立本特異的 方法과 ₁₃C-NMR 技法에 의한 잣기름의 트리아실 글리세롤의 構成脂肪酸의 分布에 관한 硏究

우효경(Woo Hyo Kyeng)(禹孝京),김성진(Kim Seung Jin)(金成眞),조용계(Joh Yong Goe)(趙鏞桂) 한국유화학회 1998 한국응용과학기술학회지 Vol.15 No.4

All the triacylglycerols including the molecular species having △^5-unsaturated fatty acids from the seeds of Pinus Koraiensis, were split into a mixture of diacylglycerols by a Grignard reagent prepared with allyl bromide without arousing acyl chains of a glycerol moiety to migration, and were also easily partially hydrolyzed to diacylglycerols by pancreatic lipase. (S)-(+)-(1-naphthyl)ethyl urethane(NEU) derivatives of the diacylglycerol mixture derived from the triacylglycerols were fractionated into sn-1, 3-, sn-1, 2- and sn-2, 3-DG-NEU by silica-HPLC and the fatty acid composition of these fractions was analysed. C_18:1ω9 is distributed evenly in the three positions of TG with C_18:2ω6 mainly located in sn-2 position, while △^5-unsaturated fatty acids such as △^5.9-C_18:2, △^5.9.12-C^18:3 and △^5.11.14-C_20:3 are exclusively present in the sn-3 position. These results could be confirmed by ^13C-NMR sepctroscopy : the signals at δ173.231 ppm and δ172.811 ppm of the carbonyl carbon of acyl moieties indicate the presence of saturated acids and/or C_18:1ω9 (oleic acid) in the α(α`)-or β- positions, and C_18:2ω6 including C_18:1:ω9 in the β-position, respectively. In addition, the resonance at δ173.044 ppm suggested a location of △^5-unsaturated fatty acid moiety in the α(α )-position.

초호열성균이 생성하는 phospholipase A₂에 관한 연구

조용계(Yong Goe Joh),우효경(Hyo Kyeng Woo),김연심(Yeon Sim Kim) 한국유화학회 1999 한국응용과학기술학회지 Vol.16 No.3

We checked the presence of phospholipase A₂(PLA)₂ which could split the ester bond at the position 2 in the glycerol backbone of glycerophospholipids, in the cells of hyperthermophiles of Pyrococcus horikoshii and Sulfolobus acidocaldarius. The results obtained are as follows; ①. Pyrococcus horikoshii cells were grown in obligate anaerobic conditions at 95℃ and they needed sulfur as energy source instead of oxygen, while Sulfolobus acidocaldarius species grew well in the aerobic medium (pH 2.5) containing yeast and sucrose at 75℃. ②. Pyrococcus horikoshii cells produced phospholipase A2 in the cell culture media although this species did not show lipase activity at least in the pH range of 1.5 ~ 3.5. Sulfolobus acidocaldarius cells produced lipase hydrolyzing triacylglycerols such as triolein, but did not split any kind of phospholipids used as substates. ③. The compound of 1-decanoyl-2-(p-nitrophenylglutaryl) phosphatidylcholine was not suitable for a substrate in this experiment, though frequently used as a subtrate for checking presence of phospholipase A₂, for its decomposi-tion in this experiment. The L-α-phosphatidylcholine-β-[N-7-nitrobenz-2-oxa-1, 3-diazol]aminohexanoyl-γ-hexadecanoyl labelled with a fluorescent material, did not show any migration of acyl chains in the molecule during the reaction with phospholipase A₂ under a hot condition. ④. Phospholipase A₂ in the cells of Pyrococcus horikoshii, showed the optimum activity at pH6.7~7.2 and 95~105℃, respectively, and was activated by addition of calcium chloride solution. Andthe phospholipase A₂ specifically hydrolyzed glycero-phospholipids such as phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl inositol, but could not split phospholipid containing ether bonds in the molecule such as DL -α-phosphatidylcholine-β-palmitoyl-γ-O-hexadecyl, DL-α-phosphati- dylcholine-β- oleoyl-γ-O-hexadecyl, DL-phosphatidylcholine-dihexadecyl.

입본특이적(立本特異的) 방법(方法)과 $^{13}C-NMR$ 수식 이미지 기법(技法)에 의한 잣기름의 트리아실 글리세롤의 구성지방산(構成脂肪酸)의 분포(分布)에 관한 연구(硏究)

우효경(Woo, Hyo-Kyeng),김성진(Kim, Seung-Jin),조용계(Joh, Yong-Goe) 한국유화학회 1998 한국응용과학기술학회지 Vol.15 No.4

All the triacylglycerols including the molecular species having ${\Delta}^5$ 수식 이미지-unsaturated fatty acids from the seeds of Pinus Koraiensis, were split into a mixture of diacylglycerols by a Grignard reagent prepared with allyl bromide without arousing acyl chains of a glycerol moiety to migration, and were also easily partially hydrolyzed to diacylglycerols by pancreatic lipase. (S)-(+)-(1-naphthyl)ethyl urethane(NEU) derivatives of the diacylglycerol mixture derived from the triacylglycerols were fractionated into sn-1, 3-, sn-1, 2- and sn-2, 3-DG-NEU by silica-HPLC and the fatty acid composition of these fractions was analysed. $C_{18:1{\omega}9}$ 수식 이미지 is distributed evenly in the three positions of TG with $C_{18:2{\omega}6}$ 수식 이미지 mainly located in sn-2 position, while ${\Delta}^5$ 수식 이미지-unsaturated fatty acids such as ${\Delta}^{5.9}-C_{18:2}$ 수식 이미지, ${\Delta}^{5.9.12}-C_{18:3}$ 수식 이미지 and ${\Delta}^{5.11.14}-C_{20:3}$ 수식 이미지 are exclusively present in the sn-3 position. These results could be confirmed by $^{13}C$ 수식 이미지-NMR spectroscopy : the signals at $^{\delta}$ 수식 이미지173.231 ppm and $^{\delta}$ 수식 이미지172.811 ppm of the carbonyl carbon of acyl moieties indicate the presence of saturated acids and/or $C_{18:1{\omega}9}$ 수식 이미지 (oleic acid) in the ${\alpha}({\alpha}- or ${\beta}$ 수식 이미지- positions, and $C_{18:2{\omega}6}$ 수식 이미지 including $C_{18:1{\omega}9}$ 수식 이미지 in the ${\beta}$ 수식 이미지-position, respectively. In addition, the resonance at $^{\delta}$ 수식 이미지173.044 ppm suggested a location of ${\Delta}^5$ 수식 이미지-unsaturated fatty acid moiety in the ${\alpha}({\alpha}-position.

역상(逆相)-HPLC와 $Ag^+$ 수식 이미지-HPLC에 의한 잣기름의 트리아실글리세롤분자종(分子種)의 상호분리(相互分離)

우효경(Woo, Hyo-Kyeng),조용계(Joh, Yong-Goe),김성진(Kim, Seung-Jin) 한국유화학회 1998 한국응용과학기술학회지 Vol.15 No.4

The lipids from the seeds of Pinus koraiensis mostly composed of triacylglycerols (TGs), in which linoleic acid (46.2 mol%) and oleic acid (25.6 mol%) are present as main components in the fatty acid composition. Surprisingly, they also have unusual fatty acids with ${\Delta}^5$ 수식 이미지-double bond systems such as ${\Delta}^{5.9.12}-C_{18:3}$ 수식 이미지 (16.0 mol%), ${\Delta}^{5.9}-C_{18:2}$ 수식 이미지 (2.3 mol%) and ${\Delta}^{5.11.14}-C_{20:3}$ 수식 이미지 (0.8 mol%). Saturated fatty acids of palmitic, stearic and arachidic acid were present in less than 8.0 mol%. TG was resolved into 17 fractions by reverse-phase HPLC according to so-called partition number (PN) suggested by Plattner, in which TG molecules with ${\Delta}^{5}$ 수식 이미지-NMDB acyl chains eluted later than did those with ${\Delta}^{9}$ 수식 이미지-MDB acyl radicals. $Ag^+$ 수식 이미지-HPLC separated the TG into 14 fractions more clearly than did those with ${\Delta}^{9}$ 수식 이미지-MDB acyl radicals. $Ag^+$ 수식 이미지-HPLC separated the TG into 14 fractions more clearly than did reverse-phase HPLC, and the complexity of ${\Delta}^{5.9.12}-C_{18:3}$ 수식 이미지 moiety with silver ion impregnated in the column bed was in the level between ${\Delta}^{9.12.15}-C_{18:3}$ 수식 이미지 ($C_{18:3{\omega}3}$ 수식 이미지) and $C_{18:2{\omega}6}$ 수식 이미지 (${\Delta}^{9.12}-C_{18:2}$ 수식 이미지). In the $Ag^+$ 수식 이미지-HPLC, it was found that the molecular species having a given-numbered double bonds widely spreaded in the acyl chains eluted earlier than those concentrated in one acyl chain. The main molecular species are $(C_{18:2{\omega}6})_2/{\Delta}^{5.9.12}-C_{18:3}$ 수식 이미지 (14.8 mol%), $C_{18:1{\omega}9}/C_{18:2{\omega}6})_2$ 수식 이미지 (12.8 mol%) and $C_{18:1{\omega}9}/C_{18:2{\omega}6}/{\Delta}^{5.9.12}-C_{18:3}$ 수식 이미지 (10.9 mol%).

대추의 果肉脂質에 存在하는 脂肪酸의 同定과 熟成에 따른 그 組成의 變化

우효경(Hyo Kyeng Woo),김성진(Seong Jin Kim),박성혜(Sung Hea Park),조용계(Yong Goe Joh) 한국유화학회 2001 한국응용과학기술학회지 Vol.18 No.1

In search for several fatty acid with unusual structure in vegetable oils, we have found that unknown peaks were shown on GLC in the analysis of fatty acids of the lipids from the pulp of ripened jujube (Zizypus jujuba var. inermis) fruits. These fatty acids were identified as a series of cis-monoenoic acids with ω-5 double bond system such as C14:1ω5, C16:1ω5 and C18:1ω5, including ω-7 fatty acid as C16:1ω7 and C18:1ω7, by GLC, solid-phase extraction silver ion-column chromatographic, GLC-mass spectrometric and IR techniques. First of all, total fatty acid methyl esters were resolved into saturated and branched fatty acid, monoenoic acid, dienoic acid, and trienoic acid fraction, respectively, with 100% dichloromethane (DCM), DCM/acetone (9:1, v/v) 100% acetone, and acetone/ acetonitrile (97:3, v/v) solvent system. Unknown fatty acids were included in the monoenoic fraction and were confirmed to have cis-configuration by IR. Picolinyl esters of monoenoic fatty acids gave distinct molecular ion peak and dominant diagnostic peaks, for example, m/z 317, 220 and 260 fragment for cis-C14:1ω5, m/z 345, m/z 248 and 288 fragment for cis-C16:1ω5 and m/z 373, m/z 276 and 316 fragment for cis-C18:1ω5. In this way the occurrence of cis-C16:1ω7 and cis-C18:1ω7 could be deduced from the appearance of prominent fragments as m/z 345, 220 and 260, and m/z 373, 248 and 280. Level of total ω-5 fatty acids amounted to about 30% in the fatty acid composition with the predominance of C16:1ω5 (18.7~25.0%), in the semi-ripened and/or ripened samples collected in September 14 (C16:1ω5 ; 18.7%, C14:1ω5 ; 3.6% and C18:1ω5 ; 3.0%), September 22 (C16:1ω5 ; 25.0%, C14:1ω5 ; 1.4% and C18:1ω5 ; 2.6%), and October 7 (C16:1ω5 ; 24.7%, C14:1ω5 ; 7.7% and C18:1ω5 ; 2.5%). However, the lipids extracted from unripened jujube in July and August contain these unusual fatty acids as low as negligible. It could be observed that the level of ω-5 fatty acids in the pulps increased sharply with an elapse of ripening time of jujube fruits. Other monoenoic fatty acids with ω-7 series, C16:1ω7 (palmitoleic acid) and C18:1ω7 (cis-vaccenic acid) could be detected. And in the lipids of the kernel and leaf of jujube, none of ω-5 fatty acids could be detected.

重水素化, Pentafluorobenzyl化와 GLC-Mass Spectrometry에 의한 Conjugate Trienoic Acid含有 Triacylglycerol 分子種의 立体特異的 分析

우효경(Hyo Kyeng Woo),김성진(Seong Jin Kim),조용계(Yong Goe Joh) 한국유화학회 2001 한국응용과학기술학회지 Vol.18 No.3

CTA ester bonds in TG molecules were not attacked by pancreatic lipase and lipases produced by microbes such as Candida cylindracea, Chromobacterium viscosum, Geotricum candidium, Pseudomonas fluorescens, Rhizophus delemar, R. arrhizus and Mucor miehei. An aliquot of total TG of all the seed oils and each TG fraction of the oils collected from HPLC runs were deuterated prior to partial hydrolysis with Grignard reagent, because CTA molecule was destroyed with treatment of Grignard reagent. Deuterated TG (dTG) was hydrolyzed partially to a mixture of deuterated diacylglycerols (dDG), which were subsequently reacted with (S)-(+)-1-(1-naphthyl)ethyl isocyanate to derivatize into dDG-NEUs. Purified dDG-NEUs were resolved into 1, 3-, 1, 2- and 2, 3-dDG-NEU on silica columns in tandem of HPLC using a solvent of 0.4% propan-1-o1 (containing 2% water)-hexane. An aliquot of each dDG-NEU fraction was hydrolyzed and (fatty acid-PFB ester). These derivatives showed a diagnostic carboxylate ion, (M-1)⁻, as parent peak and a minor peak at m/z 196 (PFB-CH₃)⁻ on NICI mass spectra. In the mass spectra of the fatty acid-PFB esters of dTGs derived from the seed oils of T. kilirowii and M. charantia, peaks at m/z 285, 287, 289 and 317 were observed, which corresponded to (M-1)⁻ of deuterized oleic acid (d₂-C18:0), linoleic acid (d₄-C18:0), punicic acid (d₆-C18:0) and eicosamonoenoic acid (d₂-C20:0), respectively. Fatty acid compositions of deuterized total TG of each oil measured by relative intensities of (M-1)⁻ ion peaks were similar with those of intact TG of the oils by GLC. The composition of fatty acid-PFB esters of total dTG derived from the seed oils of T. kilirowii are as follows ; C16:0, 4.6 mole % (4.8 mole %, intact TG by GLC), C18:0, 3.0 mole % (3.1 mole %), d₂C18:0, 11.9 mole % (12.5 mole %, sum of C18:1ω9 and C18:1ω7), d₄-C18:0, 39.3 mole % (38.9 mole %, sum of C18:2ω6 and its isomer), d₆-C18:0, 41.1 mole % (40.5 mole %, sum of C18:3 9c,11t,13c, C18:3 9c,11t,13r and C18:3 9t,11t,13c), d₂-C20:0, 0.1 mole % (0.2 mole % of C20:1ω9). In total dTG derived from the seed oils of M. charantia, the fatty acid components are C16:0, 1.5 mole % (1.8 mole %, intact TG by GLC), C18:0, 12.0 mole % (12.3 mole %), d₂-C18:0, 16.9 mole % (17.4 mole %, sum of C18:1ω9), d₄-C18:0, 11.0 mole % (10.6 mole %, sum of C18:2ω6), d₆-C18:0, 58.6 mole % (57.5 mole %, sum of C18:3 9c,11t,13t and C18:3 9c,11t,13c). In the case of Aleurites fordii, C16:o ; 2.2 mole % (2.4 mole %, intact TG by GLC), C18:0 ; 1.7 mole % (1.7 mole %), d₂-C18:0 ; 5.5 mole % (5.4 mole %, sum of C18:1ω9), d₄-C18:0 ; 8.3 mole % (8.5 mole %, sum of C18:2ω6), d₆-C18:0 ; 82.0 mole % (81.2 mole %, sum of C18:3 9c,11t,13t and C18:3 9c,11t,13c). In the stereospecific analysis of fatty acid distribution in the TG species of the seed oils of T. kilirowii, C18:3 9c,11t,13r and C18:2ω6 were mainly located at sn-2 and sn-3 position, while saturated acids were usually present at sn-1 position. And the major molecular species of (C18:2ω6)(C18:3 9c,11t,13c)2 and (C18:1ω9)(C18:2ω6)(C18:3 9c,11t,13c) were predominantly composed of the stereoisomer of sn-1-C18:2ω6, sn-2-C18:3 9c,11t,13c, sn-3-C18:3 9c,11t,13c, and sn-1-C18:1ω9, sn-2-C18:2ω6, sn-3-C18:3 9c,11t,13c, respectively, and the minor TG species of (C18:2ω6)2(C18:3 9c,11t,13c) and (C16:0)(C18:3 9c,11t,13c)2 mainly comprised the stereoisomer of sn-1-C18:2ω6, sn-2-C18:2ω6, sn-3-C18:3 9c,11t,13c and sn-1-C16:0, sn-2-C18:3 9c,11t,13c, sn-3-C18:3 9c,11t,13c. The TG of the seed oils of Momordica charantia showed that most of CTA, C18:3 9c,11t,13r, occurred at sn-3 position, and C18:2ω6 was concentrated at sn-1 and sn-2 compared to sn-3. Main TG species of (C18:1ω9)(C18:3 9c,11t,13t)2 and (C18:0)(C18:3 9c,11t,13t)2 were consisted of the stereoisomer of sn-1-C18:1ω9, sn-2-C18:3 9c,11t,13t, sn-3-C18:3 9c,11t,13t and sn-1-C18:0, sn-2-C18:3 9c,11t,13t,

Reversed - phase 및 Ag⁺ -HPLC 에 의한 Conjugate Trienoic Acid 含有 Triacylglycerol 分子種의 相互分離

김성진(Seong Jin Kim),우효경(Hyo Kyeng Woo),조용계(Yong Goe Joh) 한국유화학회 2001 한국응용과학기술학회지 Vol.18 No.3

Conjugate trienoic acids (CTA) occurred in triacylglycerols (TGs) of the seed oils of Trichosanthes kirilowii, Momordica charantia and Aleurites fordii, and they were easily converted to their methyl esters in a mixture of sodium methoxide-methanol without any structural destruction. The main fatty acids in triacylglycerol (TG) fraction of the seed oils of Trichosanthes kirilowii are C18:2ω6 (32.2 mol %), C18:3 9c.11t,13c (38.0 mol %) and C18:1ω9 (11.8 mol %), followed with C16:0 (4.8 mol %) and C18:0 (3.1 mol %). The TG fraction was resolved into 20 TG molecular species according to the partition number (PN) by reversed-phase (RP)-HPLC. The main TG species were DTc2, MDTc and D2Tc, of which amounts reached 63 mol % of total TG molecular species. The TG sample was fractionated into 11 fractions according to the number of double bond in the molecule by Ag⁺-HPLC and the species of DTc2, MDTc and D2Tc were also eluted as main components. The TG species containing CTA showed unusual behaviours in the order of elution by HPLC ; first, TG moleular species of DTc2 (D ; dienoic acid, Tc ; punicic acid, Tci ; α-eleostearic acid, M ; monoenoic acid, St ; stearic acid) was eluted earlier than Mtc2, although they have the same PN number of 40, and, secondly, the species of DTci2 with eight double bonds was eluted earlier than that of D2Tci with seven double bonds. Intact TG of the seed oils of Momordica charantia contained mainly fatty acids such as C18:3ω9c,11t,13t (57.7 mol %), C-18:1ω9 (17.4 mol %), C18:0 (12.3 mol %) and C18:2ω6 (10.6 mol %), and was classified into 13 fractions by RP-HPLC. The main TG species were as follows ; MTci2 [(C18:1ω9)(C18:3 9c,11t,13t)2, 39.1 mol %] and StTci2 [(C18:0)(C18:3 9c,11t,13t)2, 33.9 mol %] comprising about 73 mol % of total TG species, accompanied by DTci2 [(C18:2ω6)(C18:3 9c,11t,13t)2, 7.3 mol %], D2Tci [(C18:2ω6)2(C18:3 9c,11t,13t), 3.6 mol %] and MDTci [(C18:1ω9)(C18:2ω6)(C18:3 9c,11t,13t), 3.5 mol %]. Simple TG species of Tci3 [(C18:3 9c,11t,13t)3] was present in a small amount of 1.4 mol %, but other simple TG species were not detected. The TG was also resolved into 11 fractions according to the number of double bond by Ag⁺-HPLC, and the species were mainly occupied by MTci2 [(C18:1ω9)(C18:3 9c,11t,13t)2, 39.4 mol %] and StT-ci2 [(C18:0)(C18:3 9c,11t,13t)2, 35.4 mol %] DTci2 species with eight double bonds was also developed faster than D2Tci one with seven double bonds as indicated in the analysis of TG of the seed oils of T. kirilowii, and MTci2 species with cis, trans, trans-configurated double bond was eluted earlier than MTc2 species with cis, trans, cis-configurated double bond. The main components of fatty acid in total TG fraction isolated from the seed oils of of Aleurites fordii were in the following order ; C18:3 9c,11t,13t (81.2 mol %) > C18:2ω6 (8.5 mol %) > C18:1ω9 (5.4 mol %). With resolution of the TG by RP-HPLC, eight fractions such as Tci3, Dtci2, D2Tci, MTci2, PTci2 (P ; palmitic acid), PMTci, PDTci and StTci2 (St ; scearic acid) were isolated, respectively. TG species of Tci3 [(C18:3 9c,11t,13t)3, 54.2 mol %], DTci2 [(C18:2ω6)(C18:3 9c,11t,13t)2, 15.0 mol %] and MTci2 [(C18:1ω9)(C18:3 9c,11t,13t)2, 14.8 mol %] were present as main species.