TAP c m HOA HOC T. 52(3) 372-375 T H A N G 6 N A M 2 0 I 4
NGHIEN CLfU THANH P H A N HOA HOC D A U GAN CA NHAM GOC SQUALUS JAPONICUS ISHIKAWA
Chu Quang Truyen'*, Cim Thi Inh', Hoang Thanh Huorng', N. A. Latyshev^, S. P. Kasijanov^
Pham Quoc Long'
' Viin Hda hge cdc Hgp chdt thien nhien, Viin Hdn ldm Khoa hgc vd Cong nghi Viet Nam
^Vien Sinh vgt biin A. V. Zhirmunsky, Phdn viin Viin Ddng, Viin Han ldm Khoa hgc Lien bang Nga Din Tda soan 10-01-2014
Abstract
A chemical investigation of the liver oil from the shark Squalus japonicus Ishikawa, collected in Quy Nhon bay led to the isolation of four compounds. Their structures were determined to be cholesterol (1), cholesteryl palmitate (2), palmitic acid (3) and I-O-hexadecyl glycerol (4) by mean spectroscopic methods including ESI-MS, ID- and 2D-NMR.
Keywords: Squalus japonicus. Shark liver oil, Alkyl glycerols. Sterols.
1. MO DAU 2. T H U C NGHIEM
Ci nhim gdc Squalus japonicus Ishikawa cd ngudn gdc tir Nhat Ban va phan bd d viing bien tiy bac Thai Binh Duang tir nam Nhat Ban, Trieu Tien, Trung Qudc den Viet Nam. Vimg sinh sdng chu yeu la them luc dia, ven cac dao d dp sau 52-400 m, thudng la 100-150 m [1]. Cho den nay ngoai nhifng thdng tin ve sinh hpc, hau nhu chua cd nghien ciru ve hda hpc ciia loii niy dupc cdng bd. Kit qua nghien ciiru cua chiing tdi ve sang lpc lipit sinh vat bien cho thay dau gan ci nhim gdc Squalus japonicus Ishikawa sdng d vjnh Quy Nhan - Binh Djnh cd ham luong alkyl glycerol kha cao ( - 7,6 %).
Day la Idp chat cd vai trd quan trpng trong nhieu qua trinh cua ca the sdng vdi hai chiic nang sinh hpc chinh la tham gia cau true mang te bao va dam nhan vai trd trung gian d cac dap umg te bao [2]. Tac dpng sinh ly cua cac chit nay rat da dang, ro ret nhit la dl dieu trj vi phyc hdi cho benh nhan ung thu, trj Ilfu sau xg tri va xii ly trang thii suy giam mien nhilm.
Kha nang ndi trpi khac la de tao nhu tuong dang nano va dan thudc den nai cin nhan [3]. Sy hien difn ciia l-O-octadecyl-3-O-hexadecanoyl glycerol ciing squalene tir phan doan lipit it phan cue ciia diu gan ca nham gdc da dupc chiing toi cdng bd trong mot cdng trinh gin day [4]. Bai bao nay tilp tuc md ta vifc phan lap va xie dinh cau tnic 4 hop chat mdi tim thiy la cholesterol (1), cholesteryl palmitat (2), axit palmitic (3) va 1-0-hexadecyl glycerol (4).
2.1. Nguyen lieu
Ca nham gdc Squalus japonicus dupc danh bat vio thing 02 nam 2011 tgi vinh Quy Nhan, Binh Dinh. Ten khoa hpc dupc TS. Vd Van Quang, Vien Hai Duong hpc-Nha Trang giim djnh. Bao quan gan tuai d nhiet dp -20 "C.
2.2. Thiet hi phan tich
Diem ndng chay do tren miy Mel. Temp. 3. 0 (Thermo scientific). Phd NMR ghi tren may Brucker AM 500 FT- NMR Spectrometrie. Pho ESI-MS ghi tren may Xevo- TQMS, Waters- My. Sic ky khi ghi tren may Thermo Finnigan Italia S.p.A. TRACE GC Ultra series, da cai dat phin mem nhan dang axit beo chuyen dung, cot Inowax (30m x 0,25 mm x0,25 mm) nhiet dp 210 °C, thdi gian 60 phiit. Spit flow 15 ml/phiit.
2.3. Chiet tach dau va phan l^p cac hop chat
*Chiet suit dau (lipit tong) tir gan ci theo phuang phap Bligh & Dyer [5].
*Phin lap cic hpp chit 1-4
Cho 5g dau qua ept nhdi lOOg SiO: vi nia giii lan lupt vdi «-hexan, clorofoc, clorofoc/etyl axetat l/I (v/v) va etyl axetat.
1000 mg can rira vdi clorofoc (CI) dupc chgy sic ky tren cdt Si02 vdi hf dung mdi «-hexan/etyl
TCHH, T. 52(3), 2014
axetat 95/5 (v/v) cho 4 nhSra phan dojn ClA-CID.
TO CIC bSng sSc kf nhic lai va sic ky tinh che tren c ^ nhd vdi h§ dung m6i n-hexan/etyl axetat 85/15 (v/v) nhSn tagc chat 1 (32 mg).
Phan (to?n C1B dm?c nia ^iai ti6p tren ept AI2O3 viiri w-hexan vk clorofoc. Ph4n c3n clorofoc (C2) du^c ch^y sSc ky tren cot SiOa voi he dung moi n-hexan/etyl axetat 95/5 (v/v) va tinh che tilp bang ket tinh l^i trong axeton nhan dugc chit 2(11 mg).
1000 mg can riJra giai boi h6n hop clorofoc/etyl axetat (CEl) dugc ch^iy sac ky tren ept Si02 vcri h?
dung moi «-hexan/etyl axetat 90/10 (v/v) cho 5 nhom phSn doan CEIA-CEIE. Phan tach tiep CEIB tren ban mong di8u che Si02 vai he dung moi
«-hexan/ete etylic/axit axetic 60/40/1 (v/v/v) nhan dirgro chat 3 (12 mg R, 0,5). Phan tach tiS|3 CEIC bang sic k^ tr€n c6t silicagen va tinh che tiSp bSng ket tinh Igi trong axeton cr 0 °C nhan dugrc chit 4 (27
*S6li?uph6NMR Hgrp ch^t 1
^H-NMR (500 MHz, CDCI3).
5 (ppm) 5,35 (IH , brs, J= 5 Hz), 3,52 (IH, m), 2,20 (2H, m), 2,0 (2H, m), 1,0 (3H, », CHj), 0,91
Chu Quang Truyen va cgng sv (3H, d, J = 7 Hz, CHj), 0,86 (6H, d, J = 7 Hz, 2x CHj), 0,68 (3H, s, CHj).
Hop chat 2
'H-NMR (500 MHz, CDCI3).
5 (ppm) 5,34 (IH, m), 4,60 (IH, m), 2,33 (2H, m), 2,2-2,0 (4H, m), 1,60 (2H, m), 1,26 (24H, brs, 12 CH2), 1,08 (3H, s, CH,), 0,90 (3H, d, / = 7 Hz, CHj), 0,87 (9H, m, 3CHj), 0,67 (3H, m, 3CH3).
"C-NMR (125 MHz, CDCI3), xem bang 1.
Hop chat 3
'H-NMR (500 MHz, CDCI3).
8 (ppm) 2,34 (2H, q, 7 = 7 Hz), 1,63 (IH, t\,J = 7 Hz), 1,26 (24H, bre) va 0,87 (3H, t, y = 7 Hz, CH3).
Htfp chat 4
' 'H-NMR (500 MHz, CDCI3).
5 (ppm) 3,86 (IH, m), 3,72 va 3,62 (2H, dd, J - 5 & 11 Hz), 3,52 (2H, m), 3,45 (2H, m), 2,98 (IH, brs, OH), 2,64 (IH, brs, OH), 1,57 (2H, q , y = 7 Hz), 1,26 (24H, brs), va 0,88 (3H, t, J = 7 HZ, CH3).
"C-NMR (125 MHz, CDCI3).
S (ppm) 72,5, 71,87, 70,5, 64,22, 31,91, 29,7- 29,3,22,67 va 14,09
Vi tri C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Bang 1: So Hgrp chat 1
37,2 31,6 71,8 42,2 140,7 121,7 31,8 31,8 50,1 36,6 21,1 39,8 42,3 56,7 24,3 28,3 56,1 11,9 19,3 35,7 18,7 36,1 23,8 39,5 28 0 22,5 22,8
ieu pho '^C-NMR ciia cac hcrp Hop chat 2
37,0 31,7 73,7 42,3 139,8 122,5 31,9 31,7 50,1 36,6 21,1 39,7 42,3 56,7 24,3 28,2 56,1 11,9 19,3 35,8 18,7 36,2 23,8 39,5 28,0 22,5 22,7
Vi tri C (tiep) 2'
r
3' 4'-13'
14' 15' 16'
chat 1 va 2 (do trong CDClj, 125 MHz) Hop chat 2
173,4 34,7 25,0 29,1-29,7
31,9 22,7 14,1
Nhanh 3-0-hexadecanoyl [6]
173,9 34,4 25,0 29,1-29,7
31,9 22,7 14,1
TAP CHI HOA HOC T. 52(3) 372-375 THANG 6 NAM 2014
1. R - H 2. R = CH3(CH2)uCO
3. KET QUA VA THAO LUAN
Hpp chit 1 cd dang tinh thi hinh kim, mau tring, d.n.c. 148 "C. Cic pic gia phan tir d m/z 387,3 [M+H]"" va 385,2 [M-H]"" tren phd ESI cho bilt khoi lupng phan tu la 386. Phd 'H-NMR cd dang die tnmg cua cac hpp chit steroh vdi su cd mat cic singlet ciia hai nhdm metyl gdc tai 6H 1,00 va 0,68 ppm cung cac doublet cua ba nhdm metyl tai 5H 0,91 (3H, d , J = 7 H z ) v i 0 , 8 6 p p m ( 6 H , d, J ' - 7 H z ) . Su hien dien cic tin hifu d 6H 5,35 (IH, brs, J = 5 Hz) va 3,52 ppm (IH,m) chung td cd mat mpt ndi ddi bj thi 3 lin va mpt nhdm hydroxy. Ket luan tucmg tu cung cd duoc d phd '^C-NMR va DEPT. Sd lifu cic phd nay cho biet phan td cd 27 nguyen tir cacbon, trong so dd cd 5 cacbon metyl, mpt cacbon bac 4 olephinic tai 5c 140,7, mdt cacbon metin olephinic tgi 122,7 va mpt cacbon oximetin tai 71,8 ppm.
Tuang tic H-C ciia proton metilen lien ke ndi ddi (5H 2,2 ppm) vdi cacbon d 5c 140,7 va 71.8 ppm ciing tuang tic cua cac proton metyl d 5H 1,00 (3H, s, H-19) vdi cacbon bac 4 olephinic tai 6c 140,7 ppm tren phd HMBC da khang dinh cau tnic A ' - 3 ^ - hydroxy.
Hing sd vat ly, khdi lupng phan tu va cac sd lieu phd cho phep du doin hpp chat phan lap dupc la cholesterol. Ciu true nay dupc khang djnh qua su so sinh trirc tiep vdi chat chuan.
Hpp chit 2 ket tinh trong axeton cho tinh the hinh kim, d.n.c.73 °C. Phd ESI cd pic [M+H]^ d m/z 625,83 ung vdi khdi lupng phan tu la 624,8. Tren pho ' H - N M R ngoii nhung tin hieu tucmg tu nhu phd cua chat 1 cdn cd them nhCrng tin hifu proton cua
mdt nhdm o-metilen (gin k l cacbon cacbonyl) tgi 5H 2,33 (2H, d, J= 7,5 Hz), mpt nhdm ^-metilen tgi SH U60 ( 2 H , m), mpt nhdm metyl tai 5H 0,88 (3H, t, J=
7 Hz) va mpt tin hieu don rpng tai 5H 1.26 ppm (24H, brs) ung vdi su hien dien cua mpt axit beo co mach cacbon dai va khdng phan nhanh. Dieu nay cung cho thiy ro khi so sanh so lifu phd "C-NMR cua hai hpp chat 1 va 2 (xem bing 1). Dp chuyin dich hda hpc cOa cac nguyen td cacbon tu C-1 den C-27 diu triing khdp, ngoai trir C-3. Tren phd ciia 2 cdn xuit hifn cac tin hifu cua cacbon cacbonyl tgi 5c 173,4, mdt cacbon metyl tgi 5c 14,1 v i nhieu cacbon metilen trong vung 5c 22,7-34,7 ppm. Sir chuyin dich vl phia trudng cao cua cacbon cacbonyl cung nhu su chuyen dich v l phia trudng thap cua C-3 tgi 5c 73,7 va H-3 tgi 6H 4,60 ppm la do cd s^r hinh thanh lien kit este. Sd lifu phd ciia nhanh acyl hoan loan triing khdp vdi sd lifu phd cua nhinh 3-0- hexadecanoyl d cic hpp chit I -O-alkyl-3-0- hexadecanoyl trong tai lifu [6] (bang I). Kit hpp vdi sd lieu phd khdi da xac djnh dupc hop chit 2 la 3-0- hexadecanoyl cholesterol (cholesteryl palmitat).
Hpp chit 3 dupc xac djnh la axit hexadeeanoic (axit palmitic) qua cac sd lifu phd 'H-NMR, phan tich sic ky khi va so sanh true tieii vdi chat chuin.
Hpp chit 4 cd dang tinh the hinh khdi, d.n.c.
66X. Pho khdi ESI-MS cho cic pic gia phan tuf d m/z 339,21 [M+Na]" va 315,22 [M-H]* ung vdi khii luong phan tu la 316.
Kit hop vdi cac phd NMR xie dinh dupc cdng thuc phan tii la C19H40O3. Tren cac phd ' H - vi "C- NMR kit hpfp DEPT cd mat cac tin hifu ung vol proton cua cac nhdm oximetin va oxmetilen d SH 3,86 (lH,m), 3,72 ( l H , d d , y = 5,0 & 11,0 Hz, Ha), 3,62 (1H, dd, / = 3,0 & 11,0 Hz, Hb), 3,52 ( 2H, m) va 3,45 ppm (2H, m) ciing tin hifu ciia cic cacbon tuong ung tai 5c 70,5, 64,22, 72,5 va 71,5 ppm. Ket hop vdi su hifn difn cua 2 tin iiifu dan rdng ciia proton trong cac nhdm hydroxy tai 5H 2,64 va 2,98 ppm cd the nhin ra 4 la mdt dan xuit thi mdt lan cua glycerol. Su cd mat mdt multiplet d 8H 3,86 ppm ung vdi H-2 chung td vang mgt nhdm the d C-2 [7].
Tuong tie H-C tren HMBC cua proton metilen tgi 5H 1,57 (2H, q, J = 7 Hz) vdi cacbon d 5c 71,8 ciing cac tin hifu tren phd ' H - N M R tai 5H 1,26 (26H, brs, I3CH2) v i 0,88 ppm (3H, t, 7 = 7 Hz, CHj) xac nhgn su hifn difn mpt gdc rupu beo no mach dai vJ khdng phan nhanh. Tuang tic HMBC cua proton oximetilen d 5H 3,45 (H-T) vdi cacbon oximetilen 6 5C 72,5 ppm (C-1) xac nhgn cd lien ket ete gi&a glycerol v i rupu beo d C-1. Cac sd lieu pho khoi va NMR cho phep xie dinh ciu triic cua hpp chit 4 li 1-0-hexadecyI glycerol (chim^l alcohol) vi dupc khing dinh khi so sinh tryc tiep vdi chimyl alcohol chuan. Cung nhu cac chimyl alcohol thien nhien
khac bpp chit 4 cd cau hinh d C-2 l i S [2].
Chimyl alcohol d i dupc phan lip tu mpt sd loai sinh vgt bien. Tac d^ng iirc che di cin ung thu da dupc n^ien cuu in vivo [8].
4. KET LUAN
Tir dau gan c i nham gdc Squalus japonicus Ishikawa da phan Igp v i xie djnh dupc ciu triic them 4 hpp chit la cholesterol (1), cholesteryl palmitat (2), axit palmitic (3) va 1-O-hexadecyl glycerol (4). Day li cdng bo dau tien vl s\t hifn difn cac chit nay d loai Sqiuilus Japonicus.
Ldi cam ffn: Cdng trinh dugc thue hiin dual su tdi trg cua Di tdi: Nghiin ciru quy trinh sdn xudt thuc phdm chdc ndng giau hogt chdt alkylglyxeryl ete tir ngi tgng dgng vdt thuy sdn. Ma sd: 629/HD-KHCN- CNSH, Bg Nong nghiep vd Phdt triin Ndng thdn.
TAI LIEU THAM KHAO
1. L. J. V. Compagno, M. Dando & S. Fowler. Sharks of the World, Field Guides, Princeton and Oxford.
Chu Quang Truyin vd cgng s\f Princeton University Press (2005).
H. Taguchi, W. L. Armarego. Glyceryl ether monooxigenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism. In O 1998. John Wiley & Sons, Inc. Med. Res Rev., 18(1), 43-89 (1998).
T. lannitti, B. Palmieri. An update on the therapeutic roll of alkylglycerols, Mar. Drags. 8, 2267-2300 (2010).
Chu Quang Truyin, Cim Thj Inh, Hoing Thanh Huong, N. A. Latyshev, S. P. Kasijanov, Phgm Qu6c Long. Phdn lap, xdc dinh squalene va acyl glycerol monoalkyl ete tir ddu gan cd nhdm goc Squalus japonicus Ishikawa, Tap chf H6a hpc, 5I(6ABC),
122-125(2013).
E. G. Bligh, W. J. Dyer. A rapid method of total lipid extraction and purification- Canadian joumal of BiochemisUy and Physiology, 37, 911-917 (1959).
P. Radhika, V. L. Rao, H. Laatsch. Chemical constituents of marine soft coral of the genus Lobophytum, Chem. Pharm. Bull, 52(11), 1345-1348 (2004).
The Aldrich library of "C-NMR and 'H FT- NMR, Ed. C. J. Pouchert, J. Behnke 1: 168 B, 283 C, 757 B, 949 C, 950 A, 950 C, 950 B(1993).
Deniau, A. L. et al. Which alkyl glycerols from shark liver oil have antitumour activities'} Biochemie 2010.
Doi:10.106/jbiochi. 2009.12.010.
Lien h?: Chu Quang Truyen
Vifn Hda hpc cac Hpp chit thien nhien
Nha IH Vifn Han lim Khoa hpc vi Cdng nghf Vift Nam 18 Hoing Quoc Vift, Ciu Giiy, Ha Npi
Email: [email protected].