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Two new sesquiterpene glucosides from Ixseris plants
TWO NEW SESQUITERPENE GLUCOSIDES FROM IXSERIS
PLANTS
Ji-Yuan Ma,* ~"Ai-Ming He, t De-Cheng Zhang, t Zhen-Tao Wang, ~ Luo-Shan Xu, 2~ Guo-Jun Xu, ~ Tsuneo Namba 5: and Shigetoshi Kadota w ~Shanghai Representative Office, Generation Health Co., Shanghai Medical University, 200032, Shanghai, China; ~Department of Pharmacognosy, China Pharmaceutical University, No 1 Shennong Rd. Nangjing, 210038, China; w Toyama Medical & Pharmaceutical University, 230-01, Toyama, Japan)
Key Word Index--Ixeris denticulata f. pinnatipartita Kitag.;/. sonchifolia Hance; Compositae; sesquiterpene lactone glucoside; guaianolide; ixerin X; ixerin Z; cytotoxicity. Abstract The whole plant of Ixeris denticulata f. pinnatipartita and I. sonchifofia respectively afforded a new guaianolide sesquiterpene lactone glucoside named ixerin X, 8(, 15dihydroxy- 1(10),3,11 (13)-guaiatriene- 12,6-olide- 15-O-13-D-glucopyranoside (1), ixerin Z, 1(10),3,1 l(13)-guaiatriene-12,6-olide-2-one-3-O- 13 -D-glucopyranoside (2), whose structure and stereo-chemistry were determined by spectroscopic methods. 1 inhibited the growth of human breast cancer MCF7 & MDA468 cell lines.
Introduction The whole herb of I. denticulata f. pinnatipartita and 1. sonchifolia were used as antiinflammatory and haemostatic folk remedies in China. Chemical research of other species of the genus revealed the sesquiterpene lactones[ 1-7] as active principle which showed wide-spectral activities, such as cytotoxicity[8], anti-repellence & anti-feedance to some insects, etc. This paper intended to isolate any sesquiterpene lactones from I. denticulata f. pinnatipartita and/. sonchifolia from which sesquiterpene lactones had not been reported. Results and Discussion Two new sesquiterpene lactone glucosides ixerin X (1), Ixerin Z(2) were isolated from the whole herb of/. denticulata f. pinnatipartita and/. sonchifolia, respectively. Ixerin X (1), has a molecular formula of C21H2509 as indicated by its high resolution EI-MS. The presence of an tx-methylene-3,-lactone moiety was revealed by its IR absorption bands at 1750 and 1669cm "l, and this was substantiated by a pair of characteristic low field ~H NMR(CD3OD, 250MHz) signals at 56.13(1H, dd, J=3.3, 1.3Hz, H-13a), 6.23(1H, dd, J=3.3,1.3Hz, H-13b)(Table 1). An olefinic proton at 56.05(1H, brs, H-3) and vinyl methyl at 81.81(3H, brs, H-14) were also observed. 13C NMR spectrum of I showed the presence of 21 carbons(Table 1), of which one was assigned to lactone carbonyl at 8 172.0, 6 were olefinic carbons indicating the existence of 3 double bonds, 9 oxygen beating carbons including 6 from glucose moiety, others were 2 methylene, 2 methine and 1 methyl carbons at the upper field. In the lH NMR spectrum, a doublet at 8 3.75(IH, J=10.0Hz), was attributed to H-5 which coupled with H-6. The signal of 394
H-6 was at 8 3.71(1H, dd,J=10.0,12.5Hz). This indicated the trans-diaxial relationship between H5 and H6, H6 and H7. Since the H-7 in all other naturally occurring guaianolides has been assumed to be a -oriented, so, H-5 and H-6 would be a and 13 oriented in 1, respectively. Signal at 8 3.03(1H, dt, J=3.3, 12.5Hz) was assigned to H-7, the large coupling constant between H-7 and H-8 (12.5Hz) indicative of an approximate dihedral angle of around 180", allowed the assignment of H-8 as a configuration. Based on the above evidence as well as by comparison with the data of previously reported crepidiaside E[9], 1 was finally determined to be 813,15-dihydroxy- 1(10),3,11 (13)-guaiatriene12,6-olide-15-13-D-glucopyranoside. The anomeric structure of I was thought to be 13 from its coupling constant (7.SHz).
H OH
,,~~x~
H
.H O
CH2
OH
~L,, x
H~O O,/~ x
H
O
1
2
Ixerin Z (2), amorphous powder. FAB-MS and El-MS indicated the formula of C21H2609. ~H NMR (pyridine-d5, 400MHz) showed the characteristic signals at 5 6.19(1H, brs, H-13a), and 6 5.19(1H, brs, H-13b) which revealed the presence of a-methylene-y-lactone moiety. Signals for 2 vinyl methyls at 8 2.31(3H, brs, H-15), 5 2.42(3H, brs, H-14), and for a sugar moiety were also observed. A double doublet at 8 3.17(1H, J=10.0, 12.2Hz) was assigned to H-6, which coupled with H-5 at 6 3.22(1H, d,J=10.0Hz) and H-7 at 6 2.74(1H, brt, J=12.2), this indicated the trans-diaxial relationship of respective vicinal 2 protons. The stereochemistry of these protons were considered to be H-Sa, H-613, since the H-7 in naturally occurring guaianolides have a-orientation. ~3C NMR data of 2 showed the presence of 21 carbons, of the 8 unsaturated carbons, 2 carbonyls were attributed to lactone carbonyl at 6 169.1 (C-12) and a a, 13-unsaturated ketone carbonyl at 6 188.9(C-2), 6 were olefinic carbons; of 13 alkyl carbons, 7 were oxygen beating carbons including 6 derived from glucose moiety, others were 2 methines, 2 methylenes and 2 methyls. The anomeric configuration of glucose moiety was determined to be 13from JH1,-H2, value(7.0Hz). Based on above evidence, 2 was identified to be l(10),3,11(13)-guaiatriene-12,6-olide-2-one-3-O-13-D-glucopyranoside as a previously not reported compound. Ixerin X showed the inhibitory effect toward the growth of human breast cancer MCF7 and MDA468 cell lines, with the IC50 of 15 and 28~tM, respectively (Fig. 1).
395
13CNMRdata
Table 1.1H NMR &
of compounds 1" & 2** (6 values from internal TMS, J in Hz) IH NMR
1
2
138.1 s 38.1 t 131.1 d 142.0 s 52.8d 84.2 d 59.2 d 69.7 d
1
2 3 4 5 6 7 8
3.8(2H,m) 3.11 (2H,brs) 6.05(1H,brs) 3.75(1H,d,J=10.0) 3.71(1H,dd,J= 10.0,12.5) 3.03(1H,dt, J-3.3, 12.5) 3.76(1H,m, overlapped)
3.22(1H,d,J=l 0.0) 3.17(1H,dd,J= 10.0,12.2) 2.74(1H,brt, J=12.2) oc: 2.29(1H,m, overlapped) 13: 1.06(1H,dd,J=12.2) a: 2.08(1H,m) 13: 1.89(1H,m)
2 153.0 s 188.9 s
153.7s 146.1 s 52.4d 85.1 d 47.9 d 24.1 t
46.6 t
36.9 t
127.5 s 140.2 s 172.0 s 122.0 t
124.6 s 139.5 s 169.1 s 117.9t
23.0 q 68.8d
21.7 q 14.9q
6.24(1 H, d, J-7.0) 102.8 d 2' 75.1 d 77.9d 3' 4' 71.7 d 5' 78.2 d 6' 62.8 t * measured in CD30D at 250MHz for 'H and 62.6MI~Iz for i3C ** measured in pyridine-d5 at 400MHz for 1H and 100MHz for 13C
101.6 d 75.3 d 78.3 d 71.1 d 78.3 d 62.3 t
c~: 2.27(1H,dd, J=13.3, 2.5) [3" 2.62(1H,dd, J= 13.3, 11.5) 10 11 12 13 14 15 1
7
a: 6.13(1H,dd, J=3.3, 1.3) b: 6.23(1H,dd, J=3.3, 1.3) 1.83(3H, brs) a: 4.52(1H,brd, J=13.0) b: 4.68(1H,brd, J=13.0) 4.33(1H,d, J=7.8)
3,5
a: 6.19(1H, brs) b: 5.19(1H, brs) 2.42(3H,s) 2.31(3H,s)
'
2.53 2
468
_
1.51 ~_~~_~ : , 0.5 0~r: : ~
~
:
MCF7
~
~
~
,-,,
~
Ce")
~
r
'~0
Concentration (ktM) Fig. 1 Inhibitory Effect of Ixerin X on the Growth of Human Breast Cancer MCF7 and MDA468 Cell Lines
396
t
,
Experimental General. Mps: uncorr.; 1H NMR and 13C-NMR: 250MHz and 62.5MHz, respectively for 1, 400 MHz and 100MHz respectively, for 2; 2D-NMR data (~H-~H COSY, HMQC, HMBC, NOESY)" standard pulse sequences. CD3OD and pyridine-d5 were used as solvent, respectively for 1 and 2, with TMS as int. standard; EI-MS: 70ev, direct int.; FT-IR: KBr; CC: silica gel (coarse silica gel, 100--200 mesh), D-101 resin (16--50mesh); TLC: precoated silica gel plates (Merk, silica gel 60 F254). Plant material. The plant of I. denticulata f. pinnatipartita Kitag.was collected in Dabie Mountains at Luotian county, Hubei province, China, in October 1992./. sonchifolia Hance. was purchased from Nanjing Company of Traditional Chinese Medicine, in February 1991. Voucher specimen is deposited in the Herbarium of the Department of Pharmacognosy, China Pharmaceutical University. Extraction & Isolation. The air-dried whole plant of I. denticulata f. pinnatipartita (4Kg) were extracted (x3) with hot (100" ) water. After filtration, the solution was concentrated to 10 1, and precipitated by adding EtOH to a concentration of 60%. The supernatant was filtered after over night standing. The filtrate was applied to D-10 l resin column after the EtOH was removed off. The column was washed with water first, then with 20%, 40% and 85%EtOH successfully. The fraction from 40% EtOH was concentrated and was chromatographed on a silica gel column and eluted with chloroform-methanol (95:5--8:2). The eluates were monitored by TLC, and repeated chromatography afforded compound I (20mg). The crude materials of I. sonchifolia (5Kg) were extracted with hot 95% EtOH. The extract was evaporated to dryness and extracted with petroleum ether, chloroform and methanol successively. The chloroform extract was applied to silica gel column and the column was eluted with n-hexane-chloroform-methanol(1:1 :if-0:1:1). Repeated chromatography and purification gave compound 2 (25mg). Ixedn X (1), fine needles(MeOH). Mp 180-183(dec.). UV~,M~cN~xnm: 231. [Ot]2SD-38.9* (MeOH, cO. 11). IR V ~ x C m -1: 3412(-OH), 2294, 2880, 1750(=C=O), 1669, 1448, 1384,1078, 1029. EI-MS m/z: 424[M]+ (3), 317(4), 294(4), 262(13), 244[M-C6H~206]+ (100), 227(43), 181(40), 119(56). ~H NMR (CDaOD, 250MHz) & '3C NMR (CDaOD, 62.5MHz): see Table 1. Ixerin Z (2), amorphous powder. UV ~M~cN~xnm: 267, 198. [0{,]280 +3 5.1 o (MeOH, 0.11). FAB-MS m/z: 445[M+Na]+, 422[M]+. EI-MS m/z: 260 [M+I-C6HI~Os]+ (100), 245(13), 214(15), 189(53), 161(29), 151(28), 134(25), 107(22). ~n M R (pyridine-d5, 400MHz)& ~C-NMR (pyridine-dS, 100MHz): see Table 1. Bioassay. The regularly differentiated and cultured human breast cancer MCF7 and MDA468 cell lines (provided by Cancer Research Center, Nottingham University, U.K.) was incubated in a CO2 filled incubator at 37 after the culture solution was removed and EDTA added. The suspended cells was diluted by medium (RPMI 1640 500ml, by adding 50ml of bovine serum) to be the concentration of 2.5(102/hole, transferred to a 96-holed culture plate (180~tl/hole) and cultured for 4 hr. Ixerin X (l.4mg) was dissolved in 0.5ml of DMSO. The solution was 10 times diluted first, then 3 times diluted in turn with medium (RPMI 1640) to make 9 gradient concentration lines, and used as test solutions. 201.tl of the test solution was added to above mentioned 96-holed culture plate which contains cultured cells, 8 holes was 397
used for one concentration and 20~tl of the RPMT medium which was free of the sample (ixerin X) was used as control. The plate was cultured for a week. 50~tl of MTT (2 mg/ml) i.e. 3-(4,5dimethylthiazol)-2,5-diphenyl tetrazolium bromide was added to each hole, and cultured for another 4hr. The purple sediment would be observed at the bottom of the hole, the culture solution that containing MTT was removed, and 125~tl of DMSO-Glycine (100:25) buffer solution was added to each hole, the sediment was dissolved by oscillating the culture plate on a cradle. Subsequently, the absorbance of the solution was measured at 550nm, which was considered to correlate with the cell survival rate. The mean absorbance and the coefficient of variation for each concentration were calculated, and the relationship of absorbance versus concentrations was plotted, and ICs0 calculated. References 1. Hidehisa Asada, Toshio Miyase & Seigo Fukushima Chemical and Pharmaceutical Bulletin 1984; 32:1724 2. Hidehisa Asada, Toshio Miyase & Seigo Fukushima Chemical and Pharmaceutical Bulletin 1984; 32:3036 3. Hidehisa Asada, Toshio Miyase & Seigo Fukushima Chemical and Pharmaceutical Bulletin 1984; 32:3403 4. Keiichi Nishimura, Toshio Miyase, Akira Ueno, Tadataka Noro & Masanori Kuroyanagi. Chemical and Pharmaceutical Bulletin 1985; 33:3361 5. Mamoru Seto, Toshio Miyase & Seigo Fukushima, Chemical and Pharmaceutical Bulletin 1986, 34:4170 6. Tstomu Warashina, Mie Ishino, Toshio Miyase & Akira Ueno Phytochemistry 1990, 29:3217 7. Ha Sook Chung, Won Sick Woo & Sook Jalim, Phytochemistry 1994, 35(6): 1583 8. Mamuru Seto, Toshio Miyase, Kaoru Umehara, Akira Ueno, Yutaka Hirano & Norio Otani. Chemical and Pharmaceutical Bulletin 1988, 36:2423 9. Shigfru Adegawa, Toshio Miyase, Akira Ueno, Tadatake Noro & Masanori Kuroyanagi . Chemical and Pharmaceutical Bulletin 1985,33:4906
398
PLANTS
Ji-Yuan Ma,* ~"Ai-Ming He, t De-Cheng Zhang, t Zhen-Tao Wang, ~ Luo-Shan Xu, 2~ Guo-Jun Xu, ~ Tsuneo Namba 5: and Shigetoshi Kadota w ~Shanghai Representative Office, Generation Health Co., Shanghai Medical University, 200032, Shanghai, China; ~Department of Pharmacognosy, China Pharmaceutical University, No 1 Shennong Rd. Nangjing, 210038, China; w Toyama Medical & Pharmaceutical University, 230-01, Toyama, Japan)
Key Word Index--Ixeris denticulata f. pinnatipartita Kitag.;/. sonchifolia Hance; Compositae; sesquiterpene lactone glucoside; guaianolide; ixerin X; ixerin Z; cytotoxicity. Abstract The whole plant of Ixeris denticulata f. pinnatipartita and I. sonchifofia respectively afforded a new guaianolide sesquiterpene lactone glucoside named ixerin X, 8(, 15dihydroxy- 1(10),3,11 (13)-guaiatriene- 12,6-olide- 15-O-13-D-glucopyranoside (1), ixerin Z, 1(10),3,1 l(13)-guaiatriene-12,6-olide-2-one-3-O- 13 -D-glucopyranoside (2), whose structure and stereo-chemistry were determined by spectroscopic methods. 1 inhibited the growth of human breast cancer MCF7 & MDA468 cell lines.
Introduction The whole herb of I. denticulata f. pinnatipartita and 1. sonchifolia were used as antiinflammatory and haemostatic folk remedies in China. Chemical research of other species of the genus revealed the sesquiterpene lactones[ 1-7] as active principle which showed wide-spectral activities, such as cytotoxicity[8], anti-repellence & anti-feedance to some insects, etc. This paper intended to isolate any sesquiterpene lactones from I. denticulata f. pinnatipartita and/. sonchifolia from which sesquiterpene lactones had not been reported. Results and Discussion Two new sesquiterpene lactone glucosides ixerin X (1), Ixerin Z(2) were isolated from the whole herb of/. denticulata f. pinnatipartita and/. sonchifolia, respectively. Ixerin X (1), has a molecular formula of C21H2509 as indicated by its high resolution EI-MS. The presence of an tx-methylene-3,-lactone moiety was revealed by its IR absorption bands at 1750 and 1669cm "l, and this was substantiated by a pair of characteristic low field ~H NMR(CD3OD, 250MHz) signals at 56.13(1H, dd, J=3.3, 1.3Hz, H-13a), 6.23(1H, dd, J=3.3,1.3Hz, H-13b)(Table 1). An olefinic proton at 56.05(1H, brs, H-3) and vinyl methyl at 81.81(3H, brs, H-14) were also observed. 13C NMR spectrum of I showed the presence of 21 carbons(Table 1), of which one was assigned to lactone carbonyl at 8 172.0, 6 were olefinic carbons indicating the existence of 3 double bonds, 9 oxygen beating carbons including 6 from glucose moiety, others were 2 methylene, 2 methine and 1 methyl carbons at the upper field. In the lH NMR spectrum, a doublet at 8 3.75(IH, J=10.0Hz), was attributed to H-5 which coupled with H-6. The signal of 394
H-6 was at 8 3.71(1H, dd,J=10.0,12.5Hz). This indicated the trans-diaxial relationship between H5 and H6, H6 and H7. Since the H-7 in all other naturally occurring guaianolides has been assumed to be a -oriented, so, H-5 and H-6 would be a and 13 oriented in 1, respectively. Signal at 8 3.03(1H, dt, J=3.3, 12.5Hz) was assigned to H-7, the large coupling constant between H-7 and H-8 (12.5Hz) indicative of an approximate dihedral angle of around 180", allowed the assignment of H-8 as a configuration. Based on the above evidence as well as by comparison with the data of previously reported crepidiaside E[9], 1 was finally determined to be 813,15-dihydroxy- 1(10),3,11 (13)-guaiatriene12,6-olide-15-13-D-glucopyranoside. The anomeric structure of I was thought to be 13 from its coupling constant (7.SHz).
H OH
,,~~x~
H
.H O
CH2
OH
~L,, x
H~O O,/~ x
H
O
1
2
Ixerin Z (2), amorphous powder. FAB-MS and El-MS indicated the formula of C21H2609. ~H NMR (pyridine-d5, 400MHz) showed the characteristic signals at 5 6.19(1H, brs, H-13a), and 6 5.19(1H, brs, H-13b) which revealed the presence of a-methylene-y-lactone moiety. Signals for 2 vinyl methyls at 8 2.31(3H, brs, H-15), 5 2.42(3H, brs, H-14), and for a sugar moiety were also observed. A double doublet at 8 3.17(1H, J=10.0, 12.2Hz) was assigned to H-6, which coupled with H-5 at 6 3.22(1H, d,J=10.0Hz) and H-7 at 6 2.74(1H, brt, J=12.2), this indicated the trans-diaxial relationship of respective vicinal 2 protons. The stereochemistry of these protons were considered to be H-Sa, H-613, since the H-7 in naturally occurring guaianolides have a-orientation. ~3C NMR data of 2 showed the presence of 21 carbons, of the 8 unsaturated carbons, 2 carbonyls were attributed to lactone carbonyl at 6 169.1 (C-12) and a a, 13-unsaturated ketone carbonyl at 6 188.9(C-2), 6 were olefinic carbons; of 13 alkyl carbons, 7 were oxygen beating carbons including 6 derived from glucose moiety, others were 2 methines, 2 methylenes and 2 methyls. The anomeric configuration of glucose moiety was determined to be 13from JH1,-H2, value(7.0Hz). Based on above evidence, 2 was identified to be l(10),3,11(13)-guaiatriene-12,6-olide-2-one-3-O-13-D-glucopyranoside as a previously not reported compound. Ixerin X showed the inhibitory effect toward the growth of human breast cancer MCF7 and MDA468 cell lines, with the IC50 of 15 and 28~tM, respectively (Fig. 1).
395
13CNMRdata
Table 1.1H NMR &
of compounds 1" & 2** (6 values from internal TMS, J in Hz) IH NMR
1
2
138.1 s 38.1 t 131.1 d 142.0 s 52.8d 84.2 d 59.2 d 69.7 d
1
2 3 4 5 6 7 8
3.8(2H,m) 3.11 (2H,brs) 6.05(1H,brs) 3.75(1H,d,J=10.0) 3.71(1H,dd,J= 10.0,12.5) 3.03(1H,dt, J-3.3, 12.5) 3.76(1H,m, overlapped)
3.22(1H,d,J=l 0.0) 3.17(1H,dd,J= 10.0,12.2) 2.74(1H,brt, J=12.2) oc: 2.29(1H,m, overlapped) 13: 1.06(1H,dd,J=12.2) a: 2.08(1H,m) 13: 1.89(1H,m)
2 153.0 s 188.9 s
153.7s 146.1 s 52.4d 85.1 d 47.9 d 24.1 t
46.6 t
36.9 t
127.5 s 140.2 s 172.0 s 122.0 t
124.6 s 139.5 s 169.1 s 117.9t
23.0 q 68.8d
21.7 q 14.9q
6.24(1 H, d, J-7.0) 102.8 d 2' 75.1 d 77.9d 3' 4' 71.7 d 5' 78.2 d 6' 62.8 t * measured in CD30D at 250MHz for 'H and 62.6MI~Iz for i3C ** measured in pyridine-d5 at 400MHz for 1H and 100MHz for 13C
101.6 d 75.3 d 78.3 d 71.1 d 78.3 d 62.3 t
c~: 2.27(1H,dd, J=13.3, 2.5) [3" 2.62(1H,dd, J= 13.3, 11.5) 10 11 12 13 14 15 1
7
a: 6.13(1H,dd, J=3.3, 1.3) b: 6.23(1H,dd, J=3.3, 1.3) 1.83(3H, brs) a: 4.52(1H,brd, J=13.0) b: 4.68(1H,brd, J=13.0) 4.33(1H,d, J=7.8)
3,5
a: 6.19(1H, brs) b: 5.19(1H, brs) 2.42(3H,s) 2.31(3H,s)
'
2.53 2
468
_
1.51 ~_~~_~ : , 0.5 0~r: : ~
~
:
MCF7
~
~
~
,-,,
~
Ce")
~
r
'~0
Concentration (ktM) Fig. 1 Inhibitory Effect of Ixerin X on the Growth of Human Breast Cancer MCF7 and MDA468 Cell Lines
396
t
,
Experimental General. Mps: uncorr.; 1H NMR and 13C-NMR: 250MHz and 62.5MHz, respectively for 1, 400 MHz and 100MHz respectively, for 2; 2D-NMR data (~H-~H COSY, HMQC, HMBC, NOESY)" standard pulse sequences. CD3OD and pyridine-d5 were used as solvent, respectively for 1 and 2, with TMS as int. standard; EI-MS: 70ev, direct int.; FT-IR: KBr; CC: silica gel (coarse silica gel, 100--200 mesh), D-101 resin (16--50mesh); TLC: precoated silica gel plates (Merk, silica gel 60 F254). Plant material. The plant of I. denticulata f. pinnatipartita Kitag.was collected in Dabie Mountains at Luotian county, Hubei province, China, in October 1992./. sonchifolia Hance. was purchased from Nanjing Company of Traditional Chinese Medicine, in February 1991. Voucher specimen is deposited in the Herbarium of the Department of Pharmacognosy, China Pharmaceutical University. Extraction & Isolation. The air-dried whole plant of I. denticulata f. pinnatipartita (4Kg) were extracted (x3) with hot (100" ) water. After filtration, the solution was concentrated to 10 1, and precipitated by adding EtOH to a concentration of 60%. The supernatant was filtered after over night standing. The filtrate was applied to D-10 l resin column after the EtOH was removed off. The column was washed with water first, then with 20%, 40% and 85%EtOH successfully. The fraction from 40% EtOH was concentrated and was chromatographed on a silica gel column and eluted with chloroform-methanol (95:5--8:2). The eluates were monitored by TLC, and repeated chromatography afforded compound I (20mg). The crude materials of I. sonchifolia (5Kg) were extracted with hot 95% EtOH. The extract was evaporated to dryness and extracted with petroleum ether, chloroform and methanol successively. The chloroform extract was applied to silica gel column and the column was eluted with n-hexane-chloroform-methanol(1:1 :if-0:1:1). Repeated chromatography and purification gave compound 2 (25mg). Ixedn X (1), fine needles(MeOH). Mp 180-183(dec.). UV~,M~cN~xnm: 231. [Ot]2SD-38.9* (MeOH, cO. 11). IR V ~ x C m -1: 3412(-OH), 2294, 2880, 1750(=C=O), 1669, 1448, 1384,1078, 1029. EI-MS m/z: 424[M]+ (3), 317(4), 294(4), 262(13), 244[M-C6H~206]+ (100), 227(43), 181(40), 119(56). ~H NMR (CDaOD, 250MHz) & '3C NMR (CDaOD, 62.5MHz): see Table 1. Ixerin Z (2), amorphous powder. UV ~M~cN~xnm: 267, 198. [0{,]280 +3 5.1 o (MeOH, 0.11). FAB-MS m/z: 445[M+Na]+, 422[M]+. EI-MS m/z: 260 [M+I-C6HI~Os]+ (100), 245(13), 214(15), 189(53), 161(29), 151(28), 134(25), 107(22). ~n M R (pyridine-d5, 400MHz)& ~C-NMR (pyridine-dS, 100MHz): see Table 1. Bioassay. The regularly differentiated and cultured human breast cancer MCF7 and MDA468 cell lines (provided by Cancer Research Center, Nottingham University, U.K.) was incubated in a CO2 filled incubator at 37 after the culture solution was removed and EDTA added. The suspended cells was diluted by medium (RPMI 1640 500ml, by adding 50ml of bovine serum) to be the concentration of 2.5(102/hole, transferred to a 96-holed culture plate (180~tl/hole) and cultured for 4 hr. Ixerin X (l.4mg) was dissolved in 0.5ml of DMSO. The solution was 10 times diluted first, then 3 times diluted in turn with medium (RPMI 1640) to make 9 gradient concentration lines, and used as test solutions. 201.tl of the test solution was added to above mentioned 96-holed culture plate which contains cultured cells, 8 holes was 397
used for one concentration and 20~tl of the RPMT medium which was free of the sample (ixerin X) was used as control. The plate was cultured for a week. 50~tl of MTT (2 mg/ml) i.e. 3-(4,5dimethylthiazol)-2,5-diphenyl tetrazolium bromide was added to each hole, and cultured for another 4hr. The purple sediment would be observed at the bottom of the hole, the culture solution that containing MTT was removed, and 125~tl of DMSO-Glycine (100:25) buffer solution was added to each hole, the sediment was dissolved by oscillating the culture plate on a cradle. Subsequently, the absorbance of the solution was measured at 550nm, which was considered to correlate with the cell survival rate. The mean absorbance and the coefficient of variation for each concentration were calculated, and the relationship of absorbance versus concentrations was plotted, and ICs0 calculated. References 1. Hidehisa Asada, Toshio Miyase & Seigo Fukushima Chemical and Pharmaceutical Bulletin 1984; 32:1724 2. Hidehisa Asada, Toshio Miyase & Seigo Fukushima Chemical and Pharmaceutical Bulletin 1984; 32:3036 3. Hidehisa Asada, Toshio Miyase & Seigo Fukushima Chemical and Pharmaceutical Bulletin 1984; 32:3403 4. Keiichi Nishimura, Toshio Miyase, Akira Ueno, Tadataka Noro & Masanori Kuroyanagi. Chemical and Pharmaceutical Bulletin 1985; 33:3361 5. Mamoru Seto, Toshio Miyase & Seigo Fukushima, Chemical and Pharmaceutical Bulletin 1986, 34:4170 6. Tstomu Warashina, Mie Ishino, Toshio Miyase & Akira Ueno Phytochemistry 1990, 29:3217 7. Ha Sook Chung, Won Sick Woo & Sook Jalim, Phytochemistry 1994, 35(6): 1583 8. Mamuru Seto, Toshio Miyase, Kaoru Umehara, Akira Ueno, Yutaka Hirano & Norio Otani. Chemical and Pharmaceutical Bulletin 1988, 36:2423 9. Shigfru Adegawa, Toshio Miyase, Akira Ueno, Tadatake Noro & Masanori Kuroyanagi . Chemical and Pharmaceutical Bulletin 1985,33:4906
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