Tieu ban: HOAT C H A T SINH HQC B I ^ N _ ^ 6 4 5
PHAN LAP VA TINH CHE FUCOXANTHIN MOT CAROTENOIT CO GIA TRI Tir RONG NAU KHANH HOA
Tran Thj Thu Thuy*, Nguyen Thj Minh Hang, Tran Quoc Toan, Nguyen Quyet Chien Viin Hod hpc, Viin Khoa hpc vd Cong nghi Viet Nam
I. M O DAU
Rong bien la san vat cua bien cd frii lugng ddi dao, tiem an nhifu ich lgi cd the khai thac cho hifn tai va tuong lai. Cac loai rong bien cung cap cac thanh phan cd gia trj dinh dudng nhu protein, carbohydrat, lipit va vitamin. Hon nira chiing con chiia nhifu hgp chat thien nhien co boat tinh sinh hgc co gia trj, trong do co cac hgp chat carotenoit [I].
Trong so khoang 600 hgp chat carotenoit tim thay trong ty nhien, gan day hgp chat fucoxanthin dugc gidi khoa hgc danh cho nhieu sy quan tam vi tinh nang lam thyc pham b6 sung, nhung trudc bet vi nhirng phat hifn d Nhat Ban ve kha nang ch6ng bfnh beo phi ciia n6 [2, 3].
Dugc biet fucoxanthin cd frong mgt so loai rong nau, dac bift la loai rong nau thyc pham Undaria pinnatifida ciia Nhat Ban, chiing toi da tien hanh khao sat mgt s6 loai rong nau ciia Vift Nam. Trong c6ng trinh nay chiing t6i nghien ciru vifc phan lap fucoxanthin tir mgt loai rong nau thu hoach nam 2008 d thanh ph6 Nha Trang, Khanh Hoa va nhan dang no bang cac phuang phap pho.
IL THlTC N G H I E M
Dung mdi chiet va xii ly mau dfu thugc loai tinh khiet phan tich, hoac dugc chung cat lai trudc khi sir dyng. Dung mdi dimg cho sac ky long cao ap thugc loai tinh khiet HPLC (Merck).
Cac hda chat khac deu thugc loai tinh khiet phan tich.
P h i UV-VIS dugc ghi fren quang p h i kl GBC Insfrument-2855; Phi APCI-MS dugc ghi d chi dg do difn tich duong (positive mode) tren may LC-MS Agilent 1100 series HPLC-DAD (Agilent Technologies, Wilmington, USA); Cac ph6 NMR dugc ghi tren may Bruker Avance 500 MHz (Dure) vdi dung mdi la CDCI3 va chat chuan ngi la TMS.
s i c ky ban m6ng (TLC) dugc tien hanh fren ban Kieselgel 60 F254 de nhom (Merck KGaA, Darmstadt, Dire). Sic ky Idng hifu nang cao (HPLC) dugc chay tren may LC-MS Agilent 1100 series HPLC-DAD, cot Zorbax SB-18 (150 x 3mm, 5)im).
1, Mau rong nau
Mau rong nau, dugc thu hai tai viing bifn Khanh Hoa nam 2008 va phai kho, do TS Biii Minh Ly, Vifn frudng Vifn Nghien ciru va Ung dyng C6ng nghf Nha Trang cung cap. Co hai miu, mgt miu co dang la h^p hinh kim, mgt mau co dang la rgng.
2. Xur ly va chiet nguyen lieu
Miu rong nau kh6 dugc rira sach, de rao nude, nghien nho bang each dgi N2 long va nghien fren cii i-nlc. Sau khi ngam fron^ dimethylsulfo.xide (DMSO) d nhift do phong frong 2-3 phiit, nguyen lifu dugc rira lai 2 lan bang nude cat va df rao nude. Ngam va chiet nhieu lin trong axeton cho din khi djch chiet hau nhu khong con mau. Co can bdt djch chiet (con 1/2 thf tich).
646 Tuyen tgp H$i nghj KHOA HQC T O A N Q U 6 C V £ SINH HQC BI^N VA PHAT TRI^N B £ N VLTNG
phan bo lai frong n-hexan/nudc. Tach rieng pha hexane, lam kh6 fren Na2S04 va lgc. Sau khi co can dung m6i d nhift do < 40°C, can chilt thu dugc dugc lgc nhanh qua cot MgO/Celite (1/2) de loai bdt chlorophyll rli dua Ifn cgt sic ky silica gel (hf nia giai la hexan/axeton gradien ndng dg). Phan doan mau vang dam co Rf = 0,23 (hexan/axeton: 3/1) dugc tach rieng, co can va tinh chi fren cgt silicagel vdi hf dung moi rira giai la hexan/axeton (4/1-2/1). Phan doan co chiia fucoxanthin dugc thu hii khi ty If dung moi giai hap la 2/1. Sau khi cd can dung mdi dudi ap suit giam d nhift do < 40°C, fiicoxantbin thu dugc dugc ket tinh l^i bang each boa tan frong mgt lugng toi thieu axeton, them hexan vao cho den khi dung djch van dye va lam lanh dung djch mgt each tir tir nhift do phong xu6ng den -20°C. Dnc: 167-I68°C.
UV-VIS (hexan) A ^ , 264, 340, 450 nm.
IR (KBr) v ^ , 3440 (OH), 1929 (allene), 1734 (OAc), 1658 (conj. CO) va 1608 (C=C) cm"'.
APCI-MS m/z 659,9 [M-^H]^
'H-NMR (500 MHz, CDCI3) ^0,95 (3H, s, H-16); 1,03 (3H, s, H-17); 1,06 (3H, s, 17');
1,21 (3H, s, H-18); 1,34-(3H, s, H-18'); 1,34 (IH, m, H-2 ax); 1,38 (3H, s, H-16'); 1,42 (IH, m, H-2' ax); 1,49 (IH, m, H-2 eq); 1,51 (IH, t, J = 12 Hz, H-4' ax); 1,78 (IH, dd, H-4 ax); 1,80 (3H, s, H-19'); 1,93 (3H, s, H-19); 1,98 (6H, s, H-20, 20'); 1,99 (IH, m, H-2' eq); 2,03 (3H, s, H-22'); 2,26-2,33 (2H, m, H-4 & 4' eq); 2,60 &v 3,65 (2xlH, d, J = 18 Hz, H-7); 3,80 (IH, m, H-3); 5,37 (IH, m, H-3'); 6,04 (IH, s, H-8'); 6,11 (IH, d, J = 11 Hz, H-10'); 6,26 (IH, d, J = 11,5 Hz, H-14'); 6,34 (IH, d, J = 15 Hz, H-12'); 6,40 (IH, d, J = 11,5 Hz, H-14); 6,57 (IH, dd, J
= 11; 15Hz, H-11); 6,60 (IH, dd, J = 11 Hz, H-11'); 6,63 (IH, dd, J = 14; 11,5 Hz, H-15); 6,67 (IH, d, J = 15 Hz, H-12); 6,74 (IH, dd, J = 14; 11,5 Hz, H-15'); 7,14 (IH, d, J = 11 Hz, H-10).
"C-NMR (125 MHz, CDCI3) ^11,80 (CH3, C-19); 12,73 (CH3, C-20); 12,88 (CH3, C-20');
13,98 (CH3, C-19'); 21,13 (CH3, C-18); 21,37 (CH3, C-22'); 25,01 (CH3, C-16); 28,11 (CH3, C- 17); 29,18 (CH3, C-16'); 31,23 (CH3, C-18'); 32,06 (CH3, C-I7'); 35,14 (C, C-I); 35,75 (C, C- 1'); 40,80 (CH2, C-7); 41,65 (CH2, CA); 45,26 (CHj, C-4'); 45,44 (CHj, C-2'); 47,09 (CH2, C- 2); 64,25 (CH, C-3); 66,16 (C, C-5); 67,11 (C, C-6); 68,05 (CH, C3'); 72,62 (C, C-5'); 103,35 (CH, C-8'); 117,51 (C, C-6'); 123,36 (CH, C-Il); 125,67 (CH, C - I I ' ) ; 128,50 (CH, C-10');
129,40 (CH, C-15); 132,14 (CH, C-14'); 132,48 (CH, C-15'); 132,52 (C, C-9'); 134,52 (C, C-9);
135,39 (C, C-13); 136,61 (CH, C-14); 137,07 (CH, C-12'); 138,04 (C, C-I3'); 139,07 (CH, C- 10); 145,01 (CH,G-12); 170,43 (C=0, C-2I'); 197,86 (C=0, C-8); 202,35 (=C= C-7').
HI. KET QUA VA THAO LUAN - 1. Chiet tach va tinh che fucoxanthin
Nam 2008, chiing toi nhan dugchai miu rong nau thu hoach d viing biln Khanh Hda, mgt mau CO dang la hep hirih kim, mgt miu co dang la rgng. ^k phat hifn sy cd mat cua carotenoit, cac mau dugc chilt xuit thii vdi cac dung mdi khac nhau, djch chilt dugc kilm fra bing sic ky ban mong, cac carotenoit dugc nhan biet tren ban mong bdi miu sic ty nhien, sy hifn miu dudi
^^". ^ ^^ ^^ ^'^" '^.^" ^^"8 thulc thii ceri sunphat. Kft qua cho thiy, chi khi sir dung dung moi chiet la DMSO kit hgp vdi axeton chiing toi mdi phat hifn thiy sy cd mat cua mgt lugng dang ke carotenoit trong miu rong nau la rgng, thanh phin chu ylu la fiicoxantbin. Lugng carotenoit phat hifn thay frong miu la hep, hinh kim thip ban nhilu. Vi thf, miu rong nau la rgng dugc sir dyng lam nguyen lifu dl chift tach fucoxanthin.
Qua frinh chiet mau rong nau nhu dugc md ta trong phin tiiyc nghifm cho djch chilt carotenoit tho. Tiep theo, djch chilt dugc c6 lai roi phan b l trong n-hexan/nudc, cac carotenoit tap frung trong pha hexan. Sau khi cd can dung mdi d nhift do < 40°C, can chilt thu dugc dugc
Tieu ban: HOAT C H A T SINH HOC Bi^N 647 lgc nhanh qua cgt MgO/Celite (1/2) de loai bdt chlorophyll r6i dua len cgt sac ky silica gel (hf rira giai la hexanyaxeton, gradient nong do). Phan doan mau vang dam co Rf = 0,23 (hexan/axeton 3:1) dugc tach rieng, co can va tinh che tren cgt silicagel vdi he dung moi nia giai la hexan/axeton (che do gradient 4/1 - 2/1). Phan doan co chiia fucoxanthin dugc thu hoi khi ty If dung m6i giai hap la 2/1. Fucoxanthin thd dugc ket tinh lai trong hexan/axeton, ket qua thu dugc tinh the hinh kim, mau vang, dnc. 167-168°C.
2. Nhan dang fucoxanthin bang cac phuomg phap pho
Fucoxanthin la mgt hgp chat carotenoit, co c6ng thiic phan tii la C42H58O6, ten hf th6ng la (3S,3'S,5R,5'R,6S,6'R)-3'-(acetyloxy)-6',7'-didehydro-5,6-epoxy-5,5',6,6',7,8-hexahydro-3,5'- dibydroxy-8-oxo-P,P-carotene. Fucoxanthin cd cimg khung cau tnic vdi cac carotenoit P- caroten, lutein, zeaxanthin, astaxanthin, ..., dac frung bdi hai vong 1,1,5-trimethylcyclohexan gan d hai dau cua mgt mach hd g6m 4 don vj isopren noi lien tiep vdi nhau theo kieu duoi - dau - dudi - du6i - dau - du6i. Ngoai hf polyen lien hgp ciia mach hd, fucoxanthin co cac yfu to cau tnic dac trung la lien ket epoxy giira C-5 va C-6, lien kft alien giira C-7', C-6' va C-8'. Cac yeu t6 cau tnic khac la: hai nhom OH d C-3 va C-5', mgt nhom C=0 d C-8, va mgt nhom acetoxy d C-3'. Fucoxanthin cd 5 cacbon bat ddi d C-3, 3', -5, -5', -6, va mgt trung tam chiral d C-6', -7', -8' xung quanh lien ket alien. Hgp chat lutein cd nhieu yeu td cau tnic phii hgp vdi fucoxanthin, co the diing de so sanh khi giai cau tnic.
18' _ 2 1 ' 2 ?
H 0 / , , / ^ > » 0 C 0 C H 3
Astaxanthin
D I nhan dang fucoxanthin thu dugc, chiing t6i ghi cac ph6 UV, MS, ID va 2D NMR. Ket qua ghi pho dugc trinh bay fren cac hinh: Hinh 1 (ph6 UV), Hinh 2 (ph6 EI-MS), Hinh 3 (phi 'H NMR), Hinh 4 (phi '^C NMR), Hinh 5 (phi COSY), Hinh 6 (phi HMBC).
648 Tuyen tjp HOi nghj KHOA HQC TOAN QU6C Vfe SINH HQC BI^N vA P H A T T R I ^ N B £ N VCTNG
Hinh 1. Pho UV cua m l u fucoxanthin thu Hinh 2. Ph6 MS ciia m l u fucoxanthin thu
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1 1 1 . . . . 6 5 4
Jwli ll
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Hinh 3. Pho 1H NMR (500 MHz, CDCI3) cua m l u fucoxanthin thu du-^c.
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Hinh 4. Pho 13C NMR (125 MHz, CDC!3) cua m l u fucoxanthin thu du-^c.
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Hinh 5. Phd COSY cua m l u fucoxanthin thu du-gc.
650 Tuyen tap HQi nghi KHOA HQC T O A N Q U O C V £ SINH HQC B | £ N V A P H A T TRifeN B £ N VCfNG
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Hinh 6. Pho HMBC cua mlu fucoxanthin thu du'g'C.
Kit qua gan phi NMR dugc trinh bay fren bang 1. Cac dii' kifn ph6 thu dugc phii hgp vdi cac dir kien da dugc c6ng bo [4, 5, 6, 7, 8].
c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Bang
C CH2
CH CH2
C C CH2 C = 0
c
CH CH CH C CH CH CH3 CH3 CH3 CH3 CH3
1. S d l i f u pho 1H N M R (500 M H z ) VE 5H ( J = H Z )
1,34m (ax) &1,49m (eq) 3,80m
1,78 dd (ax) & 2,33m (eq)
2,60 & 3,65 d (18)
7,14 d(11) 6 , 5 7 d d ( 1 1 ; 1 5 )
6,67(15) 6,40 d (11,5) 6,63(11,5:14)
0,95 s 1,03 s 1,21 s 1,93 s 1,98 s
6c 35,14 47,09 64,25 41,65 66,16 67,11 40,80 197,86 134,52 139,07 123,36 145,01 135,39 136,61 129,40 25,01 28,11 21,13 11,80 12,73
1 1 3 C N M R c u a F u c o x a n t h i n t r o n g C D C I 3 C
r
2' 3' 4' 5' 6' T 8' 9' 10' 11' 12' 13' 14' 15' 16' 17' 18' 19' 20' 2 1 ' 22'
C CH2
CH CH2
C C C CH
C CH CH CH C CH CH CH3 CH3 CH3 CH3 CH3 C = 0 CH3
8 H ( J = H Z )
1,42m (ax) & 1,99m (eq) 5,37m
1,51 t ( 1 2 , a x ) & 2 , 3 3 m ( e q )
6,11 d ( 1 1 ) 6,60 dd (11) 6,34 (15) 6,26 d (11,5) 6,74(11,5:14)
1,38 s 1,06 s 1,34 s 1,80 s 1,98 s 2,03 s
8c 35,75 45,44 68,05 45,26 72,62 117,51 202,35 103,35 132,52 128.50 125,67 137,07 138,04 132,14 132,48 29,18 32,06 31,23 13,98 12,88 170,3 21,37
Tieu ban: HOAT C H A T SINH HQC BifeN 6 5 1
Tu lifu khoa bgc cho bilt fucoxanthin la chat mau di kem frong the luc lap ciia rong nau va hau het cac loai tao heterokont, lam cho chung co mau nau va xanh la cay tham. Fucoxanthin la mgt hgp chit thien nhien co tilm nang img dyng vi co nhieu boat tinh dang chii y. Gi6ng nhu mgt s6 carotenoit khac, fucoxanthin co boat tinh chong oxi boa [9]. Doi vdi bfnh ung thu, mgt so nghien curu cho thiy fucoxanthin xuc tien cd hifu qua qua trinh apoptosis frong cac tf bao bach cau va ung thu rugt ket [10, 11], ire chf manh qua trinh tang trudng cua cac te bao ung thu tuyen tien lift [12]. Cac nghien cuu ve sy chuyen boa va tinh chat dinh dudng fren chugt ciia trudng DH Hokkaido cho thay fucoxanthin xiic tien qua trinh "dot chay" chat beo frong cac te bao md ciia md adipose nhd ca che tang cudng bieu hifn cua enzym thermogenin thyc hifn qua trinh sinh nhift [2]. Hoat tinh nay lien quan chat che vdi sy co mat cua nhom alien trong cau triic cua fiicoxantbin. Mgt nghien ciru gan day tren chugt cho thay, thiic an cd b6 sung 0,1 va 0,2%
fucoxanthin lam cho glucose frong mau va lugng m6 md trang (WAT) khoang byng giam ro rft [13]. Chia kbda cho cac boat tinh nay cua fucoxanthin la kha nang xiic tien UCPl, dieu hda PPARy va P3-AR frong WAT, ciing nhu tiet che cac adipokine nhu TNF-a [9].
Trong mgt so cdng trinh nghien ciiu trudc day, chiing t6i da nghien ciiu astaxanthin va mgt sd carotenoit khac trong bai san ciia Viet Nam [14, 15]. Vdi vifc phan lap dugc fiicoxantbin tir rong nau, chiing toi hy vgng ddng gop them dugc mgt phan cho vifc nghien cuu khai thac ngu6n hgp chat thien nhien co gia frj nay. Chiing tdi cung mong mu6n tiep tyc vifc nghien curu df tim ra fucoxanthin vdi ham lugng cao trong cac loai rong nau va rong bifn khac ciia Viet Nam, ciing nhu xay dyng dugc cac phucmg phap phan tich chinh xac va tifn lgi de djnh lugng chiing trong nguyen lifu.
Ldi cam on: Cac tac gia chan thanh cam an TS Biii Minh Ly va cac cgng sy cua Vifn Nghien cuu va U'ng dyng Cdng nghf Nha Trang da thu thap va cung cap cac mau rong nau Khanh Hoa. Cdng trinh nay dugc thyc hien vdi sy hd trg tai chinh cua Hgi dong khoa hgc ty nhien.
IV. TAI LIEU THAM KHAO
1. Trin Dinh Toai, Chau Van Minh, Rong biln dugc lifu Vift Nam, Nxb Khoa hgc Ky thuat 2005.
2. Maeda H, Hosokawa M, Sashima T, Funayama K, Miyashita K, Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCPl expression in white adipose tissues, Biochem. Biophys. Res. Commun., 2005, 332(2), 392-7.
3. Maeda H, Tsukui T, Sashima T, Hosokawa M, Miyashita K, Seaweed carotenoid, fucoxanthin, as a multi-functional nutrient, Asia PacJ Clin Nutr, 2008, 17 Suppl 1,196-9.
4. J. A. Haugan, T. Aakermann, S. Liaaen-Jensen, Isolahon of Fucoxanthin and Peridinin, Methods Enzymol., 213, 231-245 (1992).
5 X. Yan, Y. Chuda, M. Suzuki, T. Nagata, Fucoxanthin as the major antioxidant in Hijikia fiisiformis, a common edible seaweed, Biosci. Biotechnol. Biochem., 63 (3), 605-607 (1999).
6. J. A. Haugan, S. Liaaen-Jensen, Improved isolation procedure for fiicoxantbin, Phytochemistry, 28 (10), 2797-2798 (1989).
7. G. Englert, T. Bjorland, S. Liaaen-Jensen - ID and 2D NMR study of some allenic carotenoids of the fucoxanthin series, Magne. Res. Chem., 28, 519-528 (1990).
8. Y.Yamano, C. Tode, M. Ito - Carotenoids and related polyenes. Part 3. First total synthesis of fucoxanthin and halocynthiaxanthin using oxo-metallic catalyst, J. Chem. Soc, Perkin Trans. I, 1895-1904(1995).
6 5 2 Tuyen t i p HQi nghj KHOA HQC T O A N Q U 6 C V £ SINH HQC B I £ N V A P H A T T R I ^ N B £ N VCTNG
9. Y. Mine, (Editor), K. Miyashita (Editor), F. Shahidi (Editor), Nufrigenomics and Proteomics in Health and Disease: Food Factors and Gene hiteractions, Wiley-Blackwell 2009, pp. 145 -
160.
10. Kotake-Nara E, Terasaki M, Nagao A., Characterization of apoptosis induced by fucoxanthin in human promyelocytic leukemia cells, Biosci Biotechnol Biochem., 2005, 69(1), 224-7.
11. Das SK, Hashimoto T, Shimizu K, Yoshida T, Sakai T, Sowa Y, Komoto A, Kanazawa K., Fucoxanthin induces cell cycle arrest at GO/Gl phase in human colon carcinoma cells through up-regulation ofp21WAFl/Cipl, Biochim Biophys Acta., 2005, 1726(3), 328-35.
12. Kotake-Nara E, Kushiro M, Zhang H, Sugawara T, Miyashita K, Nagao A., Carotenoids affect prohferation of human prostate cancer cells, 7. Nutr., 2001, 131(12), 3303-6.
13. Maeda H, Hosokawa M, Sashima T, Miyashita K, Dietary combination of fucoxanthin and fish oil attenuates the weight gain of white adipose tissue and decreases blood glucose in obese/diabefrc KK-Ay mice, J Agric Food Chem., 2007, 55(19), 7701-6.
14. Hoang Thj Huf An, Trin Thj Thu Thuy, Nguyfn Quylt Chiln - Phan lap va tinh che Astaxanthin tir vo t6m. Tap chi Hod hpc, 45(6A), 226-230 (2007).
15. Le Lan Anh, Nguyen-Quyet Chiln, Hoang Thj Huf An - Djnh lugng carotenoit frong mgt so loai giap xac thuy san Vift Nam bing sac ky Idng hifu nang cao. Tap chi Hod hpc, 45(6A), 269-274 (2007).
SEGREGATING AND REFINING F U C O X A N T H I N - A V A L U E A B L E CAROTENOIT FROM P H A E O P H Y T E IN K H A N H H O A P R O V I N C E
Tran Thi Thu Thuy, Nguyen Thi Minh Hang, Tran Quoc Toan, Nguyen Quyet Chien SUMMARY
From a species of brown seaweed collected in the sea of province Khanh-Hoa of Vietnam fiicoxantbin has been isolated, purified and characterized by UV, MS, ID and 2D NMR specfroscopy.
Key words: fucoxanthin, brownseaweed, Khanh-Hoa Vietnam.
