JP5933338B2 - Method and composition for producing cyclic sodium phosphatidate - Google Patents
Method and composition for producing cyclic sodium phosphatidate Download PDFInfo
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- JP5933338B2 JP5933338B2 JP2012117412A JP2012117412A JP5933338B2 JP 5933338 B2 JP5933338 B2 JP 5933338B2 JP 2012117412 A JP2012117412 A JP 2012117412A JP 2012117412 A JP2012117412 A JP 2012117412A JP 5933338 B2 JP5933338 B2 JP 5933338B2
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- Prior art keywords
- sodium
- cyclic
- phosphatidate
- phospholipase
- phospholipid
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- -1 cyclic sodium phosphatidate Chemical class 0.000 title claims description 62
- 239000011734 sodium Substances 0.000 title claims description 59
- 229910052708 sodium Inorganic materials 0.000 title claims description 56
- 239000000203 mixture Substances 0.000 title claims description 13
- 238000000034 method Methods 0.000 title description 16
- 150000003904 phospholipids Chemical class 0.000 claims description 41
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 238000004519 manufacturing process Methods 0.000 claims description 27
- 102000011420 Phospholipase D Human genes 0.000 claims description 24
- 108090000553 Phospholipase D Proteins 0.000 claims description 24
- 125000004122 cyclic group Chemical group 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 17
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 claims description 16
- 239000003960 organic solvent Substances 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 239000007795 chemical reaction product Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 159000000000 sodium salts Chemical class 0.000 claims description 11
- 244000068988 Glycine max Species 0.000 claims description 9
- 235000010469 Glycine max Nutrition 0.000 claims description 9
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 9
- 102100037611 Lysophospholipase Human genes 0.000 claims description 9
- 108010058864 Phospholipases A2 Proteins 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical group OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 239000002738 chelating agent Substances 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 7
- 241000187362 Actinomadura Species 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 32
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 30
- 238000006243 chemical reaction Methods 0.000 description 18
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 18
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
- 238000003756 stirring Methods 0.000 description 11
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 9
- 239000008351 acetate buffer Substances 0.000 description 8
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 235000014113 dietary fatty acids Nutrition 0.000 description 6
- 239000000194 fatty acid Substances 0.000 description 6
- 229930195729 fatty acid Natural products 0.000 description 6
- 150000004665 fatty acids Chemical class 0.000 description 6
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 5
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 5
- 102000002322 Egg Proteins Human genes 0.000 description 5
- 108010000912 Egg Proteins Proteins 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 235000013345 egg yolk Nutrition 0.000 description 5
- 210000002969 egg yolk Anatomy 0.000 description 5
- 239000000787 lecithin Substances 0.000 description 5
- 229940067606 lecithin Drugs 0.000 description 5
- 235000010445 lecithin Nutrition 0.000 description 5
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000008347 soybean phospholipid Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 241000187747 Streptomyces Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- KHJWSKNOMFJTDN-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium Chemical compound [Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KHJWSKNOMFJTDN-UHFFFAOYSA-N 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 159000000007 calcium salts Chemical class 0.000 description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 235000009973 maize Nutrition 0.000 description 2
- 229960003330 pentetic acid Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 241000187361 Actinomadura sp. Species 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 241000187180 Streptomyces sp. Species 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000467 phytic acid Substances 0.000 description 1
- 229940068041 phytic acid Drugs 0.000 description 1
- 235000002949 phytic acid Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
- C07F9/106—Adducts, complexes, salts of phosphatides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Cosmetics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
本発明は、環状ホスファチジン酸ナトリウムの製造方法、並びに上記製造方法により得られる環状ホスファチジン酸ナトリウムを含有する組成物に関する。 The present invention relates to a method for producing cyclic sodium phosphatidate and a composition containing cyclic sodium phosphatidate obtained by the above production method.
環状ホスファチジン酸（以下、ｃPAと略すこともある）は、がん細胞の転移および浸潤を阻害する等の生理活性を有することが知られ（非特許文献１）、抗腫瘍剤を含む医薬品や機能性食品としての用途が期待されており、また、生体内のヒアルロン酸合成促進作用を有することから、化粧品に添加されている。 Cyclic phosphatidic acid (hereinafter sometimes abbreviated as cPA) is known to have physiological activities such as inhibiting metastasis and invasion of cancer cells (Non-patent Document 1), and includes pharmaceuticals and functions including antitumor agents. It is expected to be used as a functional food, and has an action of promoting hyaluronic acid synthesis in vivo, so it is added to cosmetics.
従来、このような環状ホスファチジン酸の製造方法としては、化学的に合成する方法（特許文献１及び２）や、リゾ型リン脂質にホスフォリパーゼDを作用させることによる酵素反応を利用した方法（特許文献３及び４）が知られている。環状ホスファチジン酸は脂質であり水に不溶であるため、ナトリウム塩などの水溶性の塩とすることが必要であり、化学的に合成した環状ホスファチジン酸を、水素化ナトリウムや水酸化ナトリウムなどの強塩基で処理し、ナトリウム塩に変換する方法で環状ホスファチジン酸ナトリウムが調製されている。 Conventionally, as a method for producing such cyclic phosphatidic acid, a method of chemically synthesizing (Patent Documents 1 and 2) or a method using an enzyme reaction by causing phospholipase D to act on lyso-type phospholipid ( Patent Documents 3 and 4) are known. Since cyclic phosphatidic acid is a lipid and insoluble in water, it is necessary to use a water-soluble salt such as a sodium salt, and chemically synthesized cyclic phosphatidic acid is converted into a strong compound such as sodium hydride or sodium hydroxide. Cyclic sodium phosphatidate has been prepared by treatment with a base and conversion to the sodium salt.
環状ホスファチジン酸は不安定であることから、上述の科学的合成法に記載されたような強塩基を用いる方法は好ましくなく、また、化学的合成法では環状ホスファチジン酸を高収率および高純度で得ることは困難であり、温和で簡便な方法で環状ホスファチジン酸ナトリウムを高収率かつ高純度で調製する方法が求められていた。 Since cyclic phosphatidic acid is unstable, a method using a strong base as described in the above-mentioned scientific synthesis method is not preferable, and in the chemical synthesis method, cyclic phosphatidic acid is obtained in high yield and high purity. It was difficult to obtain, and a method for preparing cyclic sodium phosphatidate in high yield and high purity by a mild and simple method has been demanded.
本発明者らは、環状ホスファチジン酸ナトリウムの調製方法について鋭意検討した結果、有機溶媒及び／又は水を含む系においてリゾ型リン脂質（但し、水素添加物を除く）にホスホリパーゼDを作用させて得られる反応物に、塩化ナトリウムを添加した後、反応物から溶媒を除去することにより、極めて穏和な条件下で簡便な方法で、高収率かつ高純度で環状ホスファチジン酸ナトリウムを調製することができることを見出した。本発明は係る知見に基づいてなされたものである。 As a result of intensive studies on a method for preparing cyclic sodium phosphatidate, the present inventors obtained phospholipase D on lyso-type phospholipids (excluding hydrogenated products) in a system containing an organic solvent and / or water. After adding sodium chloride to the reaction product, the solvent can be removed from the reaction product to prepare cyclic sodium phosphatidate in a high yield and high purity by a simple method under extremely mild conditions. I found. The present invention has been made based on such knowledge.
すなわち、本発明によれば以下の発明が提供される。
（１） 有機溶媒及び／又は水を含む系においてリゾ型リン脂質（但し、水素添加物を除く）にホスホリパーゼDを作用させて得られる反応物に、ナトリウム塩を添加した後、反応物から溶媒を除去することを含む環状ホスファチジン酸ナトリウムの製造方法。
（２） ナトリウム塩が塩化ナトリウムである、（１）に記載の環状ホスファチジン酸ナトリウムの製造方法。
（３） 有機溶媒及び／又は水を含む系においてリゾ型リン脂質にホスホリパーゼDを作用させて得られる反応物に、キレート剤の存在下においてナトリウム塩を添加する、（１）又は（２）に記載の環状ホスファチジン酸ナトリウムの製造方法。
That is, according to the present invention, the following inventions are provided.
(1) After adding a sodium salt to a reaction product obtained by allowing phospholipase D to act on a lyso-type phospholipid (excluding a hydrogenated product) in a system containing an organic solvent and / or water, the solvent is removed from the reaction product. A process for producing cyclic sodium phosphatidate, comprising removing
(2) The method for producing cyclic sodium phosphatidate according to (1), wherein the sodium salt is sodium chloride.
(3) To a reaction product obtained by allowing phospholipase D to act on lyso-type phospholipid in a system containing an organic solvent and / or water, a sodium salt is added in the presence of a chelating agent, (1) or (2) The manufacturing method of cyclic phosphatidic acid sodium of description.
（４） キレート剤がＥＤＴＡである、（３）に記載の環状ホスファチジン酸ナトリウムの製造方法。
（５） リゾ型リン脂質が大豆由来リン脂質あるいは卵黄由来リン脂質あるいはトウモロコシ由来リン脂質にホスホリパーゼA2を作用させて得られたリゾ型リン脂質である、（１）から（４）の何れか１項に記載の環状ホスファチジン酸ナトリウムの製造方法。
（６） リゾ型リン脂質が大豆由来リン脂質あるいは卵黄由来リン脂質あるいはトウモロコシ由来リン脂質にホスホリパーゼA2を作用させて得られた反応物から、リゾ型リン脂質を単離精製することなく有機溶媒及び／又は水を含む系に溶解し、ホスフォリパーゼDを作用させる、（１）から（５）の何れか１項に記載の環状ホスファチジン酸ナトリウムの製造方法。
(4) The method for producing cyclic sodium phosphatidate according to (3), wherein the chelating agent is EDTA.
(5) The lyso-type phospholipid is a lyso-type phospholipid obtained by allowing phospholipase A2 to act on soybean-derived phospholipid, egg yolk-derived phospholipid, or maize-derived phospholipid, and any one of (1) to (4) The manufacturing method of cyclic | annular sodium phosphatidic acid of description to term.
(6) An organic solvent and a lyso-type phospholipid can be obtained from a reaction product obtained by allowing phospholipase A2 to act on soybean-derived phospholipid, egg yolk-derived phospholipid, or maize-derived phospholipid without isolating and purifying lyso-type phospholipid. The method for producing cyclic sodium phosphatidate according to any one of (1) to (5), wherein the phospholipase D is dissolved in a system containing water.
（７） （１）から（６）の何れか１項に記載の環状ホスファチジン酸ナトリウムの製造方法により得られる、４０％以上の純度を有する環状ホスファチジン酸ナトリウム含有組成物。
（８） （１）から（６）の何れか１項に記載の環状ホスファチジン酸ナトリウムの製造方法により得られる、1mg/mL以上の環状ホスファチジン酸ナトリウムを含有する溶液。
（９） 環状ホスファチジン酸ナトリウムを含有する溶液が、水溶液である、（８）に記載の溶液。
(7) A cyclic sodium phosphatidate-containing composition having a purity of 40% or more obtained by the method for producing cyclic sodium phosphatidate according to any one of (1) to (6).
(8) A solution containing 1 mg / mL or more of cyclic sodium phosphatidate obtained by the method for producing cyclic sodium phosphatidate according to any one of (1) to (6).
(9) The solution according to (8), wherein the solution containing cyclic sodium phosphatidate is an aqueous solution.
本発明によれば、有機溶媒及び／又は水を含む系においてリゾ型リン脂質にホスホリパーゼDを作用させることで、極めて穏和で簡便な方法で、高収率かつ高純度で環状ホスファチジン酸ナトリウムを製造できる。本発明によれば、反応物をナトリウム塩で処理するだけで、環状ホスファチジン酸を単離、精製することなく、環状ホスファチジン酸ナトリウムを調製することができるため、大豆などの天然物を原料とした環状ホスファチジン酸ナトリウムの製造法として特に有用である。本発明の製造条件によって得られる環状ホスファチジン酸ナトリウムは、特に大豆由来のリン脂質あるいは卵黄由来リン脂質あるいはトウモロコシ由来リン脂質を原料とした場合、室温での水溶性が高く、特に精製することなく水溶液を調製することができる。 According to the present invention, phospholipase D is allowed to act on lyso-type phospholipids in a system containing an organic solvent and / or water, thereby producing cyclic sodium phosphatidate in a very mild and simple method with high yield and high purity. it can. According to the present invention, it is possible to prepare cyclic sodium phosphatidic acid by treating the reaction product with a sodium salt without isolating and purifying cyclic phosphatidic acid, so that natural products such as soybeans are used as raw materials. It is particularly useful as a method for producing cyclic sodium phosphatidate. Cyclic sodium phosphatidic acid obtained by the production conditions of the present invention is highly water-soluble at room temperature, especially when soybean-derived phospholipids, egg yolk-derived phospholipids or corn-derived phospholipids are used as raw materials. Can be prepared.
以下、本発明についてさらに詳細に説明する。
本発明は、有機溶媒及び／又は水を含む系においてリゾ型リン脂質（但し、水素添加物を除く）にホスホリパーゼDを作用させて得られる反応物に、塩化ナトリウムを添加した後、反応物から溶媒を除去することを特徴とする。
Hereinafter, the present invention will be described in more detail.
In the present invention, sodium chloride is added to a reaction product obtained by allowing phospholipase D to act on a lyso-type phospholipid (excluding a hydrogenated product) in a system containing an organic solvent and / or water. It is characterized by removing the solvent.
環状ホスファチジン酸ナトリウムの調製に関しては、水素添加大豆由来リゾホスファチジルコリンを水に溶解し、酢酸ナトリウム緩衝液および２Mの塩化ナトリウムを添加、ホスホリパーゼDと反応させた後、水酸化ナトリウム水溶液で中和、クロロホルム/メタノールで抽出し、有機溶媒層を回収して溶媒を溜去し、環状ホスファチジン酸ナトリウムを得たとの記載がある（特許文献４）が、得られた物質の脂肪酸組成が記載されているのみであり、ナトリウム含有量などの記載はなく、環状ホスファチジン酸ナトリウムであることが明確に示されていない。 For preparation of cyclic sodium phosphatidate, hydrogenated soybean-derived lysophosphatidylcholine was dissolved in water, sodium acetate buffer and 2M sodium chloride were added, reacted with phospholipase D, neutralized with aqueous sodium hydroxide, chloroform There is a description that the organic solvent layer was recovered by extracting with methanol / methanol, and the solvent was distilled off to obtain cyclic sodium phosphatidate (Patent Document 4), but only the fatty acid composition of the obtained substance is described. There is no description such as sodium content, and it is not clearly shown to be cyclic sodium phosphatidate.
本発明者らはホスホリパーゼDの酵素反応はカルシウムイオンが存在しなくても十分に進行し、塩化ナトリウムは酵素反応を阻害することを見出した。更にリゾ型リン脂質を有機溶媒及び／又は水を含む系に溶解し、酢酸緩衝液を添加して、ｐHを5.5〜6.5に調整し、ホスホリパーゼDの酵素反応を行なうことにより、反応を効率よく進行させるとともに、反応終了後に、ナトリウム塩の水溶液を添加することで、環状ホスファチジン酸ナトリウムを収率良く製造することに成功した。また、また、大豆由来のリン脂質あるいは卵黄由来リン脂質あるいはトウモロコシ由来リン脂質を原料としホスホリパーゼA2を作用させてえられるリゾ型リン脂質を単離精製することなく、連続してホスホリパーゼDの反応を行なう場合は、反応液中に少量のカルシウムイオンが残存し、環状ホスファチジン酸のカルシウム塩が精製するが、反応終了後にナトリウムイオンと共にキレート剤を併用することで高純度の環状ホスファチジン酸ナトリウムを得ることができる。 The present inventors have found that the enzyme reaction of phospholipase D proceeds sufficiently even in the absence of calcium ions, and sodium chloride inhibits the enzyme reaction. Furthermore, lyso-type phospholipids are dissolved in a system containing an organic solvent and / or water, an acetate buffer solution is added, pH is adjusted to 5.5 to 6.5, and the reaction of phospholipase D is performed efficiently. As the reaction proceeded, an aqueous sodium salt solution was added after completion of the reaction to successfully produce cyclic sodium phosphatidate in a high yield. In addition, phospholipase D can be reacted continuously without isolating and purifying lyso-type phospholipids obtained by using phospholipase A2 from phospholipids derived from soybean, egg yolk or corn. When performing, a small amount of calcium ion remains in the reaction solution, and the calcium salt of cyclic phosphatidic acid is purified. After the reaction is completed, a high-purity sodium phosphatidate can be obtained by using a chelating agent together with sodium ion. Can do.
本発明において用いられるナトリウム塩としては、塩化ナトリウム、硫酸ナトリウム、酢酸ナトリウム、硝酸ナトリウム等を使用できるが、好ましくは塩化ナトリウム、硫酸ナトリウム、より好ましくは塩化ナトリウムである。好ましく使用できる塩化ナトリウム濃度は0.5M〜3Mであるがより好ましくは2〜3Mである。 As the sodium salt used in the present invention, sodium chloride, sodium sulfate, sodium acetate, sodium nitrate and the like can be used, preferably sodium chloride, sodium sulfate, and more preferably sodium chloride. The sodium chloride concentration that can be preferably used is 0.5M to 3M, more preferably 2 to 3M.
本発明において用いられるキレート剤としては、エチレンジアミン四酢酸ナトリウム（EDTA）、ジエチレントリアミン五酢酸、グリコールエーテルジアミン四酢酸、クエン酸、酒石酸、フィチン酸等であるが、好ましくはエチレンジアミン四酢酸ナトリウム（EDTA）、ジエチレントリアミン五酢酸、グリコールエーテルジアミン四酢酸、最も好ましくはEDTAである。 Examples of the chelating agent used in the present invention include ethylenediaminetetraacetic acid sodium (EDTA), diethylenetriaminepentaacetic acid, glycol etherdiaminetetraacetic acid, citric acid, tartaric acid, phytic acid and the like, preferably ethylenediaminetetraacetic acid sodium (EDTA), Diethylenetriaminepentaacetic acid, glycol etherdiaminetetraacetic acid, most preferably EDTA.
本発明において用いられる有機溶媒としては、クロロホルム、メチレンクロライド、トルエン、エチルエーテル、酢酸エチル、ヘキサン等を使用できるが、好ましくは、クロロホルム、メチレンクロライド、トルエン、より好ましくはクロロホルム、トルエンである。 As the organic solvent used in the present invention, chloroform, methylene chloride, toluene, ethyl ether, ethyl acetate, hexane and the like can be used, preferably chloroform, methylene chloride and toluene, more preferably chloroform and toluene.
本発明において用いられるリゾ型リン脂質は、ホスホリパーゼDの基質特異性を考慮すると、リゾホスファチジルコリン（LPC）であることが好ましく、大豆由来のリン脂質あるいは卵黄由来リン脂質あるいはトウモロコシ由来リン脂質を原料とし、ホスホリパーゼA2をさせて得られるLPCが最も好ましい。本発明で用いるリゾ型リン脂質としては、水素添加リゾ型リン脂質は除外され、水素未添加のリゾ型リン脂質が使用される。 In consideration of the substrate specificity of phospholipase D, the lyso-type phospholipid used in the present invention is preferably lysophosphatidylcholine (LPC), which uses soybean-derived phospholipid, egg yolk-derived phospholipid, or corn-derived phospholipid as a raw material. LPC obtained by adding phospholipase A2 is most preferable. As the lyso-type phospholipid used in the present invention, hydrogenated lyso-type phospholipid is excluded, and lyso-type phospholipid not added with hydrogen is used.
本発明において用いられるホスホリパーゼDは、上記リゾ型リン脂質に作用させた場合に、ｃPAを生成するものであれば特に限定されるものではないが、ストレプトマイセス エスピー（Streptomyces sp.）またはアクチノマヂュラ エスピー（Actinomadula sp.）に由来するホスホリパーゼDが特に好ましく用いられる。 The phospholipase D used in the present invention is not particularly limited as long as it produces cPA when allowed to act on the lyso-type phospholipid. However, Streptomyces sp. Or Actinomadura sp. Phospholipase D derived from (Actinomadula sp.) Is particularly preferably used.
リゾ型リン脂質とホスホリパーゼDとの反応は、酵素が活性を発現できる条件であれば、特に限定されないが、好ましくはリゾ型リン脂質をクロロホルムなどの有機溶媒に溶解し、酢酸緩衝液を添加してｐHを５．０から７．０に調整し、１０〜１００単位/mlのホスホリパーゼDを添加して、２５℃〜５０℃に加温し連続的に攪拌しながら５〜３０時間程度反応させることにより行なう。反応終了後2〜３Mの塩化ナトリウム、０．１から０．３MのEDTAを添加して連続的に攪拌した。反応物を遠心分離（３０００回転、５分間）にかけ有機溶媒層を回収し溶媒を溜去すれば環状ホスファチジン酸ナトリウムを粉末固形物として得ることができる。反応終了後必要に応じてメタノールなどの有機溶媒を添加して有機溶媒層を回収しても良い。更に高純度の環状ホスファチジン酸ナトリウムを得る場合には、シリカゲルや吸着樹脂などを用いて精製すれば良い。 The reaction between lyso-type phospholipid and phospholipase D is not particularly limited as long as the enzyme can exhibit its activity. Preferably, however, lyso-type phospholipid is dissolved in an organic solvent such as chloroform and an acetate buffer is added. Adjust the pH from 5.0 to 7.0, add 10-100 units / ml phospholipase D, warm to 25 ° C to 50 ° C, and react for about 5 to 30 hours with continuous stirring. By doing. After completion of the reaction, 2-3M sodium chloride and 0.1 to 0.3M EDTA were added and stirred continuously. If the reaction product is centrifuged (3000 rpm, 5 minutes), the organic solvent layer is recovered and the solvent is distilled off, cyclic sodium phosphatidate can be obtained as a powder solid. After completion of the reaction, an organic solvent such as methanol may be added as needed to recover the organic solvent layer. Further, when obtaining high-purity sodium phosphatidate, it may be purified using silica gel, an adsorption resin, or the like.
本発明によれば、上記した本発明による環状ホスファチジン酸ナトリウムの製造方法により得られる、４０％以上の純度を有する環状ホスファチジン酸ナトリウム含有組成物が提供される。純度は、好ましくは４５％以上、より好ましくは５０％以上である。本発明で言う純度とは、標準品(９７％純度品）をコントロールとして、薄層クロマトグラフのスポットをデンシトメーターにより測定し、面積比で定量した結果として得られる純度である。標準品(９７％純度品）は、実施例３で得た高純度環状ホスファチジン酸ナトリウムを再クロマトにより精製したものであり、実施例５に記載の脂肪酸組成を有するものである。 According to the present invention, there is provided a cyclic sodium phosphatidate-containing composition having a purity of 40% or more obtained by the above-described method for producing cyclic sodium phosphatidate according to the present invention. The purity is preferably 45% or more, more preferably 50% or more. The purity referred to in the present invention is a purity obtained as a result of measuring a spot of a thin-layer chromatograph with a densitometer using a standard product (97% purity product) as a control and quantifying it with an area ratio. The standard product (97% purity product) is a product obtained by purifying the high-purity cyclic sodium phosphatidate obtained in Example 3 by rechromatography, and has the fatty acid composition described in Example 5.
更に本発明によれば、上記した本発明による環状ホスファチジン酸ナトリウムの製造方法により得られる、1mg/mL以上の環状ホスファチジン酸ナトリウムを含有する溶液が提供される。環状ホスファチジン酸ナトリウムの濃度は1mg/mL以上であれば特に限定されず、2mg/mL以上、3mg/mL以上、又は5mg/mL以上でもよい。 Furthermore, according to this invention, the solution containing 1 mg / mL or more of cyclic sodium phosphatidate obtained by the manufacturing method of the cyclic sodium phosphatidate according to this invention mentioned above is provided. The concentration of cyclic sodium phosphatidate is not particularly limited as long as it is 1 mg / mL or more, and may be 2 mg / mL or more, 3 mg / mL or more, or 5 mg / mL or more.
以下の実施例で本発明を説明するが、本発明は、これらの実施例によって何ら限定されるものではない。 The present invention will be described with reference to the following examples, but the present invention is not limited to these examples.
実施例１；環状ホスファチジン酸ナトリウム塩の製造方法
大豆リン脂質（レシチン含量：７０％）（水素未添加物）１０ｇを０．３Ｍ塩化カルシウムを含有する１００ｍＬの１Ｍ酢酸バッファー（ｐＨ６．５）で溶解させた後、６０００単位のストレプトマイセス属由来のホスホリパーゼA2を添加し、４０℃で１８時間攪拌して反応させた。反応液をpＨ２．５に調整して酵素を失活させた後、１００ｍＬのクロロホルム、５０ｍＬのメタノールを添加して十分攪拌混合し脂質成分を抽出した。クロロホルム層を集め、ロータリーエバポレータで減圧乾固させた。固形分に１００ｍＬのアセトンを加えリン脂質を沈殿させ遊離脂肪酸を除去した。沈殿物５gを４０ｍＬのクロロホルムに溶解させ、１Ｍ酢酸バッファー（pＨ５．５）１０ｍＬを加え、更に１５００単位のアクチノマジュラ属由来のホスホリパーゼDを添加して４０℃で１８時間攪拌しながら反応を行った。反応液に２０ｍＬの３Ｍ塩化ナトリウム、２０ｍＬの０．１Ｍ EDTA溶液を添加して４０℃で１時間攪拌を行った。更に２０ｍＬのメタノールを添加して十分攪拌した後、３０００回転、５分間遠心分離してクロロホルム層を集めた。この溶液をロータリーエバポレータで減圧乾固させ環状ホスファチジン酸ナトリウム塩３．８ｇを得た。収率はレシチン含量７０％（１０g中７ｇ）から環状ホスファチジン酸Naを３．８ｇを得たので５４．３％であった。環状ホスファチジン酸ナトリウム塩の純度分析は、シリカゲルプレートを用い、クロロホルム：メタノール：酢酸：５％二亜硫酸ナトリウム（１００：４０：１２：５、V/V）で展開後、５％酢酸銅：８％燐酸：２％硫酸混合液に短時間浸漬し風乾後、１８０℃で約１０分加熱した後、生成したスポットをスキャナー（アトー社製）法によって行った。即ち、標準品(９７％純度品）をコントロールとして、薄層クロマトグラフのスポットをデンシトメーターにより測定し、面積比で定量した。上記工程で得られた生成物中環状ホスファチジン酸ナトリウム塩の純度は５４％であった。
Example 1: Method for producing cyclic phosphatidic acid sodium salt 10 g of soybean phospholipid (lecithin content: 70%) (hydrogen-free product) was dissolved in 100 mL of 1 M acetate buffer (pH 6.5) containing 0.3 M calcium chloride. Then, 6000 units of phospholipase A2 derived from Streptomyces was added, and the mixture was reacted by stirring at 40 ° C. for 18 hours. The reaction solution was adjusted to pH 2.5 to inactivate the enzyme, and then 100 mL of chloroform and 50 mL of methanol were added and mixed with sufficient stirring to extract lipid components. The chloroform layer was collected and dried under reduced pressure on a rotary evaporator. 100 mL of acetone was added to the solid content to precipitate phospholipids and remove free fatty acids. Dissolve 5 g of the precipitate in 40 mL of chloroform, add 10 mL of 1M acetate buffer (pH 5.5), add 1500 units of phospholipase D derived from the genus Actinomadura, and perform the reaction while stirring at 40 ° C. for 18 hours. It was. 20 mL of 3 M sodium chloride and 20 mL of 0.1 M EDTA solution were added to the reaction solution, and the mixture was stirred at 40 ° C. for 1 hour. Further, 20 mL of methanol was added and sufficiently stirred, and then centrifuged at 3000 rpm for 5 minutes to collect a chloroform layer. This solution was dried under reduced pressure using a rotary evaporator to obtain 3.8 g of cyclic phosphatidic acid sodium salt. The yield was 54.3% because 3.8 g of cyclic phosphatidic acid Na was obtained from a lecithin content of 70% (7 g in 10 g). Purity analysis of cyclic phosphatidic acid sodium salt was carried out using silica gel plates with chloroform: methanol: acetic acid: 5% sodium disulfite (100: 40: 12: 5, V / V), 5% copper acetate: 8% After being dipped in a phosphoric acid: 2% sulfuric acid mixed solution for a short time, air-dried, and heated at 180 ° C. for about 10 minutes, the generated spots were formed by a scanner (manufactured by Atto Corporation) method. That is, using a standard product (97% purity product) as a control, the spot of the thin layer chromatograph was measured with a densitometer and quantified by the area ratio. The purity of cyclic phosphatidic acid sodium salt in the product obtained in the above step was 54%.
実施例２；キレート剤を用いない環状ホスファチジン酸ナトリウム塩の製造方法
大豆リン脂質（レシチン含量：７０％）（水素未添加物）１０ｇを０．３Ｍ塩化カルシウムを含有する１００ｍＬの１Ｍ酢酸バッファー（ｐＨ６．５）で溶解させた後、６０００単位のストレプトマイセス属由来のホスホリパーゼA2を添加し、４０℃で１８時間攪拌して反応させた。反応液をpＨ２．５に調整して酵素を失活させた後、１００ｍＬのクロロホルム、５０ｍＬのメタノールを添加して十分攪拌混合し脂質成分を抽出した。クロロホルム層を集め、ロータリーエバポレータで減圧乾固させた。固形分に１００ｍＬのアセトンを加えリン脂質を沈殿させ遊離脂肪酸を除去した。沈殿物５gを４０ｍＬのクロロホルムに溶解させ、１Ｍ酢酸バッファー（pＨ５．５）１０ｍＬを加え、更に１５００単位のアクチノマジュラ属由来のホスホリパーゼDを添加して４０℃で１８時間攪拌しながら反応を行った。反応液に２０ｍＬの３Ｍ塩化ナトリウムを添加して４０℃で１時間攪拌を行った。更に２０ｍＬのメタノールを添加して十分攪拌した後、３０００回転、５分間遠心分離してクロロホルム層を集めた。この溶液をロータリーエバポレータで減圧乾固させ環状ホスファチジン酸ナトリウム塩３．７ｇを得た。収率はレシチン含量７０％（１０g中７ｇ）から環状ホスファチジン酸Naを３．７ｇを得たので５２．９％であった。環状ホスファチジン酸ナトリウム塩の純度分析は、実施例１記載の方法で行なった。本工程で得られた生成物中環状ホスファチジン酸ナトリウム塩の純度は５３％であった。
Example 2: Method for producing cyclic phosphatidic acid sodium salt without using a chelating agent 10 g of soybean phospholipid (lecithin content: 70%) (hydrogen-free product) in 100 mL of 1 M acetate buffer (pH 6) containing 0.3 M calcium chloride 5), 6000 units of phospholipase A2 derived from the genus Streptomyces was added, and the mixture was reacted at 40 ° C. for 18 hours with stirring. The reaction solution was adjusted to pH 2.5 to inactivate the enzyme, and then 100 mL of chloroform and 50 mL of methanol were added and mixed with sufficient stirring to extract lipid components. The chloroform layer was collected and dried under reduced pressure on a rotary evaporator. 100 mL of acetone was added to the solid content to precipitate phospholipids and remove free fatty acids. Dissolve 5 g of the precipitate in 40 mL of chloroform, add 10 mL of 1M acetate buffer (pH 5.5), add 1500 units of phospholipase D derived from the genus Actinomadura, and perform the reaction while stirring at 40 ° C. for 18 hours. It was. 20 mL of 3M sodium chloride was added to the reaction solution and stirred at 40 ° C. for 1 hour. Further, 20 mL of methanol was added and sufficiently stirred, and then centrifuged at 3000 rpm for 5 minutes to collect a chloroform layer. This solution was dried under reduced pressure using a rotary evaporator to obtain 3.7 g of cyclic phosphatidic acid sodium salt. The yield was 52.9% because 3.7 g of cyclic phosphatidic acid Na was obtained from a lecithin content of 70% (7 g in 10 g). The purity analysis of cyclic phosphatidic acid sodium salt was carried out by the method described in Example 1. The purity of cyclic phosphatidic acid sodium salt in the product obtained in this step was 53%.
実施例３；高純度環状ホスファチジン酸ナトリウム塩の製造方法
実施例１で得られた環状ホスファチジン酸ナトリウム塩５００ｍｇを５ｍＬの１０％メタノールを含むクロロホルムに溶解させ、シリカゲルカラムにかけ、同一溶媒で展開、更に２０％メタノールを含むクロロホルムで展開し、１０ｍＬの画分に分取した。実施例１に示したTLC法によって環状ホスファチジン酸ナトリウム塩を含有する画分を確認して集め、ロータリーエバポレータで減圧乾固させ環状ホスファチジン酸ナトリウム塩の粉末３２０ｍｇを得た。本試料の環状ホスファチジン酸ナトリウム塩の純度は９５％であった。
Example 3: Method for producing high purity cyclic phosphatidic acid sodium salt 500 mg of cyclic phosphatidic acid sodium salt obtained in Example 1 was dissolved in 5 mL of chloroform containing 10% methanol, applied to a silica gel column, developed with the same solvent, and further developed. The mixture was developed with chloroform containing 20% methanol, and fractionated into 10 mL fractions. Fractions containing cyclic phosphatidic acid sodium salt were confirmed and collected by the TLC method shown in Example 1, and dried under reduced pressure using a rotary evaporator to obtain 320 mg of cyclic phosphatidic acid sodium salt powder. The purity of the cyclic phosphatidic acid sodium salt of this sample was 95%.
実施例４；イオンクロマト法によるナトリウム含量の分析
実施例１及び実施例２で得た環状ホスファチジン酸ナトリウム塩について、イオンクロマト法によってナトリウム含量を測定した。カラムはIonPac CS14（日本ダイオネクス社製）、移動相は10ｍＭメタンスルホン酸水溶液を用い、流速１．０ｍＬ/分、カラム温度は３０℃、注入量は１０μＬとし、検出は電気伝導度検出器で行なった。標準溶液は陽イオン混合標準液II（Li+ 0.5mg/L, Na+ 2mg/L, NH4 + 2mg/L, K+ 5mg/L, Ca2+ 5mg/L, Mg2+ 5mg/L）を用いた。カラム温度は３０℃、注入量は１０μＬとした。分析の結果、環状ホスファチジン酸と等モルのナトリウムを検出したことから１ナトリウム塩であることがわかった。
Example 4: Analysis of sodium content by ion chromatography The sodium content of the cyclic phosphatidic acid sodium salt obtained in Examples 1 and 2 was measured by ion chromatography. The column is IonPac CS14 (manufactured by Nippon Dionex Co., Ltd.), the mobile phase is 10 mM methanesulfonic acid aqueous solution, the flow rate is 1.0 mL / min, the column temperature is 30 ° C., the injection volume is 10 μL, and the detection is performed with the conductivity detector. It was. Standard solution is cation mixed standard solution II (Li + 0.5 mg / L, Na + 2 mg / L, NH 4 + 2 mg / L, K + 5 mg / L, Ca 2+ 5 mg / L, Mg 2+ 5 mg / L) Was used. The column temperature was 30 ° C. and the injection volume was 10 μL. As a result of the analysis, equimolar sodium was detected with cyclic phosphatidic acid, and it was found to be a monosodium salt.
実施例５；
実施例３で得られた高純度環状ホスファチジン酸ナトリウムの脂肪酸組成をガスクロマトグラム法により分析した。試料２０ｍｇ／ｍＬになるように塩酸メタノールに溶解し、６５℃で３０分間加温した。室温に戻した後、棟梁の水、次いで棟梁のヘキサンを加えて十分に攪拌混合した。３０００回転、５分間遠心分離を行い、ヘキサン層２μＬをキャピラリーカラムに注入し構成脂肪酸を分析した。分析は以下である。
脂肪酸 含量（％）
パルミチン酸 ２３．９
ステアリン酸 ５.４
オレイン酸 ８．６
リノール酸 ５３．４
リノレン酸 ４．９
その他の脂肪酸 ３．８
Example 5;
The fatty acid composition of the high purity cyclic sodium phosphatidate obtained in Example 3 was analyzed by gas chromatogram method. The sample was dissolved in hydrochloric acid methanol so as to be 20 mg / mL, and heated at 65 ° C. for 30 minutes. After returning to room temperature, the water of the master beam and then hexane of the master beam were added and mixed thoroughly with stirring. Centrifugation was performed at 3000 rpm for 5 minutes, and 2 μL of the hexane layer was injected into the capillary column to analyze the constituent fatty acids. The analysis is as follows.
Fatty acid content (%)
Palmitic acid 23.9
Stearic acid 5.4
Oleic acid 8.6
Linoleic acid 53.4
Linolenic acid 4.9
Other fatty acids 3.8
実施例６；環状ホスファチジン酸ナトリウム水溶液の状態
実施例１及び実施例２で得た環状ホスファチジン酸ナトリウムの１ｍｇ/ｍＬの水溶液を調製し、６６０ｎｍでの吸光度を測定した。実施例１で得た試料の吸光度は０．０５、実施例２で得た試料の吸光度は０．１５であった。実施例１で得た試料では、キレート剤を用いた製造方法で得た試料であることから、カルシウム塩が存在せずに透明になった。
Example 6: State of cyclic sodium phosphatidate aqueous solution A 1 mg / mL aqueous solution of cyclic sodium phosphatidate obtained in Example 1 and Example 2 was prepared, and the absorbance at 660 nm was measured. The absorbance of the sample obtained in Example 1 was 0.05, and the absorbance of the sample obtained in Example 2 was 0.15. In the sample obtained in Example 1, since it was a sample obtained by a production method using a chelating agent, it became transparent without the presence of a calcium salt.
比較例１；環状ホスファチジン酸ナトリウム水溶液の状態
実施例１に記載した大豆リン脂質の代わりに水素添加大豆リン脂質を用い、実施例１の記載と同様の操作により環状ホスファチジン酸ナトリウムを得た。この環状ホスファチジン酸ナトリウムの１ｍｇ／ｍＬの水溶液を調製し、６６０ｎｍでの吸光度を測定した。吸光度は０．３３であった。
Comparative Example 1: State of cyclic sodium phosphatidate aqueous solution Cyclic sodium phosphatidate was obtained by the same procedure as described in Example 1 except that hydrogenated soybean phospholipid was used instead of soybean phospholipid described in Example 1. A 1 mg / mL aqueous solution of this cyclic sodium phosphatidate was prepared, and the absorbance at 660 nm was measured. Absorbance was 0.33.
実施例７：環状ホスファチジン酸ナトリウム塩の製造方法
大豆リン脂質（レシチン含量：７０％）（水素未添加物）１０ｇを０．３Ｍ塩化カルシウムを含有する１００ｍＬの１Ｍ酢酸バッファー（ｐＨ６．５）に溶解させた後、６０００単位のストレプトマイセス属由来のホスホリパーゼA2を添加し、４０℃で１８時間攪拌して反応させた。反応液をpＨ２．５に調整して酵素を失活させた後、ｐＨを５．５に調整した。次いでこの反応液に１５００単位のアクチノマジュラ属由来のホスホリパーゼＤを添加して４０℃で１８時間攪拌しながら反応を行った。この反応液にヘキサン１００ｍＬを添加し室温で３０分間攪拌抽出を行った。遠心分離によりヘキサン層を集め、これに１０ｍＭのＥＤＴＡ、０．１Ｍ酢酸緩衝液ｐＨ６．５、０．５Ｍ塩化ナトリウムからなる水溶液５０ｍＬを加え室温で３０分間攪拌した後、遠心分離によりヘキサン層を集め、ロータリーエバポレーターで濃縮させた。これにアセトン５０ｍＬを加え十分攪拌し遠心分離によりアセトン層を除去した。この操作を２回繰り返し遊離脂肪酸を除去した。アセトン不溶物を集め減圧乾燥して環状ホスファチジン酸ナトリウム３．６ｇを得た。
Example 7: Method for producing cyclic phosphatidic acid sodium salt 10 g of soybean phospholipid (lecithin content: 70%) (hydrogen-free product) was dissolved in 100 mL of 1 M acetate buffer (pH 6.5) containing 0.3 M calcium chloride. Then, 6000 units of phospholipase A2 derived from Streptomyces was added, and the mixture was reacted by stirring at 40 ° C. for 18 hours. The reaction solution was adjusted to pH 2.5 to deactivate the enzyme, and then the pH was adjusted to 5.5. Subsequently, 1500 units of phospholipase D derived from the genus Actinomadura was added to the reaction solution, and the reaction was performed while stirring at 40 ° C. for 18 hours. Hexane 100mL was added to this reaction liquid, and 30 minutes stirring extraction was performed at room temperature. The hexane layer was collected by centrifugation, and 50 mL of an aqueous solution consisting of 10 mM EDTA, 0.1 M acetate buffer pH 6.5, 0.5 M sodium chloride was added thereto and stirred at room temperature for 30 minutes, and then the hexane layer was collected by centrifugation. And concentrated on a rotary evaporator. Acetone 50 mL was added thereto and stirred sufficiently, and the acetone layer was removed by centrifugation. This operation was repeated twice to remove free fatty acids. Acetone insolubles were collected and dried under reduced pressure to obtain 3.6 g of cyclic sodium phosphatidate.
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