N g h i e n cmi Y hpc Y Hpc TP. Hb Chi M i n h * Tap 20 * So 6 * 2016
PHAN LAP, XAC DINH CAU TRUC CAC APORFHINE ALKALOID Tl/ CAY TO XANH {CASSYTHA HUFORMIS L.)
Biii The'Vinh*, Bodn Nam Trung*, Tran CdngLudn**
TOM
TATMuc tieu nghien ciiu: Gdp phdn tieu chudn hda dugc lieu to xanh, ch^g nham l&n v&i ta hdng; nghiin cihi ndy Hen hdnh khdo sdt dqc diem vi hgc vd hoa hgc, dqc biit Id nhom aporphinoid dm ta xanh.
Ddi tugng nghien cdu: Cdy ta xanh dugc thu hdi tqi tinh Long An, duac dinh danh bai phdng Tdi nguyen vd Dtegc lieu, Trung t&m S&m vd Dugc liiu Tp. H8 Chi Minh. Phan lap vd xdc dinh c&u true bang ky thudt sac ky vdky thuqt phd'.
Ket qud: Qua sdc ky cot silicagel pha thu&n dd phan lap vd xdc dinh diegc cdu true cua 2 aporphine alkaloid.
Ket luan- Phan lap vd xdc dfnh dugc c&it tnic ciui 2 aporphine alkaloid Id Cassythidine vd Thalicminine.
Titkhoa: Taxanh, aporphine alkaloid, cassythidine, thalicminine ABSTRACT
ISOLATION AND STRUCTURE IDEN'FIFICATION OF APORPHINOID ALKALOID FROM CASSYTHA FRJFORMIS L.
Bui The Vinh, Doan Nam Trung, Tran Cong Luan* Y Hoc TP. Ho Chi Minh * VoL 20 - No 6 - 2016:186 -190 Objectives: This study was made to develope the marker compound contribuHng a standardizaHon of this plant.
Materials and methods: Isolation and structural eluddation based on diromathography and spectrum Results- By using chromathography and spectrum technique, two aporphine alkaloid were isolated and eluadated.
Conclusions: Two novel aporphinoid alkaloid from Cassytha filifrrmis L. named Cassythidine and Thalicminine were isolated by using the repeated column chromatography on total alkaloid residue. Their structure was eluadated based on the NMR - spedroscopic data.
Key words: Cassytha filifrrmis L, cassythidine, thalicminine, aporphinoid alkaloid.
DAT VAN DE nhom hpp chat alkaloid voi eau tao la khung Ta xanh {Cassytha filifrrmis L.) thuoc chi aporphine. Tren the gioi, nhom aporphine
alkaloid ciia ta xanh da diroc nghien ciiu ve hoat Cassytha. hp Long nao (Lauraceae). CM Cassytha ^ ^^ j , ^ ^ ^ ^ ^ ^^ ^^^ ^„^, y ^ ^.^ ,- d, b,et trong hg vi la day leo ban ky sinh co la ^. ^ ^ „ _ ^ ^ ^ ^^ ^ hypanosoma, heu giant ftanh vay. Trong chi gom khoang 20 ^ g ^ g , , . ^g^ ^»^„, (, Vj.^ j^J^ ^^^ ^ loai, phan bo chu yeu a vune nhiet doi, nhat la n . - T.- ' - ^ i,' . -
,- ' . r - , . A , Cl uui, luuii la ^ nghien cuu nao ve nhom aporphme Aushalia CO 15 loai. O Viet Nam, chi nay co 2 loai ^^^.^ ^^ ,„ ^ ^ _ ^ ^ ^ ^ ^ ^ ^ ^ la lo xanh (Cassytha filiformis L.) va lo xanh chi ^~, ^ ^ ^ ^ ^ ^ ^ j ^^, j j ^ ^^ ^^ ^^
Cassytha capillaris Meissn.)M aporphine alkaloid lOr to xanh, gop pKan dinh Thanh phan hoa hoc chinh cua to xanh la
Tning tam Sam v& Dir^ lieu ** D^i hgc T3y Do.
Tac gid lien lac. DS.ahlVni Vinh fir: 0918 0918 64 Email: [email protected]
Y Hpc TP. H o Chi Minh * Tap 20 * So 6 * 2016 Nghien cmi Y hpc
huong cho nhirng nghidn ciiu sau hon ve hoa hpc va hoat tinh sinh hpc tren doi tuong nay.
VATlIEU-PHUtlNGPHAPNGHIENCUtJ Vat lieu nghien ciiu
To xanh dupe thu hai d tinh Long An, mau thu trong thoi ky ra hoa, qua khoang thang 7-8.
Mau dupe dinh danh biM phong Tai nguyen Dupe lieu, Trung tam Sam va Dupe lieu Tp.H6 Chi Minh. Mau thu hai dupe rua sach, cat nho, phoi kho va xay thanh bpt de nghien ciiu.
Phuong phap
Phuong phap ehung: Pho MS ghi tren may HR-MS mierOTOF-Q E 10187. Pho ^H, ^^c NMR dupe ghi tren may Bruker AM500 FT- NMR, do trong MeOH. Sac ky lop m6ng dupe thuc hien tren ban mong trang san silica gel 6OF254 (Merck), he dung moi khai trien CHCb : MeOH : NH4OH dd (9 : 1 : 1 gipt). Silica gel 60 F2S4 (0,063-0,200 mm) (Merck) dupe dimg trong sac ky cot.
Chiet xuat: 4,2 kg bpt to xanh dupe kiem hoa bing dung dich NH4OH 15% den p H 10.
Them CH2CI2 den khi ngap be mat bpt ta xanh. Ngam trong khoang 12 h, thinh thoang CO khuay trpn, Ipc thu dieh chiet. Kiem tra su hien dien ciia alkaloid trong dich chiet bang phuang phap sac ky lop mong, sau do tien hanh co quay giam ap thu cao chiet, dung moi CH2Cb thu hoi dupe sii dung de chiet tiep ba dupe lieu. Quy trinh lap l?i cho den khi dich chiet khong con alkaloid. Cao alkaloid tong thu dupe la 380 g. 100 g cao dupe phan doan qua sac ky cot (SKC) pha finh la diaion HP-20, rua giai vai dumg moi nuoc, MeOH nuoc 20%, 40%, 60%, 80%, 100% thu nhiing phan doan sach han. Phan doan tap trung nhieu alkaloid nhat (MeOH 100%, 67 g) dupe tien hanh nhieu Ian SKC silica gel voi he dung moi CH2Cb : MeOH voi dp phan cue tang dan (100:0; 99:1; 98:2 den 95:5) thu dupe khoang 10 mg chat V2 va 80 mg chat V4 tinh s?ch.
KET QUA VA BAN LUAN Chat V2 (thalicminine)
Chat V2 dupe co lap duoi dang tinh the mau vang cam, tan trong CHCb, MeOH, SKLM bang he dung moi CHCb: MeOH : NHiOH dd (9 : 1 : 0,2) cho vet tat quang 6 UV 254 nm, hi?n hinh bang thuoc thii Dragendorff xuat hien vet tron mau cam dam vai Rf=0.4.
Bi^ gidi kei (fud pho chat V2 (thalicminine) Pho 'H-NMR eho tin hieu cpng huong iing v6i su hien dien ba nhom proton methoxy 5H [3,814 (3H; s; 3-OMe); 4,026 (3H; s; 10-OMe);
4,284 (3H; s; 11-OMe)], mpt proton methylen a vimg truang thap [5H 6,223; (2H; O-CH2-O)], bon proton methine cua nhan thom 5H [8,123 (H-4);
8,855 (H-5); 8,35 (d; J=8,5 Hz; H-8); 7,181 (d; J=8,5 Hz; H-9)].
Cac dii li#u pho 'KI-NMR, DEPT cho thay V2 CO tong so 20 carbon cho tin hieu pho tii 20 ppm d a i 180 ppm. Trong do co 12 carbon b | e IV (C) trong do 1 tin hieu 181,48 la nhom ceton (C-7), 4 carbon bac m (CH), 1 carbon bae H (CH2) va 3 carbon b?c I (CHs). Tat ca 12 carbon bac IV va 4 carbon bac HI deu dich chuyen 6 vung truong thap (6c tli 110 ppm den 180 ppm).
Co 1 carbon bac II [6c 101,75 ppm] dae tnmg ciia carbon methylen dioxid O-CH2-O ciia khung aporphine.
Trong nhom methyl, co 3 nhom co dp dai hoa hpe dac tnmg eiia nhom melhoxy [6c 61,15 ppm; 6H 3,814; 3H; 3-OMe], [6c 56,10 ppm; 6H 4,02; 3H; lO-OMe] va [6c 60,21 ppm; 6H 4,28; 3H;
11-OMe].
Voi cae du lieu pho NMR nhu tren ta co the du doan V2 la mot alkaloid co mot nhom the -O- CH2-O-, ba nhom -OMe, mot nhom C=0 mot nhom -N=. Tham khao cac tai Heu cong bo dac diem pho NMR co the du doan dupe V2 la khung oxoaporphine.
Dua vao pho HMBC, hai proton ciia nhom methylen dioxid (6H 6,22) co tucmg tac voi hai carbon bac IV 6c (137,38 va 149,73; la C-1 va C-2).
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Nghien cihi Y hpc Y Hpc TP. m Chi Minh * T3p 20 * S^ 6 * 2016
Proton methoxy (6H 4,284; 3-OMe) cho tuong quan vai carbon bae IV (6c 136,21), nen carbon nay la C-3, tuang tu hai carbon methoxy con lai 10-OMe va 11-OMe cho tuong quan voi 2 carbon bae IV (6c 145,85 va 6c 157,91) nen 2 carbon nay Ian lupt la C-10 va C-11, trong cau tnic khung oxoaporphine co 2 proton methine nhan tham tai vi tri 4 va 5; dua tren pho ^H-NMR cho 2 tin hieu (6H 8,12 va 6H 8,85), hai tin hieu nay dupe de nghi la eiia H-4 va H-5, tren pho COSY ciing cho fliay tin hieu tuong quan ciia 2 proton nay. Chiic C=0 ciia khung oxoaporphine dupe de nghi tai vi tri C-7 (6c 181,48). Tren pho HMBC, mpt tin Bdng 1. Kei qud pho NMR ciui chai V2 5 (ppm), J (Hz),
hieu proton methine ciia nhan thom (6H 8,35) cho tuomg quan vol C-7 nen tin hieu nay la ciia H-8;
H-8 eho tucmg quan voi mpt carbon bac IV (6c 126,85) nen carbon nay dupe de nghj la C-7*.
Dong thoi H-8 ghep voi mot proton methine nhan thom (6H 7,181; J=S,5 Hz) nen proton nay la H-9, ghep cap vi tri orto. H-9 cho tuong quan voi C-10 va C-11 (6C 145,85 va 6c 157,91).
Tu ket qua pho NMR 1 va 2 chieu, ket hpp doi chieu voi cac tai lieu tham khao, chung to:
nhan dinh V2 la thahcminine, mot alkaloid cau tao khung oxoaporphine.
CDCb, m-NMR (500 MHz), '^C-NMR (125 MHz).
Cariion 1 1a 1b 2 3 3a 4 6 6 7 7a 8 9 10 11 11a 3-OMe 10-OMe 11-OMe O-CH2-O
"C-NMR 149,50 100.32 123,36 137,38 136,21 126,85 118,41 143,82 144,50 181,48 126,86 126,13 111,68 145,85 157,91 128,81 61,16 56,10 60,21 101,75
DEPT C C
c c c c
CH CH C C C CH CH C C C CHs CHa CH, CHz
'H-NMR
- -
8,123 (br) 8,855 (br)
8,35(d,J=8,5H2) 7,181 (d,J=8,5 Hz)
- -
4,284 (s) 3,814 (s) 4,026 (s) 6,223 (s)
COSY
H-5 H-4
H-9 H-8
HMBC
C-11: C-10; C-9:C-7a C-11; C-10: C-8
C-3 C-10 C-11 C-1, C-2
"C-NMR Thalicminine [51 148,6 102.9 122,2 136,4 135,9 130,6 118,9 144,2 145,2 181,2 125,1 109,4 108,1 153,9 148,9 128.2 60.2 56.2 56,1 102,2
^Vk,
Hinh 1. Cdng thiec cdu tqo cita V2 (thalicminine)
Chat V4 (cassj^thidine)
Ch§ft V4 duac CO lap duoi dang tinh the mau cam d$m, tan trong CHCb, MeOH, SKLM bang he dung moi CHCb : MeOH: NH4OH dd (9 : 1 : 0,2) cho vet tat quang 6 UV 254 nm, hien hinh bang thuoc thu Dragendorff xuat hien vet tron mau cam dam voi Rf=0.5.
Bien gidi kei qud pho chat V4 (cassythidine) Pho ^H-NMR eho tm hieu cpng huong iing vai su hien dien hai rthom proton methoxy 5H
Y Hpc TP. Hb Chi Minh * Tap 20 * So 6 * 2016 Nghien cuu Y hpc
methylen trong do eo 2 nhom 6 vimg tnrang thap [5H 6,037; 5H 5,952 (O-CH^O, A; O-CH2-O, B)] va [5H 5,933; 6H 5,910 (O-CTh-O, A; O-CH2-O, B)], so nhom con lai 6 vimg truang cao [2,630;
2,697 (H-4A; H-4B); 3,401; 2,925 (H-5A, H-5B);
2,763; 2,674 (H-7A, H-7B)], ba nhom proton methine 5H [3,853 (lH;H-6), 6,707 (IH; H-8), 7,564 (1H;H-11)].
Cac dii lieu pho ^C-NMR, DEPT cho thay Al CO tong so 19 carbon cho tin hieu pho tir 20 ppm doi 150 ppm. Trong do co 10 carbon bac IV (C), 3 carbon bac m (CH), 5 carbon bac II (CH2) va 1 carbon bac I (CH3). Tat ca 10 carbon bac IV deu dich chuyen 6 vimg truang thap (6c tu 115 ppm doi 150 ppm). Trong do eo 5 carbon mang di to oxy (6c 143357; 135,125; 139,930; 146,511; 146,058 ppm), cac carbon con lai deu la ciia noi doi.
Trong so 3 carbon bac m co 1 carbon 6 vimg truang cao (6c 53,692 ppm; 6H 3,853 ppm), 2 carbon con lai 6 vimg truang thap (105-115 ppm).
Co 5 carbon bac H, trong do co 2 carbon bae n xuat hien vimg truang thap [6c 100,875 ppm, 6H (6,037; 5,952) ppm] va [6c 100,600 ppm, 6H (5,933; 5,910) ppm] . Day la dae trung cita nhom methylendioxid thuong gap trong cac alkaloid ciia chi Stephania. Cac carbon bac II con lai la eae carbon mang noi don xuat hien a vimg truong eao [6c 43,048 ppm; 6H (3,401; 2,925) ppm], [6c 37,087 ppm; 6H(2,763; 2,674) ppm] va [6c 23,814 ppm; &c (2,630; 2,697) ppm].
Co 1 nhom co dp dai hoa hpe dae tnmg cua nhom methoxy [6c 59,469 ppm; 6H (4,003;
3H) ppm].
Vai eae dii lieu pho NMR n h u tren ta eo the du doin V4 la mpt alkaloid eo hai nhom the methylen dioxid, mpt nhom methoxy, ba nhom methylen, mot nhom NH va mpt nhom -OH. Tham khao cac tai lieu cong bo dac diem pho NMR CO the du doan dupe V4 la khung aporphine.
Dua vao pho HMBC, hai proton ciia nhom methylen dioxid 6H (6,037 va 5,952) eo tuang
tac vai ca hai carbon bac IV 6c (143,357 va 135,125, CO the la C-1 va C-2). Proton ciia nhom methoxy 6H (4,003) tuong quan voi mpt carbon bac IV (6c 139,930) do do carbon bac IV nay la C-3 va nhom methoxy ggn tai vi tri C-3.
Ca hai proton methylen (6H 2,630; 6H 2,697) va hai proton methylen (6H 2,925; 6H 3,401) deu cho tuong tac voi mpt carbon b?c IV (6c 119,158) nen du doan carbon bac IV (6c 119,158) la C-3a. Hon niia, hai proton methylen (6H 2,630; 6H 2,697) co tuong tac mdt carbon bac IV (6c 128,505) va hai proton methylen (&H 2,925; 6H 3,401) tuong tac voi mpt carbon methine (6c53,692), nen carbon bac IV (6C 128,505) phai la vi tri C-lb, carbon methylen (6c53,692) la vi tri C-6 va hai proton methylen (6H 2,630; 6H 2,697; H-4) g§n v6i carbon (6c 23,814) la 6 vi tri C-4 va hai proton methylen (6H 2,925; 6H 3,401; H-5) gSn voi carbon (6c 43,048) la 6 vj tri C-5. Pho COSY ding cho tuang quan giiia H-4 va H-5.
Dua vao pho COSY, H-6 ghep cap voi hai proton methylen (6H 2,674; 6H 2,801), nen hai proton nay la H-7a, H-7b. Hai proton H-7a, H~
7b I^i gan voi carbon (6c 37,087) do do carbon nay la C-7.
HMBC cho thay, H-6 co tuang quan vai carbon bac IV (6c 128,505) nen carbon bac IV (6c 128,505) nay la vi tri C-lb. Tuong tu H-7 tuong quan vai carbon bac IV (6c 124,829); (6c 110,924);
(6c 108,467) va (6c 128,742) nen cdc carbon nay Ian luat la cae vi tri C-7a; C-la; C-8 va C-lla. Mpt proton methine ciia C-8 (6c 108,467; 6H 6,707) cho tuong tae voi C-7; C-7a va mot carbon bae IV (6c 146,511) nen carbon bac 4 nay la C~9. Mpt proton methine (6H 7,564) cho tuong quan voi C-la va C-lla va mpt carbon bae IV (6c 146,058) nen proton nay la H-U, ciia carbon vi tri C-11 (6c 107,103) va carbon bae IV (6c 146,058) la C-10.
Di^a tren ket qua pho ket hpp so sanh tai Heu tham khao mot cau true tuang tu nhung khac a nhom methylendioxid tai vi tri C-9 va C-10 la cassythine, eo the nhan dinh chat V4 la mpt aporphine alkaloid co ten cassythidine.
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N g h i e n cmi Y hpc Y Hpc TP. Ho Chi M i n l i • Tap 20 * S o 6 • 2016
Bang LKSttjui pho NMR cm V4. b (jrpm), J (Hz), CDCb: MeOD (9:1), 'H-NMR (500 MHz), "C-NMR (125 MHz).
Vitri 1 l a l b 2 3 3a 4 5
6 7
7a 8 9 10 11 11a 30Me O-CH2-OA
B O-CHj-OA
B C C C C C C CH2 CH2
CH CH2
CH C C CH
C
'H(a)
2.630-2,697 (m) 3,401 (m) 2,925 (td) 3.853 (dd) 2,763-2,801 (dd)
2,674 m
6,707 (s)
7.564 (s)
4,003 6,037 (d, 3 Hz) 5.962 (d; 3 Hz) 6,933 (d; 3Hz) 5,910 (d; 2 Hz)
"C(a) 143,367 110.924 128,505 135,125 139,930 119,158 23,814 43,046
53,692 37,087
124.829 108,467 146.511 146,058 107,103 128,742 69,469 100,875
100,600
HMBC
C-3a;C-1b C-3a;C-6
C-lb; C-7 C-la, C-6; C-7a, C-8; C-11a
C-7; C-7a; C-8; C-9
C-1a;C-10;C-11a
C-3 C-1; C-2
C-9; C-10
COSY
H-5 H4
H-7 H-6
" S C s , ,
Hinh 2. Cau true dia V4 (Cassythidine)
KETLUAN
Tir cao chief alkaloid tong, sii dung ky thuat sac ky cot diaion HP-20 va sihea gel pha fhuan ket hpp ky thuat pho, chiing toi da phan lap va xac dinh dupe caii true eiia 2 h o p chat aporphine alkaloid la thalicminine va cassythidine.
TAI LIEU T H A M KHAO
1 Chang FR, Chao YC, Teng CM and Wu YC (1998). Chemical constituents from Cussytha Jtlijbrmis H )oumal of natural
Hoet S, Stevigny C, Block S, Opperdoes S, Colson P, Baldeymu B, Lansiaux A, Bailly C a n d Quetin-LedQcq ] (2004) Alkaloid from Cassytha jUifarmis and Related Aporphmes: Antibypanosomal Activi^, Cytotocoadty, and biteiaction with DNA and Topoisometases Planta Med, 70, 407-413.
Maekh SKh, Gorovoi PG and Yunusov SY (1976)., Alkaloids qf Thalictrum sMftam VoL HI. Khimiya Pnrodnykh Soedinenii, Vol 4 , p 560, July-August
Stevigny C, Block S, De Pauw-Gillel MC, De Hoffinann E, Liabrcs G, Adjakidje V and Quetin-Ledercq J (2002). C ^ t o a c Aporphine Alkaloid team aissytha filijbrmis. Planta Med, 68.
1042-1044.
Vien du(?c lieu (2004). CSy thu& v& ddng vSl lam lhu& a Viit Norn lap 2. NXB Khoa hpc Vy thu^t, tr.978.
Vo Van Chi (1997). Tif dim cSy thuSc W Nam, NXB Y hijc, tr.l236.
Wu YC, Chao YC, Chang FR and Chen YY (1997) Midoids From Cassytha filijbnms Phytodiemistry, Vol 46, N a 1, pp 181- 184.
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