HQI NGHI KHOA HQC T O A N Q U O C V £ SINH T H A I V A T A I N G U Y E N SINH VAT L A N THCf 5 ^
CAC HOfP CHAT PHENOLIC PHAN LAP T l / CAY Tylophora sp.
HOANG LE TUAN ANH, BUI HUtl TAI, DUONG THJ DUNG, VU MANH HA, NGUYEN XUAN NHIEM, NGUYEN HOAI NAM, NGUYfiN XUAN CU'OfNG, CHAU VAN MINH, PHAN VAN KlfM
Viin Hoa sinh hiin, Vi$n Hdn ldm Khoa hgc vd Cong ngh^ Vi^lNam'
NGUYEN VAN DU", TRAN HUY THAI, HA TUAN ANH, BUI VAN THANH, TRAN MINH H(?I Viin Sinh thdi vd Tdi nguyen sinh vgt, Vien Hdn ldm Khoa hgc vd Cong ngh^ Viet Nam Chi Diu dai {Tylophora R. Br.) thupc hp Thien Iy (Asclepiadaceae) gdm ed khoang 60 lo^i khac nhau. Chiing thudc Ioai day leo, phan bd d viing khi hau nhiet va can nhiet ddi. 0 Viet Nam chi Dau dai cd tam loai. Mdt so loai dirac su dung trong cac bai thudc co truyen nhu Tylophora ovala (Dau dai xoan), T. indica (Dau dai an), T. tenuis (Dau dai manh)... Cac nghien ciiu ve hda hoc chi Tylophora da cdng bd phan lap cac chat thupc Idp alkaloid, secoiridoid va phenolic (Yao S. vdnnk., 2008; Cai Y. vdnnk., 1991). Tuy nhien nhiing nghien ciiu trongnu6c ve hda hpc cac loai thudc chi Tylophora cdn rat it.
Bai bao nay thdng bao ket qua phan lap va xac dinh cau true cua ba hgp chat phenolic tir djch chiet metanol cay Tylophyra sp. bao gdm hai chat dang khung lignan: Cycloolivil (1), olivil (2) va mpt chat dang khung flavan-3-ol: Calechin (3).
I. VAT LIEU VA PHUONG PHAP NGHIEN CUtJ 1. Mau thirc vat
Mau cua loai Tylophora sp. duae thu tai Vudn Qudc gia (VQG) Yok Ddn, tinh Dik Uk.
Ten khoa hoc do PGS. Vu Xuan Phuong giam dinh (hien tai van chua thu duae mau chuin cho viec xac djnh chinh xac ten khoa hpc ciia loai). Mau tieu ban, sd hipu CTTN-23 thu t^i Krdng Na, huyen Budn Ddn, tinh Dik Lik, toa dp 107''47'288"E, Xl^SS'Aiy^, duae luu gift tai Phdng Thuc vat dan tpc hgc, Vien Sinh thai va Tai nguyen sinh vat.
2. Hda chat, thiet bi
Sac ky lop mdng (TLC): Thuc hien tren ban mdng trang sin DC-AIufolien 60 F254 (Merck 1,05715), RP-18 F254S (Merck); cac vet chit dugc phat hien bing den tii ngoai d hai budc song 254nm va 365nm hoac diing thudc thii la dung dich H2SO4 10% phun dkn len ban mong, say kho rdi ha ndng tir tir den khi hien mau.
Sac ky cgt (CC): Dugc tien hanh vdi chit hip phu la silica gel pha thudng va pha dao.
Silica gel pha thudng co cd hat la 0,040-0,063mm (240-430 mesh). Silica gel pha dao YMC c6 cahat Ia30-50p.m (Fujisilisa Chemical Ltd.).
Pho cong huang lie hgt nhdn (NMR): Do tren may Bruker AVANCE 500 tai Vien Hda hpc, Vien Han lam Khoa hgc va Cdng nghe Viet Nam.
HQl NGHj KHOA HOC T O A N Q U 6 C V £ SINH THAl V A T A I NGUYEN SINH VAT L A N THQ' 5 3. Phan lap cac hop chat
Mlu cay Tylophora s^. dugc phoi, siy khd d SO^C va duge nghien thanh bpt (1,5kg).
Lirpng mlu nay dugc chiet ndng bang metanol ba lan, sau dd gdp cac djch chi€t Iai cit loai dung mfii dudi ap suit thip thu dugc 70g ca,n ehiet metanol. Cgn chiet nay dugc phan bd vao 11 nude cat rdi bd sung clorofoc, lac deu de phan ldp qua dem (3 Ian). Cit loai cloroform bing may cat quay chan khdng thu dugc phan dc^n cloroform (30g). Phin nude cdn lai tiSp tuc duge bd sung etyl axetat, lie deu rdi de phan ldp qua dem (3 lan). Cat loai etyl axetat thu duge phan doan etyl axetat (15g) phin con lai dich nude.
Can chiet etyl axetat (15g) dugc hda tan bing metanol, tim silica gel (ty le 1: 1,2, w/w), cat lo?i dung moi dSn khd, nghien mjn. Hdn hgp nay dugc phan tach thanh 3 phan dogn TTI, TT2 va TT3 tren cot sac ky silica gel pha thudng vdi he dung mdi rua giai clorofoc/metanol/nudc (2/1/0,15, v/v). Phan dogn TT2 (3g) tiep tuc duge phan tach thanh 2 phan doan nhd ban TT2A
\i TT2B tren cdt sic ky silica gel pha thudng vdi he dung mdi riia gi^i clorofoc/melanol/nudc (5/1/0,1, v/v). Hgp ehit (1) (7mg) va (2) (5mg) thu dugc sau khi tinh chS phan doan TT2B tren cOt sic ky silica gel pha dao sir dung he dung mdi riia giai metanol/nudc (1/2, v/v).
Phan doan TT2A dugc phan tach thanh 2 phan doan nhd hem TT3A va TT3B tren cdt sac ky silica gel pha dao su dung hp dung mdi metanol/nudc (1/1,5, v/v). Hgp chat (3) (6mg) Ihu dupe sau khi tinh che phan doan TT3A tren cdt sac ky silica gel pha dao sii dung he dung mdi metanol/nudc (1/4, v/v).
Thong tin ve 3 hop chat phdn lap dugc nhu sau:
Cycloolivil (1): Bgt vd djnh hinh, mau vang nhat. Cdng thirc phan lir C2oH2:i07 (M = 376);
'H-NMR (500 MHz. CDjOD-rfj) va '^C-NMR (125 MHz, CDjOD-^/j) xem bang 1.
Olivil (2); Bpt vo dinh hinh, mau vang nhat. Cdng thiic phan tir CIGH^OO (M = 300);
'H-NMR (500 MHZ, CDJOD-^J) va '^C-NMR (125 MHz, CDyOD-d,) xem bang I.
Calechin (3): Bdt vd dinh hinh, mau vang nhat. Cdng thiic phan tir CisHi^Os (M = 290);
'H-NMR (500 MHz, CDjOD-t/v) 5H (ppm): 6.86 {d,J= 1.5 Hz, H-2'), 6.77 {d, J = 8.0 Hz, H-5'), 6.74 (dd, J = 8.0, 1.5 Hz, H-6'), 5.94 (d, J= 2.5 Hz, H-8), 5.87 (d, J = 2.5 Hz, H-6), 4.57 (d, J = 7.5 Hz, H-2), 3.99 (m, H-3), 2.88 (dd, J= 16.0, 5.5 Hz, H-4a), 2.53 (dd, J= 16.0, 8.5 Hz, H-4b); "C-NMR (125 MHz, CDjOD-t^j) 5c (ppm): 157.85 (C-7), 157.58 (C-5), 156.93 (C-9), 146.26 (C-3'), 146.26 (C-4'), 132.28 (C-1'), 120.04 (C-6'), 116.11 (C-5'), 115.29 (C-2'), 100.87 (C-10), 96.35 (C-6), 95.56 (C-8), 82.88 (C-2), 68.83 (C-3), 28.51 (C-4).
Hinh 1. Cdu true hod hgc cda ba hap chdt (I)-(3)
HQI NGHI KHOA HQC TOAN QUOC V£ SINH THAI VA TAI NGUY£N SINH VAT LAN THCf 5 n . KET QUA VA THAO LUAN
Hgp chat (1) thu dugc dudi dang bdt vd dinh hinh mau vang nhat. Tren phd cgng hudng li hat nhan proton va cac tuong tac tren phd 2 chieu HSQC cho thay cac cap tin hieu cpng huong:
Tin hieu cua hai nhdm methoxy (8H 3,80/5c 56,34 va SH 3,83/5c 56,32), mgt nhdm methylen (5H 2,6; 3,23/5c 39,67), hai nhdm hydroxymethyl (5H 3,57; 3,80/5c 60,63; 5H 3,79; 3,63/Sc 69,12), hai nhdm methyl (SH 4,05/5c 44,51 va 5H 2,04/5c 47,24) va nam proton tham 6H 6.23- 6.77. Cac phan tich tren phd '"'C-NMR va DEPT cho thay trong phan lir hgp chat (1) co 20 nguyen tii carbon bao gdm: Hai nhdm methoxy, ba nhdm methylen, bay nhdm methyl (trong do cd 5 olefin methyl), lam carbon bac bdn (trong dd cd 7 olefin carbon bac 4). Qua do cho ih^y chat (1) cd cau true dang lignan. Cac tuang tac nhan dugc tren phd HMBC giira H2-7' (5H 2,6;
3,23) va C'V (6c 126,00), C-2' (8c 133,15), C-6' (5c 112,61); cimg vdi cac tuang tac HMBC giua H-7(SH 4,05) va C-1 (Sc 138,01), C-2 (5c 113,63), C-6 (5c 123,30), C-I'(5c 126,00), C-2*
(5c 133,15) da chi ra ring lignan (1) co dang 4-aryltetralin. Tuang tac HMBC gifta H2-9 {h^
3,57; 3,80) vdi C-7 (5c 44,51), C-8 (5c 47,24), C-8' (5c 74,63) cho nhom hydroxymelhylen,thir nhit the tai C-8. Tuang tir, tuang tac HMBC gitra H2-9' (5H 3,79; 3,63) vdi C-8 (5c 47,24), C-7' (5c 39,67), C-8" (5c 74,63) cung chiing td nhdm hydroxymelhy len cdn lai the tai vj Iri C-8', Ngoai ra, tin hieu ciia C-8' (lin hieu ciia nguyen tir carbon bac 4 nhan dugc tren ph6 DEPT) co do chuyen djch hda hpc 5c 74,63 ggi y ring nhom the cdn Iai tai C-8' la nhdm hydroxyl. Ti6p dd, vj tri ciia cac nhdm the trong nhan benzene cung Ian lugl dugc xac djnh dira tren phan tich pho HMBC. Cac luang lac HMBC giiia H-3' (5H 6,23, s) vdi C-7 (5c 44,51), C-l' (5c 126,00), C-4' (6c 144,85), C-5' (5c 147,02) cimg vdi cac tuang tac HMBC giiTa H-6' (5H 6,62, s) vol C-2' (5c 133,15),_C-4' (5c 144,85), C-5' (5c 147,02), C-7' (5c 39,67) cho thiy nhan benzene thfr nhai bj the tai bdn vi Iri r,2',4',5'. Them vao do, tuong tac HMBC giiia H-3'/C-5' quan sat dugc vdi cudng do manh ddng thai Iai nhan thiy tuang tac HMBC cua H-MeO (5H 3,83)/C-5' da cho thay nhdm methoxy thk tai C-5' va nhdm hydroxyl thg tai C-4'. Nhan benzene con lai bj thi tai ba vi tri 1,3,4 dugc khing dinh dua tren tuang tac HMBC giiia H-7 (5H 4,05) vdi C-2 (5c 113,65), C-6 (5c 123,30) ciing vdi tin hieu dang douplet {J= 8,5 Hz) cua H-5 (6H 6,77) quan sat dugc tren phd 1 H-NMR. Them vao do, tuong tac HMBC vdi cudng do mgnh giua H-5 va C-3 (6c 148,63) va tin hieu tuong tac HMBC ciia H-OMe (5H 3,80)/C-3 cho phep khingdjiih nhdm methoxy the tai C-3. Cudi ciing, ciu hinh tuang ddi eua chil (I) ciing dugc xem xet. Gii su vj tri C-7 cd ciu hinh beta, hSng sd tuang tac JH-T/H-S = 12,0 Hz cho thiy dang hinh hpc cua hai nhdm the C-7/C-8 cd dang trans di€u do cd nghTa la C-8 cd ciu hinh alpha. Nhom hydroxymethylen tai C-8' cd ciu hinh beta dua tren su trimg khdp vS cac dii kien pho '^C-NMR cua (1) so sanh vdi cycloolivil nhu da cdng bd (Sugiyamam vd nnk., 1993) (bang I). Nhu vay cau triic hda hgc ciia chat (1) dugc xac dinh la cycloolivil.
Hgp chat (2) phan lap dugc dudi dang bdt vd dmh hinh mau vang nhgt. Cung gidng nhu chil (1), cac tin hieu cgng hudng Iren pho ' H - va '^C-NMR ciia (2) cung cho thiy chit (2) co ciu true dang lignan vdi hai nhdm the methoxy (6H 3,87/5c 56,33 va 5H 3,87/5c 56,35). Tuy nhien, khac vdi (1), tren phd HMBC chi quan sal thiy cac tuang tac cua H-7 (5H 4,75) vdi C-1 (6c 135,33), C-2 (5c 111,54), C-6 (5c 120,8) trong khi klidng thay cac tin hieu tuang tac giiia H-7 vdi C-V, C-T, hay C-6' cho thiy d (2) khdng cd sir ddng vdng giiTa C-7 va C-2'. D6ng thcri sir xuit hien tin hieu tuong tac HMBC giira H2-9' (5H 3,63; 3,84) va C-7 (5c 85,83) cho thiy co sir ddng vdng giira C-9' va C-7.
HOI NGH! KHOA HOC TOAN QUOC V£ SINH THAI V A T A I NGUYEN SINH VAT iJVN THLf 5 Bang 1 Dir kien pho N M R cua (1) (2) va cac chat t h a m khao
No.
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8' 9' 4-OMe 3-OMe
Cycoolivii Sc' 138,5 114.0 149,2 146,2 116,1 123.6 44,9 47,6 60,9 126,5 133,6 117,4 145,3 147,5 113,0 40,0 75,0 69.5 56,4 56,4
(11 Sc'"
138,01 113,63 148,63 145,63 115,72 123,30 44.51 47,24 60,63 126,00 133,15 117,09 144,85 147.02 112,61 39,67 74,63 69,12 56,34 66,32
BH"'^ (mult, J in Hz)
6.66
6.77 (d. 8.5) 6.68 4.05 (d. 12.0) 2.04 (dt, 4.0: 12.0) 3.57(dd, 4.0, 11.0)
3.80'
6.23 (s)
6.62 (s) 2.64 (d, 16 5) 3.23 (d, 16.6)
3.63 (d, 11.0) 3.79 (d. 11.0) 3 80 (s) 3.83 (s)
Olivil 5c"
135,5 111,8 148,7 147,3 115,9 120,8 85,8 61.9 60,9 130,5 115,5 149,8 146,3 115,9 124,0 40,7 82,6 78,0 56,6 56,6
(2) 6 c ' * 135,33 111,54 149.01 147,18 115,80 120,76 85.83 61.91 60,78 130,40 115,22 148,56 146,14 115,68 123,86 40,62 82,59 77,94 56,35 56,33
5H"'' (mult, J in Hz) 7,17 (d, 1,5)
-
6,74 6,90 (dd, 8,0:1,5)
4,75 (d, 7,5) 2.32 (dt, 7,5: 6,0) 3,85 (d, 11,5:6,0) 3,76 (dd, 11,5:6,0)
6,93 (br s)
6,76 (d, 8.0) 6,74 3,01 (d, 14,0) 2,93 (d, 14,0) 3.84 (d, 9,0)
- .
3,63 (d. 9,0) 3,87 (s) 3,87 (s) Do irong "CDjOD-rfj, "125 MHz, "500 MHz, ' t a c tin liieu bj ch6ng chap.
Hinh 2. Cac lircmg lac HMBC (H -> Q cliiiili cm (IJ va (2)
HQl NGHI KHOA HQC T O A N Q U O C V £ SINH THAl VA TAl NGUYgN SINH VAT I-AN THl> 5 Vi tri cac nhdm the d nhan benzen cua (2) cung dugc khang djnh dua tren phan tich phi hai chieu HMBC va cac hing sd tuong tac spin H-H nhan dugc tren phd 'H-NMR. Trudc tien, tin hieu tuang tac HMBC giiia H-7 (5n 4,75) vdi hai olefin metin carbon C-2 (5c 111,54) va C-6 (5c 120,76) cimg vdi tin hieu dang douplet ciia H-2 (5H 7,17, 7 = 1,5 Hz), double douplet ciia H-6(5H 6,90, J=^ 8,0; 1,5 Hz) cho thay nhan benzen thii nhit bj thi tai ba vj tri 1,3,4. Han n&a tren phd HMBC ciia (2) nhan thay ea H-2 va H-6 deu cd tin hieu tuang tac m^nh vdi C-4 (5c 147,18) cho thay nhdm hydroxyl the tai vj tri C-4, qua do chiing Id nhdm metoxi th6laiC-3 (5c 149,01). Dieu nay ciing phii hgp tin hieu tuang tac HMBC nhan dugc ciia H-2/C-3 va H-OMe (5H 3,87)/C-3- Tuang tu ta cung nhan thiy vdng benzen cdn lai cung th6 dang 1,3,4 va hai nhdm the hydroxyl va metoxi ciing lan lugt the tai vj tri C-4' (5c 146,14) va C-3' (5c 148,56). Cau hinh tuong ddi d vdng furan cung dugc xem xet dira tren phan tich ph6 'H- va '•"C-NMR. Hing sd tuong tac spin ^H-T/H-S = 7,5 Hz cho thiy cac nhdm thS tai C-7/C-8 co ciu hinh £. Cau hinh tai C-8/C-8' cung xac djnh la E dua tren su phii hgp cac gia tri ph6 '"'C-NMR ciia (2) va olivil nhu cdngbd trong tai lieu (Abe F. vdnnk, 1995) (bang 1). Nhu vay cau true hoa hgc cua (2) dugc xac djnh la olivil.
Hgp chat (3) thu dugc dudi dang tinh the mau vang. Phd 'H-NMR xuat hien tin hieu ciia hai proton dvj tri me/a vdi nhau tai 6H 5,87 (lH,d, 7 = 2 , 5 Hz, H-6), 5,94 (IH, d, J = 2,5 Hz, H-8), ddng thdi phd ' H cung xac djnh su xuit hien cua mdt vdng tham the kieu 1,3,4 bdi cac tin hieu cgng hudng 5H 6,86 (IH, d, J= i,5 Hz, H-2'), 6,77 {\W,d,J= 8,0 Hz, H-5'), 6,74 (IH, dd, J = 8,0, 1,5 Hz, H-6'). Ngoai ra cdn xuit hien lin hieu ciia mgt nhdm methyl tai 5H 4,57 (IH, d, J = 7,5 Hz, H-2), mgt proton ndi vdi oxy lai 3,99 (1 H,m, H-3) va hai proton ciia nhdm methylen tai 2,53 ( l H , d d , y = 16,0,8,5 Hz, H,-4), 2,88 (IH, dd, 7 = 16,0, 5,5 Hz, Hb-4). Phd'^C-NMR xuat hien tin hieu ciJa 15 nguyen tu carbon trong dd tin hieu cua hai nhdm metin l^i 5c 82,88 yi 68,83 la phii hgp vdi vi tri C-2 va C-3 ciia khung 3-hydroxyflavan. So sanh cac dO' ki?n pho cua hgp chat (3) vdi cac dif kien phd cua chit calechin (Yao S., 2008) thay cd sir phii hgp hoar toan.
Nhu vay hgp chat (3) la calechin.
m . KET LUAN
Budc diu da phan lap va xac dinh ciu triic ciia ba hgp chit phenolic tir djch chiSt metanol cay Tylophyra sp. bao gdm hai chat dang khung lignan: Cycloolivil (1), olivil (2) va mpt chat dang khung flavan-3-ol: Calechin (3).
Lai cdm on: Cong trinh dirge hodn thdnh nhd sir ho trg kinh phi cda di ldi thugc Chuang trinh Tdy Nguyin HI cua Viin Hdn ldm Khoa hgc vd Cong nghe Viqt Nam.
TAl LIEU THAM KHAO
1. Nguyin Tien Ban (chu bien), 2005. Danh luc cac loai thirc val Viet Nam. Tap 3. NXB. Nong nghiSp. Trang 74-75.
2. Yao S., Tang C.P., Ye Y., 2008. J. Asian Nat. Prod Res., 10: 591-596.
3. Abe F., Iwase Y., Yamauchi T,, Honda K., Hayashi N., 1995. Phytochemistiy, 39: 695-699.
4. Sugiyamam., Nagayama E., Kikuchim., 1993. Phylochemislry, 33: 1215-1219.
5. Shen Z.B., Theander O., 1984. Phylochemislry, 24: 364-365.
6. Cai Y., Evans F.J., Robertsm.F., Phillipson J.D., Zenkm.H., Gleba Y.Y., 1991. Phylochemisirfi 30: 2033-2040.
HQl NGHj KHOA HQC TOAN QU6C v £ SINH T H A I V A TAI N G U Y E N SINH VAT L A N THLf 5 ISOLATION OF PHENOLIC COMPOUNDS FROM Tylophora s p .
HOANG LE TUAN ANH, BUi HUU TAI. DUONG THI DUNG, VU MANH HA, NGUYEN XUAN NHIEM, NGUYEN HOAI NAM, NGUYEN XUAN CUONG, CHAU VAN MINH, PHAN VAN KIEM, NGUYEN VAN DU, TRAN HUY THAI, HA TUAN ANH, BUI VAN THANH, TRAN MINH HOI SUMMARY
Two lignans cycloolivil (1), olivil (2), and a flavan-3-oi calechin (3) were isolated from the methanol exbact of Tylophora sp. using various chromatographic experiments. The slnicture of isolated compounds were elucidated by spectroscopic methods including 1D- and 2D-NMR as well as and dicussed in comparison with the literatures.
