녹용에 존재하는 Gangliosides의 구조분석에 관한 연구

Abstract

본 연구에서는 녹용 중에 존재하는 gangliosides 성분을 조사하였다. Folch-Suzuki 분배법에 의해 수용액층으로 분리된 당지질 성분을 DEAD-Sephadex A-25 column chromatography와 silica gel column chromatography로 정제하였다. 녹용의 당지질들을 HPTLC에서 전개시킨 후 gangliosides의 존재를 예측할 수 있었다. DEAE-Sephadex A-25 column chromatography로 분리한 결과 gangliosides는 2종류가 존재하였고, silica gel column chromatography로 정제한 결과 5개의 band로 분리되었다. HPTLC 결과 GM_(3)과 GD_(3)이 존재하였고, negative FAB-MS 결과 분자량이 각각 1263(Gl-a), 1151(Gl-b), 1167(G2), 1555(G3-a), 1442(G3-b)이었다. 보다 정확한 구조를 분석하기 위해 HPLC와 GC-MS를 사용하였다. HPLC로 gangliosides에 존재하는 당이 sialic acid(N-acetylneuraminic acid, N-glycolylneuraminic acid), galactose, glucose라는 것을 확인하였고, GC-MS결과 ceramide의 fatty acid로는 C_(l6:0), C_(l8:0), C_(l8:1), C_(l8:2), C_(22:0)이 존재하는 것을 확인하였다. FAB-MS, HPLC, GC-MS, NMR 분석 결과 Gl-a는 C_(22:0) fatty acid, eicosasphingosine(C_(20))을 가지는 Neu5Ac-GM_(3)이고, Gl-b는 C_(l6:0) fatty acid, sphingosine(C_(l8))을 가지는 Neu5Ac-GM_(3)이다. G2는 C_(l6:0) fatty acid, sphingosine(C_(18))을 가지는 Neu5Gc-GM_(3)이다. G3-a는 C_(22:0) fatty acid, eicosasphingosine(C_(20))을 가지는 Neu5Ac-GD_(3)이고, Gl-b는 C_(l6:0) fatty acid, sphingosine(C_(18))을 가지는 Neu5Ac-GD_(3)이다.;The compositions and structures of gangliosides in deer antler were investigated. Gangliosides were purified from deer antler by a combination of solvent extraction, Folch-Suzuki partition, DEAE-Sephadex A-25, and silica gel column chromatography and were characterized by fast atom bombardment mass spectrometry, proton magnetic resonace spectrometry, high performance liquid chromatography, and GC-mass spectrometry of the fatty acids. The thin layer chromatographic pattern of the ganglioside revealed the presence of GM_(3), GD_(3) with positive reactions toward both orcinol-sulfuric acid(sugar detection) and resorcinol-hydrochloric acid(sialic acid detection) spray reagents. For direct compositional analysis, samples were hydrolyzed in trifluoroacetic acid and analyzed for sialic acid and neutral sugar without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were identified using standard monosaccharides by pulsed amperometric detection. GM_(3)(Neu5Ac+Neu5Gc) and GD_(3)(Neu5Ac) existed as equal amount in deer antler. GM_(3)(Neu5Ac) and GM_(3)(Neu5Gc) were found in a 3:1 ratio, GD_(3)(Neu5Gc) was not detected. Ganglioside fractions were separated on HPTLC and column chromatography due to differences in their fatty acids and sphingosines. The ceramide moiety was composed of C_(l6:0), C_(l8:0), C_(l8:1), C_(l8:2), C_(22:0) fatty acids and C_(l8) sphingosine and C_(20) eicosasphingosine. Carbon numbers of major fatty acids were 16 and 22.