Ba dgng sinh hgc vd cdc chdt cd hogt tinh sinh hgc
STICHLOROSIDE Ai VA STICHLOROSIDE A2, HAI TRITECPEN SAPONIN PHAN LAP T O L O A I H A I SAM STICHOPHUS CHLORONOTUS Ll Th{ Vlln', Nguyin Phinmg Thio', Tr^n Thj H^ng H^nh', Phan Thj Thanh Huong',
Nguyin ^ ^ n ThanhS Nguyin Xufin Ciriyng'*, Nguyin Ho&i NamS
&S C6ng Thung', Young Ho Kjm^ ChSu VSn Minh' ' Vign Hoa sinh biin. Vign Hdn lam Khoa hpc vd Cdng nghg Vigt Nam 'Vi^ Tdi nguyen vd Moi tru&ng bien, Vign Hdn lam Khoa hgc vd Cong nghg Vigt Nam
^College of Pharmacy, Chungnam National University. Korea
Using various chromatographic methods, two triterpene saponins stichloioside Ai (1) and stichloroside A2 (2), were isolated from the methanol extract of the sea cucumber Stichopus chloronotus. Their structures were elucidated by ID and 2D-NMR experiments and comparison of their NMR data wth reported values.
Keywords. Stichophus chloronotus, Sticbopodidae, sea cucumber, triterpene saponin.
1. M 6 DAU
Ckc loii h ^ sam thuQC hp Sticbopodidae (nginh Echinodermata, lop Holothurioidea, b$
Aspidochirotida) thudng dugc si^ dyng nhu cac cac mdn 9n b6 duSng vi thu3c b6 trong y hQc ch truyen.
Trong s6 c&c loii hii sSm tbu$c hp niy, loii Stichopus chloronotus Brandt du^c tim thay d cac vCmg diy vi biln siu thu^c Thii Bhih Duong, An Dp Duong vi D^i TSy Duong [I]. Cic hpp chat tritecpen saponin li tl^nh p h ^ hda hpc chinh d3 dupe cdng t>6 ti^ loii niy [2-4].
Trong khudn khd cic nghign ctru cua nhom tic gia \k cic loii hii sam 6 Vi^t Nam, chiSng toi d i cdng b6 chi tilt qui trinh phin l$p vi xic dinh cau true ci^ hpp chat saponin m6i stichloroside F tCt loii hai sim S. chloronotus [5]. Cong trinh niy mo ti chi tiet qui trinh xac djnh cau tnic eiia hai hpp chit tritecpen saponin li stichloroside Ai (1) v i stichloroside A2 (2) tit loii hii sim niy.
2 . T H U " C N G H I $ M
2.1. Thilt bjvihdach^t
Ph6 ' H NMR (500 MHz) vi '^C NMR (125 Hz) dupe do tran miy Bruker AM500 FT-NMR vdi TMS dupe sijt dvuig lim chat chuin nOi. Ph6 khoi lupng phun mil di?n tit ^ I - M S ) dupe do tr@n miy Agilent 1260 series smgle quadrupole LC/MS. S4c kj I6ng trung ip (MPLC) dupe tiln hinh tren miy Biotage - Isolera One system (SE-751 03 Uppsala, Thyy Diin). S5c k^ cpt (CC) su dymg silica gel
CKieselgel 60, 70-230 mesh and 230-400 mesh, Merck) vi YMC RP-18 (30-50 nm. Fuji Silysia Chemical Ltd.). Sic ky l i ^ mdng (TLC) sii dvmg bin tiing sin sihca gel 60 Fzs4 (1.05554.0001, Merck) v i RP-18 F254S plates (1.15685.0001, Merck). V ^ chdt dupe phit hifn bSng cich phun deu tiiudc thu axft H2SO4 10% v i ho n6ng tCc tit den khi hi$n miu.
2.2. MSU hii sfim
Mau loii hai sim S. chloronotus Brandt dupe thu t9i khu vvrc Cit Bi, Hii Phdng, vio thing U nim 2011 v i dupe PGS. TS. ©3 Cdng Thung giim djnh.
MIU ti6u bin dupe luu t9i Vifn Hda sinh bien vi Vifn Tii nguyfn vi Mdi trudng biln.
2.3. Phan l^p cic hpp ch^t
M§u hii sSm Stichojms chloronotus hroi (6,0 k^
dupe cat nhd v i chiet bing MeOH ndng (3 lan. moi lln 6h) tiiu dupe c&n MeOH (10,45 g. A) sau khi lo^i bd dung mdi dudi dp suit giim. C$n chiet niy dupe hda vio 1^0 vi chilt phfin b l vdi n-butanol (3 l ^ mSi lan 0,7 L). H2O-CH3OH (35-65%) thu dupe 2 phan do^n nhd hon k^ hifu Fr. B3.1^3.3.
PhSn do?n ohd'B3.2 (0,27 g) dupe phfin tich ti^p bing sic k? rfiy phin tii Sq)hadex LH-20 (pha dOng CH3OH-H20,4,5/1) v i tin chl tilp bSng sic k^ cf J sihca gel pha thudng sti dyng hf dung mdi nia giii^
diclometan-CHsOH-IfcO (1,8/1/0,2) tiiu dupe hppj chat 2 (5,6 mg). Phan do?n nhd B3.1 (0,18 g) dupe phan tich tilp bing sic i ^ cOt sihca gel pha thudng |
H^i ihdo khoa hgc ky nigm 40 n&m thdnh lap Vi^n Hdn lam KH&CN Vigt Nam
- " ' ^ * - V ^ HC Glc I °^ ^ Xyl n " "
Hinh I: Clu tnic bda hpc cia cic hpp chit I vi 2
5flng 1: s l Ufu phd 'H-NMR vi '^C-NMR phin alycon cua cic hpp chit 1. 2 vi cic chit tham khio
c
I 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 28 29 30 CO Me
•8c 35,9 27,0 88,8 47,8 22,5 119,4 146,4 47,1 35,2 22,8 30,4 58J 51,0 33,9 24,6 54,3 180,0 23,9 83,3 26,7 44,0 68,1 44,9 24,3 22,3 23,2 16,9 28,4 30,9 170,8 21,4
8c''- 3633 27,20 88,99 39,53 48,10 23,34 120,03 146,61 47,40 35,60 22,93 30,30 58,47 51,26 34,29 24,84 54,20 179,79 24,07 83,19 26,99 44,10 68,31 45,36 24,56 22,18 23,21 16,99 28,49 30,96 170,75 21,35
1 SJ^'(J=H2) l,40ni/l,45m l,90m/2,08m 3,25 m
-
0,97 m 2,00 m 5,64 brs-
3,42 m-
l,45m/2,00m 1,85 m/1,92 m-
1,61 m/1,75 m 1,88 m/2,05 m 2,30 m-
1,19 s-
1,48 s 1,86 m/2,15 m 5,41m 1,25 m/1,55 m 1,55 m 0,93 d (6,5) 0,87(1(6,5) 1,06 s 1,23 s 1,06 s-
2,13 s'"c 36,22 27,14 89,16 39,46 47,91 23,21 119,89 146,45 47,27 35,45 22,78 30,12 58,33 51,11 33,73 24,59 53,93 179,68 23,94 83,02 26,84 43,09 67,75 44,48 141,54 114,16 22,17 17,21 28,59 30,79 170,38 21,04
Sc''- 36,38 27,26 89,04 39,60 48,17 23,40 120,11 146,65 47,47 35,66 23,00 30,34 58,55 51,33 34,34 24,83 54,16 179,84 24,13 83,87 27,07 43,32 67,98 44,71 141,75 114,37 22,39 17,04 28,54 30,98 170,56 21,25
2 8H''''ty=Hz) l,42m/l,47m l,92m/2,12m 3,27 m
-
1,00 m 2,03 m 5,67 brs-
3,45 m-
l,48m/l,74m l,87m/l,95m-
l,63m/l,77m 1,90 m/2,06 m 2,31m-
1,21s-
1,50 s l,96nl/2,29m 5,48 m 2,25m/2,38m-
4,84s/4,86s 1,80 s 1,08 s 1,25 s 1,08 s-
2,11s6c phin alycon ciia stichoposide E do trong pyridme-rfi [6], ""do tiong pyridine-rfs, "125 MHz, ''SOO MHz, "5c ihin aglycon ciia synallactoside Aj do trong pyridme-</s [7].
&a dang sinh hpcvi cdc chat co hogt tinh sinh hQC
Bang 2: S6 lieu pho 'H-NMR vi "C-NMR p h ^ chuoi duong ciia cac hgp chSt 1,2 va stichoposide E r ^8^^ 1 2
id-' S B ' ^ V ^ - H Z ) 6 C ^ ' 8 H ' ' ' ( / = H Z ) ~ ~
Wl ' —
r 104,6 105,11 4,70 d (7,0) 2' 82,0 83,65 3,95- 3' 74,7 76,00 3,95- 4' 76,6 77,49 4,20' 5' 63,1 64,04 3,58/4,35' Xyin
1" 103,9 106,65 5,05 d (7,0) 2" 74,6 75,95 3,97' 3" 74,6 75,70 4,15' 4" 76,6 78,68 4,20' 5" 63,6 64,04 3,58/4,35- GicI
V" 101,6 103,34 4,97 d (7,0) 2'" 72,5 73,28 3,96' 3'" 86,0 88,00 4,15' 4'" 68,8 69,78 4,00' 5'" 77,0 78,18 3,88' 6'" 61,3 62,08 4,17/4,40' MeGlcl
V" 103,9 105,60 5,21 d (7,0) 2"" 73,5 75,10 3,94' 3"" 85,9 87,87 3,651 (9,0) 4"" 69,7 70,51 4,15' 5"" 77,0 78,24 3,88' 6"" 61,3 62,18 4,20/4,40' 3""-0Me 60,0 60,73 3,81 s
aicH
I'"" 101,3 102,84 4,95 d (7,0) 2 72,5 73,11 3,96' 3""' 86,8 87,87 4,15' 4"'" 68,8 69,68 4,00' 5'"" 77,0 78,10 3,88' 6'"" 61,3 62,08 4,17/4,40' MeGlcn
1 103,9 105,60 5,19 d (7,0) 2""" 73,5 75,10 3,94' 3 85,9 87,87 3,651 (9,0) 4""" 69,7 70,51 4,15' 5""" 77,0 78,24 3,88' i""" 61.3 62,18 4,20/4,40' 3"""-0Me 60,0 60.73 3,81 s
105,19 83,23 76,11 77,56 64,11 106,77 76,06 75,78 78,70 64,89 103,46 73,36 88,08 69,86 7832 62,13 105,72 75,17 88,00 70,58 78,36 62,24 60,78 102,95 73,19 88,08 69,75 78,23 62,13 105,72 75,17 88,00 70,58 7836 62,24
< 60,78
4,74 d (7,0) 3,98' '4,00' 4,23' 3,62/3,95' 5,08 d (7,5) 4,00' 4,14' 4,25' 3,62/4,42' 5,01 d (7,5) 4,00' 4,19' 4,04' 3,90' 4,20/4,43' 5,25 d (7,0) 3,99' 3,691(9,0) 4,13' 4,25' 4,23/4,43 3,84 s 4,97 d (7,5) 3,98' 4,19' 4,04' 3,90' 4,20/4,43' 5,23 d (7,0) 4,00' 3,691(9,0) 4,13' 2,25' 4J3/4,43' 3,84 s '5c phSa chuoi dutag dia stichoposide E do trong DMSO-rf( [6], 'do trong pyridine-*, '125 MHz, '^00 MHz, tin h i ^ bj cheng lap.
CSn tan Iron^ butanol (2,42 g, B) dugc tifc htoh 1/1, v/v.) thu duffc 3 phan do?n (B1-B3). Phto dojn phin tich trtn sic k^ cOt silica gel pha thuimg rira B3 (0,45 g) duffc phin tich tify bJng MPLC sil giii gradient dichloiometiiane-Tmethanol (10/1, 3.1, dyng c$t nhoi silica gel pha dio, riia giii gradient
Hfii thio khoa hpc iy nifm 40 nim thanh lap Vien Hin lim KH&CN Vift Nam
rta giii bing diclometao-CHjOH-HiO (2,5/1/0,15) vi tinh chS Ihtal bing sic k j cSt silica gel pha dio nia giii bing CHBOH-HIO (3,5/1, v/v) thu duijc h(ip chat 1 (4,8 mg).
Stichloroside Ai (1): Chit bOt miu tring; [ajo -44 (c 0,16, MeOH); 'H-NMR (500 MHz, Pyridme- di) vi '=C-NMR (125 MHz, Pyridine-*), xem bing 1 vi 2; ESI-MS: m/z 1477 [M+Na]* (CMHUOOSB, M
= 1454).
Stichloroside A2 (2); Chit bgt mau t ^ g ; [ajo -37 (c 0,16, MeOH); 'H-NMR (500 MHz, Pyridine- rfj) v i "C-NMR (125 MHz, Pyridine-dj), xem bing 1; ESI-MS: m/z 1475 [M+Na]* (CsBiosOjj, M = 1452).
3. K£T QUA VA THAO LUAN
Hgp chit 1 diigc phan lap dudi dang chit bOt vo djnh hlnh miu tring. Cic pho NMR ciia 1 d$c trung cho m$t hgp chit tritecpen saponin, m$t ldp chit diin hlnh ciia cic loii hii sim [8]. Ph8 '>C-NMR ctia 1 xuit bi^n 68 tin hi$u cacbon, trong dd c6 30 tm hieu ctia phin khung aglycon, hai tin hi^u cua mgt nhom axetat vi 36 tin higu ciia mgt chuSi siu dem v) dufmg.
Hinh 2: Cia tirong tic COSY (—) vi HMBC (-+) chinh ciia 1
d phin aglycon, xuit hi§n cic tin hi$u ciia mOt Uen k& ddi bi till ba vi tri [5c 120,03 (CH, C-7) vi 146,61 (C, C-8)/6H 5,64 (IH, br s, H-7)], mdt nhdm cacbonyl Y-lactone [Sc 179,79 (C, C-18)], biy nhdm metyl [6c 24,07 (CHj, C-19), 26,99 (CH,, C-21), 22 18 (CH), C-26), 23,21 (CHj, C-27), 16,99 (CHs, C-28) 28,49 (CHs, C-29) vi 30,96 (CH3, C-30)/5H 1 19 (3H, s, H-19), 1,48 (3H, s, H-21), 0,93 (3H, d, /
=' 6 5 Hz, H-26), 87 (3H, d, / = 6,5 Hz, H-27), 1,06 (6H1 S, H-28 vi H-30) vi 1,23 (3H, s, H-29)], hai
nhdm oximetin [6c 88,99 (CH, C-3) va 6831 (CH, C-23)/5H 3,25 (!H, m, H-3) vi 5,41 (IH, m, H-23)].
Theo cic ^ li$u di dugc cong bS, hiu hit cic hgp chit tritecpen saponin ciia cic loii hii sim diu cd ciu tnic 18(20)-lactone v i lien ket ddi t^ C-7/C-8 hojo C-9/C-11 ft phin khung aglycon [9]. Vi?c gin cic s6 li$u phi NMR. dia 1 dugc k t ^ g dinh thim bing phi cgng hudng t£t hat nhin hai chieu COSY va HMBC. Phin tich chi tiit cac tuong tie trSn pho COSY cho phep xic dinh cic minh cau tnic nhu sau: H-1/H-2/H-3, H-5/H-6/H-7, H-9/H-11/H-12, H- 15/H-16/H-17, H-22ffl-23/H-24/H-25/H-26 vi H- 25/H-27. Trin co sft kit qui phi COSY, cCing vdi tirong tic xa HMBC n h ^ dugc giSa H-12 (5H 1,85 vi 1,92)/H-17 (5H 2,30) vi C-18 (8c 179,79), H-19 (5H 1,19) vi C-l (5c 36,33)/C-5 (5c 48,10)/C-9 (5c 47,40), H-21 (5H 1,48) vi C-17 (5c 54,20)/C-20 (5c 83,19)/C-22 (5c 44,10), H-28 (5H 1,06)/H-29 {5H 1,23) vi C-3 (5c 88,99)/C-4 (5C 39,53)/C-5 (5C 48,10) vi giaa H-30 (8H 1,06) vi C-8 (5c I46,61)/C- 13 (6c 58,47)/C-14 (5C 51,26)/C-15 (6c 34,29) cho phip xic dinh chlnh xic ciu trie hda hgc ciia phin khung tritecpen. Proton oximetin H-23 b] dich chuyen rit m^nh vi phia viing trudng tip t^i 5H 5,41 cho phip xic dinh vi tri liSn kit thudng gip cua nhdm axetat tfi C-23 d cic hgp chat saponin tii hii sim. Nh|n dinh niy dugc khing djrih bdi sv phji hgp hoto toin vi so li^u pho '^C-NMR phin aglycon cua 1 (btog 1) vdi cac si li^u tuong ^ g cua stichoposide E [6].
Ngoii ra, tr€n cic pho NMR cia 1 xuat bien cic tin hi|u cua 06 don vi dudng vdi cic tin h i ^ cacbon anome tai 5c 105,11 (C-l'). 106,65 (C-I"), 103,34 (C-l'"), 105,60 (C-l""), 102,84 (C-l'"") vi 105,60 (C-l""") cd tuong tic HSQC vdi cac proton anome hrong ling t?i 6K 4,70 (IH, d, 7 = 7,0 Hz, H-l'), 5,05 (IH, d, y = 7,0 Hz, H-l"), 4,97 (IH, d, J= Ifi Hz, H-r"), 531 (IH, d, y = 7,0 Hz, H-l'"'), 4,95 (IH, d, J" 7,0 Hz, H-l'"") vi 5,19 (IH, d, / = 7,0 Hz, H- 1'""'). So sinh s6 lieu phi '^C-NMR ciia chuoi dudng (bing 2) vdi cic so 11^ di dugc cdng b i [6, 10] kit hgp vdi kit qui phin tich cic phd cdng hudng tir hat nhan hai chiiu HSQC, HMBC vi COSY (Unh 2) cho phip xic dinh chu5i dudng li 3- a-{3-0-methyl-^-D-glucopyranosyl-(l-»3)-^-D- gIucopyraqosyl-(l-»4)-^-D-xylopyranosyl-(l->2)- [3-0-methyl-^-D-gIucopyranosyl-(l~+3)-^-D- glucopyranosyI-(l->4)]-yff-D-xylopyranoside}.
Tuong tic HMBC nhin dugc giiia proton anome H- r (5H 4,70) vi C-3 (5c 88,99) cho phip xic dinh vi tri h6n kit cia chuSi siu don vi dudng tai C-3 cia aglycoiL Nhir v|y, hgp chat 1 dugc xic dinh li
Ba dgng sinh hpc vd cdc chdt cd hogt tinh sinh hpc stichloroside Ai [3, 4] hay con dupe gpi li stichoposide E [6\.
Cic phd NMR cua hpp chit 2 hoin toin tuong t^ nhu cic ph§ ciia 1 cho p h ^ xac dinh hai hpp chat cd clu trte hda hpc tiiong tv nhau. Trgn phd '^C- NMR cua hpp chit 2 cung xuat hifn 68 thi hifu cacbon tiong dd cd 30 tin hifu cik phin khung tritecpen, 02 tin hifu ctia m$t nhdm axetat vi 36 tin hifu cik mOt chuoi siu don vi dudng. So Ufu ph6 NMR cik phin chuSi dudng d hpp chit 2 tri^g khdp vdi cic s6 Ufu tuong ung cik 1 cho pbSp khing dinh hai hpp chit cd cung clu tn^c chuoi dudng. Sv khic bift de nh^n thiy nhat li s\f xuit hifn cic tin hifu cua mft USn kit ddi hi thl hai ^i tri [5c 141,75 (C-25) v4 8c 114,37 (CH2, C-26y5H 4,84, 4,86 (mSi thl hifu IH, s, H-26)] ti^ cac phd cik hpp chit 2 thay cho mft nhdm metyl vi mft nhdm metin tren cic phd cik hpp chit 1. So sinh chi tilt so lifu pb6 "C-NMR phin aglycon cua 2 (bing 1) vdi cic s6 Ufu tuong -itag ciia hpp chit synaUactoside Ai [7] cho p h ^ dyi doan vi bi cik hgn kit ddi t?i C-25/C-26. D\r doin niy dupe IdiSng djnh th&n bing cic tuong tic COSY vi HMBC.
Nhu v$y, hpp chit 2 dupe xic rfjnh l i stichloroside A2[3,4].
Hai hpp chit 1 vi 2 dS dupe cic nhi khoa hpc Nhft Bin cdng b6 tit loii hai sim Stichopus chloronotus tii nim 1981 [3, 4], dilu niy mpt lln nOa khSng djnh tinh chlnh xic cik vife djnh danh loii hii s ^ dupe nghifn cdu trong cdng tnnh niy.
Ldi cim on. Cdng trinh ndy du^c hodn thdnh v&i sv tdi tr^ kinh phi cda Fj#n Hdn lam khoa hgc vd C6ng nghg Vi^t Nam. di tdi md sd: VAST.JB.BA3.03/13- 15.
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Liinhg: Nguyen X u ^ Cudng
Vifn Hda smh biln, Vifn Hin lam Khoa hpc vi Cdng nghf ViftNam 18 Hoing QuIc Vift, Clu Giiy. Hi Nfi, Vift Nam
E-mail; [email protected].
