time is 0.5 h.
Preferably, the organic solvent used for the back extraction is ethyl acetate, and the feed-liquid ratio is 1: 5-1: 10.
S2, dynamic axial preparative chromatography separation and purification: dissolving the sanshool enrichment completely with a mobile phase with the mass and volume of 1-3 times, preparing liquid phase separation by adopting dynamic axial compression, wherein the mobile phase is methanol and water, the detection wavelength is 254nm, collecting target components according to different retention times, decompressing, rotary-steaming and concentrating eluent, and freeze-drying to obtain each monomer compound.
Preferably, the dynamic axial compression column packing in the dynamic axial preparative liquid chromatography is selected from one or more of Nucifera C18, Hedera ODS-2, ACCHAR X5 and ACCHAR C18PE, and the inventor can realize effective separation of the hydroxysanshool through screening by Nucifera C18 and Hedera ODS-2.
Octadecyl reverse silica gel, the particle size of the silica gel is 5-10 μm, and the preferable particle size is 5 μm; the dynamic axial compression column is preferably 50mm by 250mm in gauge.
Preferably, the mobile phase ratio is methanol: water 65: 35-85: 15 (v/v).
Preferably, the flow rate of the elution is 10 to 40 mL/min.
Preferably, the concentration of the sample injection liquid is 50-150 mg/mL.
The eutectic solvent is formed by combining hydrogen bond donors (such as amide, carboxylic acid, polyalcohol and other compounds) and hydrogen bond acceptors (such as quaternary ammonium salt) through hydrogen bonds, has a melting point which is obviously lower than that of pure substances of all components, is green and environment-friendly, has no toxicity, is low in raw material cost, and can be recovered. The design of this patent synthesizes the higher DESS of extraction efficiency, combines dynamic axial preparation chromatogram, realizes the large-scale industrial preparation of serial high-purity sanshool class compound in the rattan pepper.
Compared with the prior art, the positive effects of the invention are as follows:
the DES solvent is prepared by mixing choline chloride and acids, and compared with the conventional extraction solvent, the extraction rate of the hydroxysanshool compound is remarkably improved.
The invention adopts a whole set of technology taking eutectic solvent extraction/purification-dynamic axial compression column separation as a core for the first time to simultaneously separate hydroxyl-alpha-sanshool, hydroxyl-beta-sanshool, hydroxyl-gamma-sanshool and hydroxyl-epsilon-sanshool from the zanthoxylum oil, establishes a process route for extracting, purifying and separating 4 monomer compounds from the zanthoxylum, has simple process, does not need a large number of purification steps, is convenient for recovering the solvent, has high purity, is suitable for large-scale industrial production, and has very strong use value.
Drawings
FIG. 1 is an HPLC chromatogram of the separation using DAC of example 1, wherein a, b, c, d represent hydroxy- α -sanshool, hydroxy- β -sanshool, hydroxy- γ -sanshool, hydroxy- ε -sanshool, in that order.
FIG. 2 is an HPLC chromatogram of hydroxy-alpha-sanshool prepared in example 1, with a purity of 97.63%.
FIG. 3 is an HPLC chromatogram of hydroxy- β -sanshool prepared in example 1, with a purity of 99.96%.
FIG. 4 is an HPLC chromatogram of hydroxy-gamma-sanshool prepared in example 1, with a purity of 98.57%.
FIG. 5 is an HPLC chromatogram of hydroxy- ε -sanshool prepared in example 1, with 100% purity.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The material-liquid ratios described in the present application all represent the relationship between the mass g of the material and the volume ml of the liquid, unless otherwise specified.
Example 1:
(1) eutectic Solvent (DES) preparation: mixing choline chloride and acetic acid according to a molar ratio of 1:2, sealing, heating at 80 ℃ for co-dissolving until the mixture is clear to form a DES solvent, and standing at normal temperature for later use.
(2) Extracting the sanshool compound based on the eutectic solvent: collecting 50g Zanthoxylum piperitum oil (preferably supercritical CO)2Extracting, such as oil of Zanthoxylum oil of Yao Mazi) as raw material, adding DES according to a material ratio of 1:10(w/v), performing ultrasonic-assisted extraction for 30min, centrifuging at 4000rpm for 10min, and collecting DES layer to obtain DES extract of hydroxysanshool;adding ethyl acetate into the obtained extracting solution according to the material-liquid ratio of 1:8 to recover sanshools, filtering and collecting filtrate, and performing rotary evaporation and concentration to obtain a hydroxy sanshools component enrichment product.
(3) Dynamic axial compression separation: the chromatographic column adopts Nucifera C18U (5 mu m, 50 multiplied by 250mm) to balance by a mobile phase until a chromatographic baseline is stable, the hydroxy sanshool component enrichment product is configured into 80mg/mL by the mobile phase, a sample solution is obtained by filtration, the industrial preparative chromatography separation is carried out, methanol with the volume fraction of 65% is used as the mobile phase, the flow rate is 40mL/min, the detection wavelength is 254nm, target components are collected according to different retention times, the collected solution is subjected to reduced pressure concentration at 35 ℃, and the monomer compounds are obtained by freeze drying under vacuum.
(4) The monomer compound is detected by HPLC, the purity of the hydroxy-alpha-sanshool is 97.63%, the purity of the hydroxy-beta-sanshool is 99.96%, the purity of the hydroxy-gamma-sanshool is 98.57%, and the purity of the hydroxy-epsilon-sanshool is 100.00%.
Example 2:
(1) eutectic Solvent (DES) preparation: mixing choline chloride and formic acid according to a molar ratio of 1:2, sealing, heating at 80 ℃ for co-dissolving until the mixture is clear to form a DES solvent, and standing at normal temperature for later use.
(2) Extracting the sanshool compound based on the eutectic solvent: 100g of supercritical CO is taken2Extracting the zanthoxylum oil as a raw material, adding DES according to a material ratio of 1:10, performing ultrasonic-assisted extraction for 30min, centrifuging at 4000rpm for 10min, and collecting a DES layer to obtain a sanshool DES extracting solution; adding ethyl acetate into the obtained extracting solution according to the material-liquid ratio of 1:8 to recover sanshools, filtering and collecting filtrate, and performing rotary evaporation and concentration to obtain a hydroxy sanshool component enriched product.
(3) Dynamic axial compression separation: the chromatographic column adopts Nucifera C18U (5 mu m, 50 multiplied by 250mm), the mobile phase is used for balancing until the chromatographic baseline is stable, the hydroxy sanshool component enrichment product is prepared into 100mg/mL by the mobile phase, the sample solution is obtained by filtering, the sample solution is subjected to industrial preparative chromatography separation, gradient elution is adopted, the time is 0min to 60min, the time is 65:35(v/v), the time is 60min to 80min, the time is 85:15, the flow rate is 40mL/min, the detection wavelength is 254nm, the target component is collected according to different retention times, the collected solution is subjected to reduced pressure concentration at 35 ℃ to 45 ℃, and the monomer compounds are obtained by freeze drying under vacuum.
(4) The monomer compound is detected by HPLC, the purity of the hydroxy-alpha-sanshool is 97.57 percent, the purity of the hydroxy-beta-sanshool is 96.38 percent, the purity of the gamma-sanshool is 98.30 percent, and the purity of the hydroxy-epsilon-sanshool is 97.70 percent.
Example 3
(1) Eutectic Solvent (DES) preparation: mixing choline chloride and acetic acid according to a molar ratio of 1:2, sealing, heating at 80 ℃ for co-dissolving until the mixture is clear to form a DES solvent, and standing at normal temperature for later use.
(2) Extracting the sanshool compound based on the eutectic solvent: 100g of supercritical CO is taken2Extracting the zanthoxylum bungeanum oil as a raw material, adding DES (DES) according to a material ratio of 1:10, performing ultrasonic-assisted extraction for 30min, centrifuging at 4000rpm for 10min, and collecting a DES layer to obtain a zanthoxylum bungeanum DES extracting solution; adding ethyl acetate into the obtained extracting solution according to the material-liquid ratio of 1:8 to recover sanshools, filtering and collecting filtrate, and performing rotary evaporation and concentration to obtain a hydroxy sanshool component enriched product.
(3) Dynamic axial compression separation: hedera ODS-2(5 mu m, 50 multiplied by 250mm) is adopted as a chromatographic column, a mobile phase is used for balancing until a chromatographic baseline is stable, a hydroxy sanshool component enrichment product is prepared into 100mg/mL by the mobile phase, a sample solution is obtained by filtering, the sample solution is subjected to industrial preparative chromatographic separation, methanol with the volume fraction of 65% is used as the mobile phase, the flow rate is 45mL/min, the detection wavelength is 254nm, target components are collected according to different retention times, the collected solution is subjected to reduced pressure concentration at 35-45 ℃, and freeze drying is carried out under vacuum to obtain each monomeric compound.
(4) The monomer compound is detected by HPLC, the purity of the hydroxy-alpha-sanshool is 97.24 percent, the purity of the hydroxy-beta-sanshool is 98.23 percent, the purity of the hydroxy-gamma-sanshool is 96.54 percent, and the purity of the hydroxy-epsilon-sanshool is 99.39 percent.
Comparative example 1
In contrast to example 1, no DES solvent was used, but methanol was used as extraction solvent.
Comparative example 2
Compared to example 1, urea, acetamide (amide) was used as hydrogen bond donor.
Comparative example 3
The column used acchmrom C18PE (7um, 50 x 250mm) compared to example 1. The obtained monomer compound is detected by HPLC, the purity of the hydroxy-alpha-sanshool is 90.64 percent, the purity of the hydroxy-beta-sanshool is 64.68 percent, the purity of the hydroxy-gamma-sanshool is 62.45 percent, and the purity of the hydroxy-epsilon-sanshool is 85.82 percent. Compared with examples 1, 2 and 3, the impurity content is higher, and the purity is reduced.
Test example 1
Performing spectrum detection on the substance obtained by the preparation, and performing detection by adopting High Performance Liquid Chromatography (HPLC), wherein the chromatographic conditions are as follows: mobile phase A: water, mobile phase B: methanol; 0-50 min, wherein A and B are 50: 50-25: 75; flow rate: 1mL/min, column temperature: 30 ℃; ultraviolet detection wavelength: 254nm, sample size: 10 uL. Calculating the extraction rate of each extraction method, wherein the extraction rate is equal to the percentage of the content of the sanshool compound in the extraction solvent to the total content of the two phases.
Wherein, the extraction rates of the examples 1, 2 and 3 are respectively 24 percent, 23 percent and 23 percent; compared with the example, the comparative example 1 using methanol to replace the DES solvent belongs to the traditional method for extracting amide substances in pepper/rattan pepper, the extraction rate is 20%, and the DES solvent plays a crucial role in extracting the target compound. By observing the extraction rate of the comparative example 2, wherein urea is used as a hydrogen bond donor, the extraction rate is only 15%, so that the extraction efficiency of the acid hydrogen bond donor is higher, and the method possibly results from the combination of the amide compound and the acid hydrogen bond donor, so that the mass transfer process of the amide compound to the solution is caused, and the sufficient extraction of the amide compound is promoted.
In conclusion, the DES solvent prepared by mixing choline chloride and acids is combined with ultrasonic-assisted extraction for extraction, so that the extraction effect is remarkably improved under the synergistic effect. And then combining with the method of separating by dynamic axial compression column to simultaneously separate hydroxyl-alpha-sanshool, hydroxyl-beta-sanshool, hydroxyl-gamma-sanshool and hydroxyl-epsilon-sanshool from the zanthoxylum schinifolium oil, a process route for extracting, purifying and separating 4 monomer compounds from the zanthoxylum schinifolium is established, the process is simple, a large number of purification steps are not needed, the solvent is convenient to recover, the purity is high, the method is suitable for large-scale industrial production, and the method has very high use value.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for rapidly preparing high-purity hydroxy sanshool compounds in an industrial grade is characterized by comprising the following steps:
s1, by supercritical CO2Extracting the zanthoxylum oil as a raw material, adding a eutectic solvent for ultrasonic auxiliary extraction, centrifuging after extraction is finished, and collecting a lower DES layer to obtain a DES extracting solution of sanshool compounds; recovering hydroxy sanshool components from the obtained extracting solution by an organic solvent reverse extraction method to obtain a hydroxy sanshool component enrichment product;
s2, dynamic axial preparative chromatography separation and purification: completely dissolving the enriched product of the hydroxy sanshool component by using a mobile phase with the mass volume of 1-3 times, preparing liquid phase separation by adopting dynamic axial compression, wherein the mobile phase is methanol and water, the detection wavelength is 254nm, collecting target components according to different retention times, carrying out reduced pressure rotary evaporation and concentration on eluent, and carrying out freeze drying to obtain each monomer compound.
2. The method as claimed in claim 1, wherein the eutectic solvent is prepared by mixing a hydrogen bond donor and a hydrogen bond acceptor according to a certain molar ratio, sealing, heating at 60-80 ℃ for co-dissolution until clarification to form a DES solvent, and standing at normal temperature for later use.
3. The method of claim 2, wherein the hydrogen bond donor is choline chloride; the hydrogen bond acceptor is any one of formic acid, acetic acid and lactic acid.
4. The method of claim 2, wherein the molar ratio of hydrogen bond donor to hydrogen bond acceptor is 1:1 to 2.
5. The method of claim 1, wherein in S1, the centrifugation is performed at 4000r/mi for 10 min.
6. The method of claim 1, wherein in S1, the ultrasonic-assisted treatment conditions are that the material-to-liquid ratio is 1: 5-1: 10(w/v), the extraction power is 150-200 w, and the extraction time is 0.5 h.
7. The method according to claim 1, wherein in S1, the organic solvent used for the back extraction is ethyl acetate, and the feed-liquid ratio is 1: 5-1: 10.
8. The method according to claim 1, wherein in S2, the packing of the dynamic axial preparative liquid chromatography dynamic axial compression column is octadecyl reverse silica gel, and the silica gel has a particle size of 10 to 20 μm; the dynamic axial compression column has a specification of 50mm x 250 mm.
9. The method of claim 1, wherein in S2, the mobile phase is methanol: water 65: 35-85: 15 (v/v).
10. The method of claim 1, wherein in S2, the flow rate of the elution is 10-40 mL/min; the concentration of the sample injection liquid is 50-150 mg/mL.
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| CN111909050A (en) * | 2020-07-15 | 2020-11-10 | 中山大学 | Enrichment and separation method of pepper polyene amide monomers and fragrance components |
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| CN111909050A (en) * | 2020-07-15 | 2020-11-10 | 中山大学 | Enrichment and separation method of pepper polyene amide monomers and fragrance components |
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| 韦庆益，等: "《食品生物化学实验》", vol. 2, 华南理工大学出版社, pages: 109 - 114 * |
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