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TAP CHl KHOA HQC VA C O N G NGHE Tap 47, so 1, 2009 Tr. 95-99 CAC TRITERPENE KHUNG OLEANAN VA URSAN TU' V6 CAY EBERHARDTIA AURATA (SAPOTACEAE) D O A N THI MAI HUONG, N G U Y E N V A N HUNG, MARC LITAUDON, FRANCOISE GUERITTE 1. MO DAU Cay Cdng sira vang hay cdn ggi la cay Bara vang la loai cay dai mdc cao khoang 6 - 8 m, la cd phieu bau due mgc so le dai 16 - 25 cm, rdng 4 - 6 em. Cay ed ten khoa hgc la Eberhardtia aurata (Pierre ex Dubard) Lecomte thudc hg Hdng xiem (Sapotaceae), thudng phan bd d Nam Trung Qudc va Bac Viet Nam [1, 2]. La ciia cay dugc dimg lam thudc trj ho, ho ga [1]. Trong khudn khd hgp tac Viet Phap ve nghien ciru tham thuc vat Viet Nam, chiing tdi da thir boat tinh sinh hgc djch chiet EtOAc ciia vd cay Cdng sira vang, ket qua eho thay djch chiet ire che 49% ddng te bao ung thu KB d ndng do 1 pg/mL. Cho den nay van chua cd mdt nghien cuu nao ve thanh phan hoa bgc ciia loai nay. Trong khudn khd bai bao nay, chiing tdi thdng bao ve viec phan lap dugc 4 triterpen khung oleanan va 2 triterpen khung ursan tir vd cay Cdng sira vang. 2. KET QUA VA THAO LUAN Tir can djch EtOAc sau khi tien hanh sac ki cot nhieu lan va ket tinh chiing tdi thu dugc 4 triterpen khung oleanan la P-amyrin acetate (1), laraxerol (2), taraxerone (3), 3d-3doetacosanoyloxy-12-oleanen-28-ol (4) va 2 triterpen khung ursan la gamboukokoensein A (5), axit myrianthic (6). Cau true ciia cac hgp chat tren dugc xac djnh bang su ket hgp cac phuang phap phd khdi lugng (MS), phd cdng hudng tir proton va cacbon ('H va "C-NMR) va cac phd cgng hudng tir hat nhan hai ehieu COSY, HMQC, HMBC. 95 Chat 1 dugc phan lap dudi dang tinh the mau trSng, dne 238-239 °C. Phd khdi ion hda bui dien tir (ESI-MS) cd pic ion d m/z 469 [M+H]" tuang irng vdi cdng thirc phan tir la C32H52O2. Phd '^C-NMR va DEPT cho tin hieu cua 32 cacbon trong dd cd 9 nhdm metyl, 10 nhdm metylen, 5 nhdm metin, 1 nhdm cacbonyl va 7 cacbon bac bdn. Tren phd ' H - N M R xuat hien tin hieu dac trung cho 8 nhdm metyl d 5H 0,82; 0,86; 0,86; 0,87 (2 x CH3), 0,96; 0,96; 1,12, 1 nhdm axetat (OCOCH3) d 5H 2,04 va mgt tin hieu ciia proton metin olefinic d 5H 5,17 (t, J=3,5 Hz, H12). Them vao dd, tren pho ' H - N M R cd tin hieu ctia 1 nhdm metin d vj tri C-3 da dugc acyl hoa 0 5j^ 4^49^ proton nay bj tach dudi dang triblet vdi bang sd tuong tac J= 8,0 Hz cho biet cau hinh d ciia nd. Tren pho HMBC thiy tin hieu ciia H-3 tuang tac vdi cacbon carbonyl G=0 d 5c 170,9 chirng td ring nhdm OCOCH3 g4n vdi khung terpen d vj tri C-3. Tir cac dir kien phd MS, ' H N M R , ' ^ C - N M R , H S Q C , HMBC va so sanh vdi tai lieu tham khao [3,4] cho phep xac djnh ciu triic ciia chat 1 la P-amyrin axetat. Chat 2 dugc phan lap dudi dang tinh the binh kim mau trang, diem ndng chay 270 - 27TC. Phd ' ^ C - N M R va DEPT cho tin hieu ciia 30 cae bon trong do cd 8 nhdm metyl, 10 nhdm metylen, 5 nhdm metin va 7 cacbon bac bdn. Phd khdi ion bda bui dien tir (ESl-MS) cd pic ion d m/z All [M+H], Cac dir lieu phd '^C-NMR va MS cho phep xac djnh cdng thiJC phan tu ciia chat 2 la C30H50O va eho biet day la 1 triterpen 5 vdng ciing vdi 1 lien ket ddi va 1 nhdm hydroxy trong phan tir. Phd ' H - N M R eho 8 tin hieu singlet ciia 8 nhdm metyl d 5H 0,70; 0,73; 0,81; 0,81; 0,83; 0,85; 0,87; 0,99 va dac biet la tin hieu d 5H 5,53 (dd, J=8,0; 3,0 Hz) cua metin olefinic H15 va tin hieu ciia nhdm hidroxy metin d 5H 3,07 (dd, J=9,0; 7,0 Hz, H-3). Ket hgp phd ' H NMR, '^C-NMR, phd NMR 2 chieu va so sanh vdi tai lieu tham khao [5] cho phep ket luan hgp chat 2 chinh la taraxerol. Chat 3 dugc phan lap dudi dang chat ran mau trang. Phd khdi ion hda bui dien tir (ESIMS) cd pic ion d m/z 425 [M-i-H]^ tuong ung vdi cdng thirc phan tu' la C3oH4g02. Cac du' lieu pho ciia hgp chat nay gan gidng dir lieu ciia hgp chat 2. Gidng nhu chat 2, tren phd H-NMR ciia 3 cd tin hieu ciia 8 nhdm metyl va 1 nhdm metin olefinic H-15 d 5H 5,55 (dd, J=8,0; 3,0 Hz). Khac vdi 2, tren phd ' H - N M R mat di tin hieu edng hudng ciia proton thudc nhdm hydroxymetin. Tren phd C-NMR de dang nhan thay tin hieu cdng hudng ciia 1 nhdm cacbonyl d 5c 217,5, khdng cd tin bieu ciia nhdm hydroxymetin. Nhu vay cd the ket luan rang chat nay la mdt triterpen thudc khung olean nhu chat 2 trong do nhdm hydroxymetin dugc thay the bang nhom cacbonyl. Cac dir lieu phd va tai lieu tham khao [6,7] da cho phep xac djnh cau triic eua chat nay la taraxerone. Chat 4 dugc phan lap dudi dang chat ran mau trang. Phd khdi ion hda bui dien tir (ESI-MS) cd pic ion d m/z 849 [M+H] . Tren phd 'H-NMR xuat hien tin hieu dac trung cho 8 nhdm metyl va 1 tin hieu triplet cua nhdm metylen ben canh nhdm este d 5H 2,89 (J=7,0 Hz), 2 tin hieu double doublet ciia nhdm CH2OH d 5H 3,22 (J=l 1,0; 5,5 Hz, H-28a) va 3,55 (J=l 1,0; 6,5 Hz, H28b). Them vao do, tren phd ' H - N M R cd tin hieu ciia 1 nhdm metin d vj tri C-3 da dugc este hoa d 5H 4,50, proton nay bj tach dudi dang double doublet vdi bSng so tuong tac J=10,5; 5,5 Hz cho biet cau hinh d ciia nd. Cum tin hieu chdng cheo d viing 6H 1,22-1,28 dac trung cho cac nhdm metylen mach dai. Cac dir lieu phd '''C-NMR, DEPT hoan toan phii hgp vdi cac du" lieu phd ' H - N M R da phan tich d tren, bao gdm 1 cacbonyl este d 5c 173,6, mdt nhdm metin gin vdi oxi d 6c 80,5, cac tin hieu ciia khung terpen olean va axit beo mach thing. Sau khi so sanh vdi tai lieu tham khao [8] chiing tdi di den ket luan hgp chit 4 chinh la 3d-octacosanoyloxy-12oleanen-28-ol. Hgp chat 5 dugc phan lap dudi dang chat rin mau tring. Pho khoi ion hda bui dien tir (ESIMS) cd pic ion d m/z 521 [M+H] . Phd "C-NMR va DEPT cho tin hieu ciia 30 cacbon trong do 96 cd 6 nhdm CH3, 8 nhdm CH2, 8 nhdm CH, 1 nhom cacbonyl va 7 cacbon bae bdn. Cae dir lieu phd C-NMR va MS cho phep xac djnh cdng thirc phan tu ciia chat 5 la C3oH4.i(0- \a cho biet day la 1 triterpen 5 vdng ciing vdi 1 lien ket ddi, 5 nhdm hydroxy va 1 axit trong phan tir. Tren phd 'H-NMR cd tin hieu ciia 1 broad singlet d 6H 2,51 dac trung cho H-18 ciia terpen khung ursan cd nhdm the O-R d vj tri 19 [9], ciing vdi tin hieu ciia 5 metyl dudi dang singlet d 6H 0,83 (2 xCH,): 1,06; 1,22; 1,38; 1 metyl dudi dang doublet d 5H 0,95 (d, J=6,5 Hz, CH3-3O) va 1 proton olefinic d 6H 5,31 {t, J=3,5 Hz, H-12). Ddng lhdi tren phd ' H - N M R cdn cho tin hieu ciia 2 proton thudc nhdm CHjOH d 3,41 6H (d, J=l 1,0 Hz, H-23a) va 3,54 (d, J=11.0 Hz. H-23b) ciing tin hieu ciia 3 nhdm hydroxy metin d 5H 3,50 (d, J=9,5 Hz, H-3): 3,62 (dd, J=9,5, 3,0 Hz, H-2); 3,70 (d, J=3,0 Hz, H-1). Hang sd tuong tac JH-I H-: =3.0 Hz va JH.2 HO =9.5 Hz cho phep xac djnh 3 proton metin d vj tri 1,2 va 3 dudi dang H-ip, H-2P va H-3d. Ket hgp cac dir lieu phd va so sanh vdi tai lieu tham khao [9, 10] cho phep ket luan hgp chat 5 chinh la gamboukokoensein A. Hgp chat 6 dugc phan lap dudi dang chat ran mau trang. Phd khdi ion hda bui dien tir (ESIMS) cd pic ion d m/z 505 [M+H]' tuang irng vdi cdng thirc phan tir la C30H48O6 Cac dii' lieu phd ' H - N M R ciia 6 gan gidng vdi dur lieu phd ciia hgp chat 5, khac vdi chat 5, hgp chat 6 chi cd 2 nhdm hydroxy metin d 6„ 3,62 (IH, d, J=2,5 Hz, H-3); 3,89 (IH, m, H-2). Sau khi so sanh vdi tai lieu tham khao [ 10] chiing tdi di den ket luan hgp chat 6 chinh la axit myrianthic. 3. THl/C NGHIEM VA PHU'ONG PHAP NGHIEN ClTU 3.1.Thiet bj va nguyen lieu Di^m ndng chay dugc do tren may Boetius. Phd cdng hudng tir hat nhan NMR dugc ghi tren may Bruker Avance 500 MHz vdi TMS la chat chuan ndi. Phd khdi lugng (ESI-MS) dugc do tren may sic ky Idng ghep khoi phd vdi dau dd MSD (LC/MSD Agilent series 1100), sir dung mode ESI va dau dd DAD. Vd cay Cong sira vang {Eberhardtia aurata (Pierre ex Dubard) Lecomte), dugc ThS. Dao Dinh Cudng va ThS. Nguyen Quoc Binh thu hai d Thuan Chau, San La vao thang 10/2005. Mau lieu ban cd ten VN 1575 dugc luu giir lai Vien Sinh thai va Tai nguyen sinh vat - Vien KH&CN VN. 3.2. Xir li mau thtrc vat va chiet tach Mau vd cay sau khi dugc phoi khd. nghiijn nhd (1.12 kg) dugc ngam chiet vdi dung mdi EtOH (4 lan trong 96 gid). Sau khi cit loai dung mdi dudi ap suat giam thu dugc can djch EtOH. Can djch EtOH dugc them H2O roi chi^t tiep bing EtOAc va BuOH, cit loai dung mdi thu dugc 33 g can djch EtOAc va 20 g can djch BuOH. Tir can djch EtOAc, sau khi tien hanh sic ki cot silica gel vdi he dung mdi n-he.\an-EtOAc-MeOH gradient chimg tdi thu 12 phan doan chinh F1-F12. Tir phan doan Fl, sau khi chay edt silica gel vdi he dung mdi n-hexan-EtOAc gradient cho 70 mg chat 1. Tir phan doan F6, sau khi chay cot silica gel vdi he dung mdi n-hexan-EtOAc gradient cho 10 mg chit 2. Tir phan doan F5, sau khi chay cot silica gel vdi he dung mdi nhexan-EtOAc gradient cho 7 mg chat 3 va 71 mg chit 4. Tir phan doan FIO, sau khi chay cot silica gel vdi he dung mdi CH2Cl2-MeOH gradient cho 17 mg chat 5 va 5 mg chat 6. P-amyrin acetate (1): Chat rin mau tring, dnc: 238-239°C, ESI-MS m/z: 469 [M+H]'; ' H N M R (CDCl, 500 MHz) 5 (ppm): 0,82 (3H, s, CH3), 0,86 (3H, s, CH3), 0,86 (3H, s, CH3), 0,87 (6H, s, 2 X CH,), 0,96 (3H, s, CH,), 0,96 (3H, s, CH,). 1,12 (3H, s, CH,), 2,04 (3H, s, OCOCH3), 97 4,49 (IH, L J=8,0 Hz, H-3), 5,17 (t J=3,5 Hz). "C-NMR (CDCI3, 125 MHz) 5 (ppm):15,5; 16,7; 16,8; 18,2; 21,3; 23,5; 23,6; 23,7; 25,9; 26,1; 26.9; 28,0; 28,4; 31,0; 32,5; 32,6; 33,3; 34,7; 36,8; 37,1; 37,7; 38,2; 39,8; 41,7; 46,7; 47,2; 47,5; 55,2; 80,9: 121,6; 145,2; 170,9 Taraxerol (2) ESI-MS m/z: All [M+H]"; ' H - N M R (CDCl, + CD,OD 500 MHz) 5 (ppm): 0,68 (IH, d, H-5); 0,70 (3H, s, CH3); 0,73 (3H, s, CH3), 0,81 (6H, s, 2x CH,), 0.83 (3H, s, CH,). 0,85 (3H, s, CH3), 0.87 (3H, s, CH3), 0,99 (3H, s, CH3), 1,82 (IH, dd, J=15,0, 3,0 Hz, H-16), 1,94 (IH, dL J=12,5; 3,0 Hz, H-7); 3,07 (IH, dd, J=9,0; 7,0 Hz, H-3); 5,43 (IH, dd, J=8,0; 3,0 Hz, H-15). '^C-NMR (CDCI3 + CD3OD, 125 MHz) 5 (ppm): 15,1; 15,2; 17,3; 18.6; 21.0; 25,6; 26,6; 27,7; 28,6; 29,5; 29,6; 32,9; 33,1; 33,5; 34,9; 35,6; 36,5; 37,4; 37,5; 37,6; 37,8; 38,5; 38,8; 41,1; 48,6; 55,4; 78,7; 116.6; 157,9. Taraxerone (3) : ESI-MS m/z: 425 [M+H]^; ' H - N M R (CDCI3 500 MHz) 5 (ppm): 0,83 (3H, s, CH3), 0,89 (3H, s, CH,), 0,91 (3H, s. CH,), 0,95 (3H. s, CH3), 1,06 (3H, s, CH,), 1,08 (3H, s, CH3), 1,09 (3H, s, CH3), 1,14 (3H. s, CH3), 2,34 (IH, m, H-1), 2,56 (IH, m, H-2), 5,55 (IH, dd, J=8,0; 3,0 Hz, H-15). '^C-NMR (CDCI3, 125 MHz) 5 (ppm): 14,8; 17,4; 19,9; 21.3; 21,4; 25,5; 26,1; 28,8; 29,7; 29,8; 29,9; 33,1; 33,3; 33,6; 34,1; 35,1; 35,7; 36,7; 37,5; 37,7; 37,7; 38,3; 38,9; 40,6; 47,5; 48,7; 48.8; 55.8; 117,2; 157,6; 217,5 36-Octacosanoyloxy-12-oleanen-28-ol (4) ESI-MS m/z: 849 [M+H]'; ' H - N M R (CDCI3 500 MHZ) 5 (ppm): 0,86-0,89 (15H, m, 5 x CH3), 0,94 (3H, s, CH3), 0,95 (3H, s, CH3), 1,16 (3H, s, CH3), 2,89 (2H, L J=7,0 Hz, -CH.COO-), 3,22 (IH, dd, J=l 1,0; 5,5 Hz, H-28a), 3,55 (IH, dd, J=11,0; 6,5 Hz, H-28b), 4,50 (IH, dd, J=10.5; 5,5 Hz, H-3), 5,19 (IH, L J=3,5 Hz, H-12). "CNMR (CDCI3, 125 MHz) 8 (ppm): 14,1; 15,5; 16,7; 16,7; 18,2; 22,0; 22,6; 23,5; 23,6; 25,1; 25,5; 25,9; 28,0; 29,1; 29.2; 29.3; 29.4, 29,5; 29,6; 29,6; 29,7 (CH2)n; 30,9; 31,0; 31,9; 32,5; 33,1; 34,1; 34,8; 36,8; 36,9; 37,3; 38,2; 39,8; 41,7; 42,3; 46,4; 47,5; 55,2; 69,7; 80,5; 122,3; 144,2; 173,6. Gamboukokoensein A (5) ESI-MS m/z: 521 [M+H]*; ' H - N M R (CD3OD, 500 MHz) 5 (ppm) 0,83 (6H, s, 2 x CH3), 0,95 (3H, d, J=6,5 Hz, CH3), 1,06 (3H, s, CH,), 1,22 (3H, s, CH3), 1,38(3H, s, CH3), 3,41 (IH, d, J=11,0 Hz, H-23a), 3,50 (d, J=9,5 Hz, H-3), 3,54 (IH, d, J=l 1,0 Hz, H-23b), 3.62 (IH, dd, J=9,5; 3,0 Hz, H-2), 3,70 (IH, d, J=3,0 Hz, H-1), 5,31 (IH, X, J=3,5 Hz, H-12); "C-NMR (CD3OD, 125 MHz) 5 (ppm) 13,3; 16,5; 17,6; 17,8; 19,1; 24,9; 26,6; 27,0; 27,3; 28.2; 29.6; 33,8; 39,0; 41,6; 41,9; 42,5; 43,0; 44,0; 44,1; 54,9; 71,1; 71,9; 73,6; 79,3; 81,1; 130,6; 138,8: 182,4. Axit myrianthic (6) ESI-MS m/z: 505 [M+H]*; ' H - N M R (CD3OD, 500 MHz) 5 (ppm) 0,80 (3H, s, CH3); 0,82 (3H, s, CH3); 0,95 (3H, d, J=6,5 Hz, CH3), 1,04 (3H, s, CH,), 1,22 (3H, s, CH3), 1,37 (3H, s, CH3), 3,42 (IH, d, J=11,0 Hz, H-23a), 3,56 (IH, d, J=l 1,0 Hz, H-23b), 3,62 (IH, d, J=2,5 Hz, H-3), 3,89 (IH, m, H-2), 5,32(1H, X, J=3,5 Hz, H-12). Loi cam on. Cac tac gia xin cam on ThS. Dao Oinh Cu-dng va ThS. Nguyin Quoc Binh da thu hai va xac djnh mau thtrc vat, cd Nguyen Thi Hdng Van da giiip dd thyc hien cong trinh nay. Cac ket qua nghien CU'U thu du-gc trong khudn kho hgp tac Viet - Phap v4 nghien cdu tham thuc vat Viet Nam. TAI LIEU THAM KHAO 1. Vd Van Chi - Tir dien lhuc vat thdng dung, Nha xuat ban Khoa hoc va Ky thuaL tap 1. 1035-1036. 2. Pham Hoang Hg - Cay cd Viet Nam, Nha xuit ban Tre, Tap 1, 2000, tr. 636-637. 98 3. Shunya Matsunaga, Reiko Tanaka and Masao Akagi - Triterpenoids from Euphorbia maculata, Phytochemistry 27 (2) (1988) 535-537. 4. Nakane T., Maeda Y. - Fern Constituents : Triterpenoids from Adiantum Chem. Pharm. Bull. 50(1002) 1273-1275. 5. Ahashi B. Mahato and Asish P. Kumdu - '''C spectra of pentacyelic triterpenoids - A compilation and some salient features, Phytochemistry 17 (4) (1994) 1517-1575. 6. Nobuko Sakurai, Yoshikatsu Yaguchi and Takao Inoue - Triterpenoids from Myrica rubra, Phytochemistry 26 (1) (1986) 217-219. 7. Arot Manguro, Lawrence O., Midiwo. Jacob O., Kraus, Wolfgang Triterpenoids and Steroids of Myrsine africana Leaves, Planta Medica 63 (1997) 290. 8. Wandji Jean, Tillequin Francois. Mulholland Dulcie A., Wansi Jean-Duplex, Fomum Tanee Z., Fuendjiep Victorine, Libol Franeine, Tsabang Nole Fatty Acid Esters of Triterpenoids and Steroid Glycosides from Gambeya africana, Planta Med. 68 (9) (2002) 822-826. 9. Bao-Zhi Li, Bin-Gui Wang and Zhong-Jian Jia - Pentacyelic triterpenoids from Riihus xanthocarpus, Phytochemistry 49 (8) (1998) 2477-2481. capillus-veneris. 10. Jean Wandji, Francois Tillequin, Dulcie A. Mulholland. Jovita Chi Shirri. Nole Tsabang, Rlisabeth Seguin, Philippe Verite, Franeine Libol, Z. T. Fomum Pentacyelic triterpenoid and saponins from Gambeya boukokoensis, Phytochemistry 64 (2003)845-849. SUMMARY OLEANANE- AND URSANE-TRITERPENES FROM THE BARK OF EBERHARDTIA AURATA (SAPOTACEAE) In the framework of scientific cooperation between the Institute of Chemistry (VAST, Vietnam) and the Institute of Natural Product Chemistry (CNRS, France) on phytochemistry of the Vietnamese flora, the plant Eberhardtia aurata (Pierre ex Dubard) Lecomte was selected for its cytotoxic activity on KB cell lines (49% inhibition at 1 pg/mL for the EtOAc extract of the bark). From the EtOAc extract of the stem bark of this plant, 4 oleanane triterpenes, P-amyrin acetate (1), taraxerol (2), taraxerone (3), 3d-octacosanoyloxy-12-oleanen-28-ol (4) and 2 ursane triterpenes, gamboukokoensein A (5) and myrianthic acid (6) were isolated. Their structures were elucidated by using IR, MS and NMR spectroscopic methods, including 2D NMR spectroscopy (COSY, HMQC, HMBC). Dia chi: Nhdn bdi ngdy 5 thdng 7 ndm 2008 Doan Thj Mai Huang, Nguyen Van Hiing, Vien Hda hge, Vien Khoa hgc va Cdng nghe Viet Nam. Marc Litaudon, Francoise Gueritte, Vien Hda hgc Cac hgp chat Thien nhien, Gif sur Yvette, CH Phap. 99