nghien co'u ti^p v& thanh phan hoa hoc

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TAP CHI HOA HOC

DOI: 1015625/0866-7144.2014-0063

T. 52(6)728-731 THANG 12 NAM 2014

NGHIEN CO'U TI^P V& THANH P H A N HOA HOC D A U G A N C A N H A M G O C SQUALUS JAPONICUS I S H I K A W A

TAI VjNH QUY NHON

Chu Quang Truyin^\ C i m Thi tnh', Tran Thi Tbu Thiiy', Hoang Thanh Huomg', N. A. Latysbev^, S. P. Kasijanov\ Pham Qu6c Long'

' Vien Hda hgc cdc Hgp chdt thien nhien, Vien Hdn lam Khoa hgc vd Cdng ngh? Vi?t Nam

^ Vien Sinh vdt bien A. V. Zhirmunsky, Phdnviin Viin Ddng. Vien Han lam Khoa hgc Liin Bang Nga Den Tda soan 18-9-2014; Chip nhgn dang 4-12-2014

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 ethyl (5Z, 8Z, \\Z, I4Z, 17Z) eicosapentanoate (1), glycerol 1,3-dioleate, 2-palmitate (2), l-0-(9Z-octadecenyl) 2-0-hexadecanoyl 3-C'-(9Z- octadecenoyl) glycerol (3) and selachyl alcohol (4) by mean spectroscopic methods including ESI-MS, ID- and 2D- NMR.

Keywords. Squalus japonicus, shark liver oil, alkyl glycerols.

1. MCi DAU

Ca nham gdc Squalus faponicus Ishikawa cd n^udn gdc tir Nhat Ban va phan bd d vimg bien tay bac Thai Binh Duong tir nam Nhat Ban, Trilu Tien, Trung Qudc den Viet Nam. Viing sinh sdng chii ylu la them lye dia, ven cac dao d do sau 52-400 m, thudng la 100-150 m [1]. Cho din nay ngoai nhiing thdng tin ve sinh hgc, hau nhu chua cd nghien ciru ve hda hgc cua loai nay dugc cdng bd. Kit qua nghien ciiu cua chiing tdi ve sang Igc lipit sinh vat bien cho thay dau gan ca nham gdc Squalus japonicus Ishikawa sdng d vinh Quy Nhon - Binh Djnh cd ham lugng alkyl glycerol kha cao (-7,6 %).

Day la Idp chit cd vai trd quan frgng trong nhilu qua trinh ciia co the sdng vdi hai chuc nang sinh hgc chinh la tham gia cau true mang te bao va dam nhgn vai trd trung gian d cac dap ung te bao [2]. Tac ddng sinh ly ciia cac chat nay rat da dang, ro ret nhit la de dieu trj va phyc hdi cho benh nhan ung thu, fri ligu sau xa fri va xu ly trang thai suy giam miln nhilm.

Kha nang ndi trgi khac la de tgo nhii tuong dang nano va dan thuoc din noi can nhgn [3], Su hien dien cac hgp chit l-O-octadecyl-3-O-hexadecanoyl glycerol, batyl alcohol, chimyl alcohol cimg cac hgp chat squalene, cholesterol, cholesteryl palmitat va axit palmitic da dugc chiing tdi cdng bd trong cac cdng trinh gin day [4, 5]. Bai bao nay tilp tuc md ta vi?c phan Igp va xac dinh ciu triic 4 hgp chit mdi

tim thiy la ethyl (5Z, 8Z, 112, 14Z, 17Z) eicosapentanoate (1), glycerol 1,3-dioleate 2- palmitate (2), l-(9-(9z-octadecenyl) 2-0- hexadecanoyl 3-0-(9Z-octadecenoyl) glycerol (3) va selachyl alcohol (4).

2. THUC NGH1$M 2.1. Nguyen lieu

Ca nham gdc Squalus japonicus dugc danh bat vao thang 02 nam 2011 tgi vinh Quy Nhon, Binh Djnh. Ten khoa hgc dugc TS. Vo Van Quang, Vl?n Hai Duong hgc Nha Trang giam dinh. Gan tuoi bao quan d nhi§t dg -20 °C, diu beo bao quan d nhi?t dg 4°C.

2.2. Thiet bi pban tich

Dilm ndng chay do tren may Mel. Temp.3.0 (Thermo scientific). Phd NMR ghi tren may Brucker AM 500 FT-NMR Specfrometric. Phd ESI-MS ghi tren may Xevo-TQMS, Waters-My. Sic ky khi ghi tren may Thermo Flnnigan Italia S.p.A. TRACE GC Ultra series, da cai dat phin mem nhgn dang axit beo chuyen dyng, cgt Inowax (30 m x 0,25 mm x 0,25 mm) nhiet dg 210 °C, thdi gian 60 phut, tdc dO ddng 15 ml/phut.

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TCHH, T. 52(6), 2014 2.3. Phan lap cac bop chat 1-4

Chiet xu§t dau (lipit tdng) tir gan ca theo phirgng phap Bligh & Dyer [7].

Cho 1 g d4u qua cgt nh6i 100 g SiO: va rua giai lan lugt voi «-hexan (A), clorofoc (B), clorofoc/etyl axetat 1/1 (y/v) (C) va etyl axetat (D).

Bang skc ky cgt va ban mong dieu ehe (Si02 Merck voi he dung moi «-hexan:ete etylic (6:4, v:v)) tach dugc hgp chat 1 (6 mg) co Rf ~ 0,80 ttr phan rua giai voi n-hexan (A), hgp chat 2 (41 mg) co Rf

~ 0,55 va hgp chat 3 (35 mg) co Rf ~ 0,65 tir phSn rira giai vai clorofoc (B). Hgp chat 4 (5 mg) nhan dtrgc khi thuy phan 3 bang dung dich KOH/etanol.

* Phan irng thiiy phSn cac hgp chat 3 va 4 th^rc hi^n nhu mieu ta trong tai li?u [4].

* Phan img tao dan xuat TMS va phan tich GC- MS theo tai lieu [8].

* S6 li?u ph6 NMR:

Hgp chat 1:

ESI-MS ra/z331,13 [M+H]* va 329,12 [M-H]*

'H-NMR (500 MHz, CDClj), 8 (ppm): 5,35 (lOH, ra), 4,12 (2H, m), 2,83 (8H, m), 2,32 (2H, t, J

= 7,5 Hz), 2,1 (4H, m), 1,71 (2H, m), l , 2 5 ( 3 H , t , J = 7,5 Hz), 0,96 (3H, t, J = 7,5 Hz).

"C-NMR (125 MHz, CDCI3), S (ppm): 173,6 (C-I), 132,0 (C-180), 129,04 (C-5), 128,81 (C-8), 128,59 (C-11), 128,27 (C-6 & C-9), 128,14 (C-12), 128,13 (C-14), 127,90 (C-17), 127,04 (C-15), 60,24 (C-r), 33,76 (C-2), 25,60 (C-3, C-7, C-10, C-13, C- 16), 24,8 (C-40), 20,57 (C-19), 14,26 (C-20 vaC-2').

Hgp chat 2:

'H-NMR (500 MHz, CDCI3), S (ppm): 5,35 (4H, m), 5,2 (IH, m), 4,30 (2H, dd, J = 3 & 11 Hz), 4,18 (2H, dd, J = 5 & 11 Hz), 2,01 (8H, m), 1,61 (6H, m), 1,26 (64H, m), 0,88 (9H, t,J= 7,5 Hz).

"C-NMR (125 MHz, CDCI3), 5 (ppm): 173,2 (C-r & C-r"), 173,0 (C-I"), 130 (C-9' & C-9"'), 129,7 (C-10' & C-10'"), 68,9 (C-2), 62,1 (C-1 & C- 3), 34,1 (C-2', C-2" & C-2'"), 31,9 (C-16', C-16"'&

C-14"), 27,2 (C-8' & C-1 r , C-8'" &C-11'"), 22,7 (C- 17', C-IT" va C-16"), 14,1 (C-18', C-18'" va C-16").

Hgp chat 3:

'H-NMR (500 MHz, CDCI3), <5 (ppm): 5,35 (4H, m), 5,2 (IH, m), 4,30 (IH, dd, 3 & 11 Hz), 4,18 (IH, dd, J = 5 & 11 Hz), 3,52 (2H, m), 3,45 (2H, m), 2,32 (4H, q,J= 7 Hz), 2,01 (8H, m), 1,61 (4H, m), 1,57 (2H, m), 1,26 (66H, m), 0,88 (9H, t, J = 7,5 Hz).

"C-NMR (125 MHz, CDCI3), S (ppm): 173,4 (C-1'"), 173,2 (C-1"), 130 (C-9", C-10', C-9'"& C- 10"), 71,77 (C-r), 70,1 (C-2), 69,0 (C-1), 62,8 (C- 3), 34,3 (C-2'"), 34,1 (C-2"), 31,9 (C-16', C-16'" &

C-14"'), 29,7-29,1 (C-2', C-4' - . C-7', C-12' -^ C- 15', C-4" ^ C-13", C^t'" - * C-7"', C-12'" ^ 15'"),

Chu Quang Truyen va cong sir 27.2 (C-8', C - i r , C-8'" & C-11"'), 26,0 (C-3'), 25,0 (C-3" & C-3'"), 22,7 (C-17', C-15" & C-17'"), 14,11 (C-18', C-16;'& C-18'").

Hgp chat 4:

ESI-MS ni/z 343,13 [M+H]* va 341,12 [M-H]*

'H-NMR (500 MHz, CDCI3), S (ppm): 5,35 (2H, m), 3,86 (IH, m), 3,72 (IH, dd, J = 3 & 11 Hz), 3,65 (IH, dd, y = 5 & 11 Hz), 3,52 (2H, ra), 3,45 (2H, m), 2,0 (4H, m), 1,57 (2H, ra), 1,26 (22H, m), 0,87(3H,t,y= 7,5 Hz).

"C-NMR (125 MHz, CDCI3), S (ppm): 130 (C- 9' & C-10'), 72,5 (C-1), 71,89 (C-1'), 70,45 (C-2), 64.3 (C-3), 31,9 (C-16'), 29,7-29,3 (C-2', C-4'-. C- 7', C-12'-. C-15'), 27,2 (C-8' & C-11'), 26,0 (C-3'), 22,7 (C-17'), 14,11 (C-18').

3. KET QUA VA THAO LUAN

Hgp chit 1 CO diing diu, khong raau. Pho ESI- MS cho cac pic gia phan tur a m/z 331,13 [M+H]* va 329,12 [M-H]* irng voi kh6i lugng phan tir la 330.

Su chuyen djch ve phia trugng cao cua tin hieu img vgi cacbon cacbonyl tai 5c 173,6 ciing sir hien dien cac tin hieu ciia mgt nh6m metilen cacbinol tai 5c 60,24 va 5H 4,12 (2H, q) xac nhan co mat mgt lien ket este.

Cac tin hieu ciia cacbon raetin olephin tai 5c 132,06, 129,04, 128,81, 128,59, 128,17 x (2), 128,14, 128,13, 127,90 va 127,0 cimg mgt multiplet tai 5H 5,35 (lOH, ra) irng vai 10 proton metin olephin cho biet c6 5 lien ket doi trong phan tij. Sir chuyin djch v6 phia trucmg thap cua proton

^-metilen thugc nhanh axit tai 5H 1,71 va tuang tac C—*H tren pho HMBC ciia cac cacbon metin olephin voi cac proton tai <5H 2,83, 2,10 va 1,71 chu:ng to cac noi doi thugc ve nhanh axit. Tirgng tac ciia proton metilen cacbinol tai 6H 4,12 vai cacbon cacbonyl tai 8c 173,6 va cacbon metyl tai <5c 14,26 cung xac nhan nhanh ancol chi c6 hai nguyen tir cacbon.

K6t hgp vai ph6 khdi lugng va cac pho NMR da xac dinh dugc hgp chat da phan lap la este etyl cua ragt axit beo chira 5 n6i doi co cong thuc phan tir C22H3402. So sanh vcri tu lieu pho '^C-NMR ctia dan xuat metyl este (5Z, 8Z, IIZ, 14Z, I7Z) eicosapentanoate [6] thay hoan toan triing khap voi nhanh acyl (C-1 den C-20) cua hgp chat 1. Nhu vay cau triic cua hgp chat da phan lap la etyl (5Z, 8Z, IIZ, 14Z, 17Z) eicosapentanoat (1). Cau triic nay dugc khang dinh qua pho HMBC va phan tich GC-MS co so sanh vol thu vien pho.

Hgp chat 2 dang dau raau vang nhat. Cac pho NMR CO dang ctia mgt dan xuat glyxerol the hoan toan. Sir xuat hien cac tin hieu ciia proton thuipc 3 nhom a-metilen tai 5H 2,32 (6H, t) va 3 nhom

^-metilen tai (5H 1,61 (6H, q) chimg to day la hgp

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TCHH, T 52(6), 2014

chit triacyl glycerol. Phd '^C-NMR kit hgp DEPT cd cac tin hieu ciia cacbon cacbonyl tai 6c 173,2 va 173,0, cacbon metilen cacbinol tai 5c 62,1 va cacbon metin cacbinol tgi 5c 68,9, trong dd cac tin higu d 5c 173,2 va 62,1 diu cd cudng dg manh gap ddi. Tuong tu nhu vay tren phd ' H - N M R cung cd cac tin hieu ling vdi proton cua 2 nhdm metilen cacbinol tai 5H 4,30 (2H, dd) va 4,18 (2H, dd). Nhu vay hgp chit 2 phai cd cau true ddi xiimg bac 2. Dieu nay mgt lan nda dugc khang dinh qua sir hien dien tin hi?u iing vdi 4 proton metin olephin tgi 5H 5,35 va 8 proton thugc 4 nhdm metilen liin ke ndi ddi tai 5n 2,01 trong khi tren phd '^C-NMR kit hgp DEPT chi xuat hien 2 tin hi?u cacbon metin olephin tgi 5c 130 va 129,7 cd cudng do mgnh gap ddi va 1 tin hi$u cua cacbon metilen lien ke ndi ddi tai 5c 27,2 cd cudng dg mgnh gap 4 lan. Tir nhirng dan lieu tren suy ra hgp chat 2 la mgt triglyxerit cd hai nhanh acyl d6i xiing nhau va mdi nhanh nay chira mgt lien ket ddi d vi tri tuong t^^.

Hgp chat 2 dugc thiiy phan vdi dung dich KOH/etanol [4]. Hdn hgp axit beo tgo thanh dugc phan tich bang sac ky khi vdi phan mem nhgn dang chuyen dung, ddng thdi cung so sanh true tilp vdi chat chuan. Ket qua cho hdn hgp gdm axit palmitic va axit oleic vdi ty le 1:2. Theo cac pho NMR, cac nhanh oleat se dugc gin kit vao vi tri C-1 va C-3 va ciu true ciia hgp chat da phan lap se la glycerol 1,3- dioleate 2-palmitate (2). Cau true nay hoan toan khing dinh qua phd HMBC.

Hgp chat 3 cd dang dau, mau vang nhgt. Cac phd NMR cung cho cac tin hieu cua glycerol bi thi hoan toan. Tuy nhien khac vdi hgp chit 2 cac tin hi$u ciia cacbon cacbonyl d 6c 173,4 va 173,2 cung cac nhdm a- va ^-metilen tgi 6H 2,32 (4H, q) va 1,60 (4H) cho thay chi cd 2 nhanh acyl trong phan tii. Them vao dd, ngoai cac tin hleu cua 2 nhdm metilen cacbinol tai 6c 69,0, 5H 3,52 (2H, m) va tgi 6c 62,8, 5H 4,30 (IH, dd, H-3a), 4,18 (IH, dd, H-3b) cimg mgt nhdm metin cacbinol tai 6c 70,1 va 6H 5,2 ciia gdc glycerol cdn xuit hien cac tin hieu img vdi mgt nhdm metilen cacbinol tgi 5c 71,77 va 5H 3,45 (2H, m). Tuong tac giiia cac proton nay vdi cacbon metilen cacbinol ciia gdc glycerol tai 5c 69,0, chung td nhanh alkoxy dugc gin kit vao C-I bdi lien kit ete. Vin dl cdn Igi la phai xac djnh ciu triic ciia 3 nhanh thi va vj fri gan ket cua hai nhanh acyl.

Tuong ty hgp chit 2 phd ' H - N M R ciia 3 cung xuat hien multiplet d 6H 5,35 (4H, m) irng vdi 4 proton metin olephin va d 6H 2,01 (8H, m) irng vdi proton cua 4 nhdm metilen liin kl noi ddi, xac nhan phan tir cd hai lien kit ddi. Su xuit hien d dang mgt tin hieu duy nhit vdi cudng dg manh img vdi 4 cacbon metin olephin tai 5c 130 va tin hieu iing vdi 4 cacbon metilen liin kl ndi ddi tai 5c 27,2 cho phep

Nghiin cuu tiip vi thdnh phdn hda hgc...

du doan: hogc nhanh ndi vao C-2 chiia 2 ndi ddi d vj fri bi?t lap hoac moi nhanh ndi vao C-1 va C-3 co mdt ndi ddi d vi fri gidng nhau.

Tien hanh phan iimg thuy phan hgp chit 3 bing dung dich KOH/etanol [4] va tach san phim thiiy phan bing sac ky ban mdng diiu chi (SiOj, n-hexan/benzen/HiO, 3/7/0,1, v/v/v) thanh phan dogn axit (Rf ~ 0,70) va alkyl glycerol 4 (Rf ~ 0,12).

Bang sic ky khi vdi phan mim nhgn dgng chuyen dung va so sanh true tiep vdi chat chuan dS xac dinh dugc phan doan cd Rr~ 0,70 la hdn hgp axit palmitic va axit oleic ty le 1:1. Tir dd suy ra ndi ddi cdn lai sg thugc vl nhanh alkoxy va nhanh 9Z-octadecenoyl se dugc ndi vao C-3. Nhu vay, cac hgp chit 2 va 3 chi khac nhau ve cau triic nhanh ndi vao C-I. So sanh cudng do cac tin hi^u proton frong hai chit chi co su khac bi?t d muUiplet tai 5H 1,26. 6 hgp chit 2 tin hieu nay iing vdi 64H trong khi hgp chat 3 lai iing vdi 66H. Sir chenh lech nay cung la su chenh lech so proton giira cac nhanh acyl va alkoxy cd cimg s6 nguyen tu cacbon. Kit hgfp vdi cac du lieu phi NMR cd the du doan nhanh alkoxy cung cd 18 nguyen tii cacbon va cd ndi ddi d vj tri <B9. Kit hgp vdi cac pho HMBC va COSY xac dinh dugc ciu friic cua hgp chit 3 la l-0-(9Z-octadecenyl) 2-0- hexadecanoyl3-0-(9Z-octadecenoyl).

Hgp chit 4 cd dang diu, khdng mau. Phd ESI- MS cho cac pic gia phan tii d m/z 343,13 [M+H]*va 341,12 [M-H]^ ung vol khoi lugng phan ti> la 342.

So sanh vdi pho NMR ciia 4 vdi 3 thi thiy khdng cdn cac tin hieu ciia 2 nhanh acyl. Cac tin hieu iing vdi H-2 va H-3 dich chuyin vl phia trudng cao hon d 6H 3,86 (IH, q, H-2) va 5H 3,72 (IH, dd, H-3a), 3,65 (IH, dd, H-3b) do cac lien kit este da hi thuy phan.

~-0R3

Rl R2 R3 C,7H33CO CsHsiCO C H j j C O

C,8H35 Ci5H3,CO C17H33CO Ci8H35 H H Cudng dg cac tin hi?u d 5c 130, 6H 3,53 (2H, m)

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T C H H , T. 52(6), 2 0 1 4

chi cdn irng v d i 2 n h d m metin olephin. T u o n g t u n h u v a y cac tin hieu d 6c 27,2 va 6H 2,0 (4H, q) c u n g chi cdn iing v d i 2 n h d m metilen liin k l ndi ddi. Dieu nay da x a c nhgn s u cd mat cua mgt lien ket ddi d nhanh alkoxy.

K e t h g p p h o khdi l u g n g va N M R xac dinh d u g c hgp c h i t 4 la m d t alkyl glycerol cd c d n g thiic p h a n tir la C2]H4203. Tren c o s d dii lieu p h d va t u lieu v e cac alkyl glycerol frong dau gan ciia c a c loai Squalus chiing tdi d u doan ndi ddi d u g c n i m d vj tri cu9 v a hgp chat 4 la selachyl ancol. K e t q u a phan tich G C - M S cac dan x u i t T M S ciia h g p c h i t 4 va selachyl alcohol c h u i n da k h i n g djnh k i t luan tren. T u o n g t u cac alkyl glycerol thien nhien khac, h g p chat 4 cd cau hinh S. Cau triic cua h g p chat 4 m g t lan nira da giiip khang dinh h g p chat 3 cd cau true ( 2 R ) 1-0- (9Z-octadecenyl) 2 - O - h e x a d e c a n o y l 3 - 0 - ( 9 Z - octadecenoyl) glycerol.

4. K E T L U A N

Tir dau gan ca n h a m gdc Squalus japonicus Ishikawa da p h a n lap v a x a c dinh d u g c cau friic them 4 hgp c h i t la ethyl (5Z, 8Z, IIZ, 14Z, I7Z) eicosapentanoate (1), glycerol 1,3-dioleate 2 - palmitate (2), 1 - 0 - ( 9 Z - o c t a d e c e n y l ) 2-O- hexadecanoyl 3 - 0 - ( 9 2 - o c t a d e c e n o y l ) glycerol (3) va selachyl alcohol (4). H g p chat 3 lan dau tien d u g c phan lap t u thien nhien. D a y la lan dau tien 4 h g p chat nay cd mat d loai Squalus japonicus d u g c cdng bd.

Loi cam o n . Cdng trinh dirge thuc hien duoi su tdi tro cua Be tdi: "Nghien ciru quy trinh san xudt thuc pham chia: ndng giau hogt chdt alkylglyxeryl ete tir

Chu Quang Truyen va cdng s ndi tang ddng vdt thuy san". Md sd: 629/HD- KHCN-CNSH Bd Ndng nghiep vd Phdt trien Ndng thdn.

TAI LIEU THAM KHAO

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3 lannitti, T., palmieri, B. An update on the therapeutic roi ofalkylglycerols. Mar. Drugs, 8, 2267-2300 (2010) t . Chu Quang Truyen, Cam Thi fnh, Hoang Thanh

H u o n g , N. A. Latyshev, S. P. Kasijanov, Pham Q u i c Long. Phdn lap, xdc dinh squalene vd acyl glycerol monoalkyl ete tic ddu gan cd nhdm gdc Squalus japonicus Ishikawa, Tap chi Hoa hoc, 51(6ABC),

122-125(2013)

5. Chu Quang Truyin, C i m Thj Inh, Hoang Thanh Hucrng, N. A. Latyshev, S P. Kasijanov, Pham Qu6c Long. Nghien cuu thdnh phdn hoa hoc ddu gan cd nhdm gdc Squalus japonicus ishikawa. Tap chi Hoa hoc, 52, 372-375(2014).

u, John A. Findlay and Ashok D. Patil, Atibacterial constituents of the diatom navicula delognei, Joumal ofNafriral products, 47(5), 815-818 (1984), 7. Bligh E.G & Dyer W.J. A rapid method of total lipid

extraction and purification- Canadian Joumal of Biochemistty and Physiology, 37, 911-917 (1959).

8, KacbaHOB C. n . , JlaxbiQieB H. A., CBexameB B.H, H ap. 0apMaKOJioeuHecKue s^^peKmu OUOJIOSUICCKU aKmueHoU doOaeKu h nume ua ocuoee MOHoaiiKumjiuiiepuHoe, TnxooKeaHCKHH MeaHUHHCKHii JKypHan, (2), 32-35 (2010).

Lien he: C h u Q u a n g T r u y e n

Vien Hda hgc c a c H g p chat thien nhien Vien Han lam K h o a hgc va C d n g ngh$ V i e t N a m N h a 1H, sd 18, H o a n g Q u d c Viet, Cau G i i y , H a N d i E-mail: q u a n g t r u y e n 6 9 @ g m a i l . c o m .