429
Cytotoxic Activity of Cardenolides from Beaumon Cia brevituba Stems1 N. Kaneda2. H. Chai2. J. M. Pezzuto2, A. D. Kinghorn25. N. R. Farnsworth2, P. Tuchinda3, I Udchachon3. T. Santisuk4, and V. Reutrakul3 Plant Anticancer Agents, XLIX. For Part XLVIII. see Ref. (1) Program for Collaborative Research in the Pharmaceutical Sciences. Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago. Chicago, Illinois 60612. U.S.A. 'Department of Chemistry, Faculty of Science. Mahidol University. Bangkok 10900. Thailand The Forest Herbarium. Royal Forestry Department. Bangkok 10400. Thailand Address for correspondence
ferometer. NMR spectra ( ppm. J Hz) were obtained with TMS as
Abstract
internal standard, using Varian XL-300 (300MHz 'H-NMR, 75.6 MHz '3C-NMR spectra) and Nicolet NT-360 instruments
Five known cardenolides, digitoxigenin
(90.8MHz selective INEPT experiments). 1H-'H COSY and 'H-'3C HETCOR spectra were also obtained on the Varian XL-300 instrument, using standard Varian pulse sequences. Mass spectra were measured on a Varian MAT 112S double-focusing mass spectrometer (70eV). Cl-MS and FAB-MS spectra were recorded on a Finnegan MAT 90 instrument.
(1), oleandrigenin (2), digitoxigenin a-i.-cymaroside (3), digitoxigenin fi-gentiobiosyl-a-L-cymaroside (4). and '6-digitoxigenin $-o-glucosyl-a-i.-cymaroside (5). were isolated from the stems of Beaumontia brevituba Oliver
by cytotoxicity-directed fractionation monitored by a cultured human lung cancer cell line. The cytotoxic activ-
ity of these compounds was evaluated with a panel of twelve human and murine cancer cell lines. The lignan glycoside, syringaresinol f3-o-glucoside, was obtained for the first time in the form of its levo-enantiomer.
Key words
Plant material The stems of B. brevituba Oliver (Apocynaceae) were collected in December 1989, in Saraburi Province. Thailand. and identified by one of us (T.S.). A voucher specimen representing this collection has been deposited at the Herbarium of the Royal Forestry Department. Bangkok. Thailand.
Extraction and isolation
Beaumontia brevituba, Apocynaceae, cardenolides, cytotoxicity, lignan, structure elucidation.
Introduction The stems of Beau,nontia brevituba Oliver (Apocynaceae), a medium-sized tree native to South and Southeast Asia, were collected in Thailand as part of a continuing collaborative search for naturally occurring antineoplastic agents (1). The genus Beaumontia is known to contain cardenolides, and such compounds have been reported previously from the seeds, fruits and leaves of B. brevituba (2), the leaves of B. rnurtonii (2), and the seeds of B. grandflora (3). In this communication, we report the identification of five known cardenolides and a lignan, (—)syringaresinol fi-n-glucoside, which were isolated from the stems of B. brevituba. Four of the cardenolides exhibited a
general cytotoxic effect when evaluated with several human and murine canccr cell lines.
Materials and Methods
Air-dried powdered stems (14.8 kg) of B. brerituba were exhaustively extracted with MeOH (60 liters) at room temperature to afford 1.4kg of residue on removal of solvent under reduced pressure. This extract was taken up in MeOH (300 ml), and the resultant suspension formed by adding H2O (2 liters) was partitioned successively with Et20 (3 x 2 liters) and nBuOH (3 x 1.5 liters). The Et20 layer was concentrated to dryness, and a residue (260 g), which exhibited an ED50 of 0.7 zg/ml against
a human lung cancer cell line (Lul), was chromatographed batchwise over silica gel (Merck Silica gel 60. 70—2 30 mesh) with CHC13, toluene-acetone (10: 1). and CHCI3-acetone (5: 1), to afford
1 (340mg), 2 (250mg), and 3 (780mg). The n-BuOH layer was evaporated to dryness and the residue (140 g), which also showed cytotoxic activity against Lul (ED50, 0.4ig/ml). was subjected to silica gel column chromatography, using CHCI3-MeOH-H20 (40:8: 1) and EtOAc-MeOH-H20 (20:4: 1) as eluents, to afford 4 (13.2g), 5 (52mg), and 6 (280mg). Compounds 1—5 gave a greenish-blue or a purple color in daylight after spraying with 10%H2S04/H20(110°C, 10mm). Compounds 1—5 were identified, respectively, as
digitoxigenin, oleandrigenin, digitoxigenin a-L-cymaroside, digitoxigenin /3-gentiobiosyl-a-L-cymaroside, and z '-digitoxigenmn /3-o-glucosyl-a-L-cymaroside by comparison of their physical and spectroscopic data (m.p.. [a]0, IR. UV, 'H- and '3C-
NMR, MS) with those reported in the literature (2—4). (—)-
Melting points (uncorrected) were determined on a Kofler micro hot-stage. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. UV spectra were taken on a Beckman DU-7 spectrometer, and IR spectra on a Nicolet MX-1 inter-
Syringaresinol /3-o-glucoside (6) was crystallized from MeOH, and exhibited the following data: colorless prisms; m.p. 183—185°C; — 19.0° (c 0.20, MeOH); IR vcm: 3440, 1600, 1520, 1460, 1220, 1120, 1080, 820; UVAnm (log a): 219 (4.31), 236 (s/i)
Downloaded by: National University of Singapore. Copyrighted material.
Received: August 22, 1991
430 Planta Med. 58(1992)
N. Kaneda et al.
(4.18), 271 (3.41). 280(3.30); 'll-NMR, UCNMR and FAB-MS data
147.1 ppm) and one methoxy carbon peak (a = 56.3 ppm)
identical with the (+)-enantiomer (5. 6). Enzymic hydrolysis
in the '3C-NMR spectrum. Therefore, it was considered
(hesperidinase. Sigma Chemical Co.. St. Louis. Missouri. U.S.A.) of 6 gave a product identical (m.p.. lal,)) with (-)-syringaresinol (7).
that this aglycone was a 3,7-dioxabicyclol3.3.Oloctane type
The EtO and n-BuOFI extracts of B. bre,'ituba were tested for cytotoxic activity against eight cell lines: BCI (human breast cancer), Co12 (human colon cancer). llT-1080 (human fibrosarcoma), KB (human nasopharyngeal carcinoma). KB-V1 (vinblastine-resistant KB), Lul (human lung cancer), Me12 (human melanoma) and P-388 (murine lymphocytic leukemia), by procedures as described previously (8—10). The cytotoxic activity of chromatographic fractions obtained from the EtA) and BuOll extracts was monitored by l.u I cytotoxicity, for which the extracts showed the greatest potency. Pure compounds 1—6 isolated in this manner were tested for cytotoxicity against the above-mentioned
cell lines as well as three additional cell lines, A-431 (human epidermoid carcinoma), LNCaP (human prostate cancer) and ZR75-1 (human breast cancer). These evaluations were performed by
published procedures (9, 10). In addition, antimitotic potential was assessed using cultured ASK cells (11). Each test compound was evaluated in duplicate at seven concentrations (20.0, 4.0, 0.8, 0.16,0.032.0.0064, and 0.00128 pg/mI).
Results and Discussion
By cytotoxicity-directed fractionation, active compounds, 1—3 and 4 were isolated from the Et20 and n-BuOH extracts of B. brevituba stems, respectively. Two additional components of the n-BuOH extract. 5 and 6. were not active with any of the cancer cell lines available to this study. Analysis of the Cl-MS and 13C-NMR spectra of compounds 1 and 2 suggested them to be cardenolides, and they were individually identified, in turn, as digitoxigenin and oleandrigenin, by data comparison with
published values for these cardenolides (4, 12). The structures of the cardiac glycosides 3—5 were, in turn, con-
firmed by comparison with reported data (m.p.. [a]1), '1-land 13C-NMR, FAB-MS) as digitoxigenin a-i.-cymaroside, digitoxigenin /3-gentiobiosyl-a-i-cymaroside. and A 16-digitoxigenin fi-o-glucosyl-a-i.-cymaroside, which were obtained recently from the leaves and fruits of B. brevituba (2) while the present study was underway. For compound 6, a molecular formula of C25H36013 was consistent with its observed FAB-MS and 13C-NMR spectral data, suggesting it
to be a mono-glucoside. The presence of a simple ben-
glucoside. although this glycoside does not appear to have been obtained previously as the (—)-enantiomer.
The cytotoxic activity of isolates 1—6 was evaluated with ten human cancer cell lines and murine lymphocytic leukemia in cell culture (P-388), and was com-
pared with analogous data for three clinically used anticancer agents in Table 1. As summarized in the table, com-
pounds 1—4 were cytotoxic against most of the human cancer cell lines utilized, but not active against P-388. In contrast, A °'-digitoxigenin (-i-glucosyl-a-i.-cymaroside (5) and (—)-syringaresinol /3-n-glucoside (6) were inactive against all of the human cancer cell lines employed. None of the test compounds showed demonstrable antimitotic activity in the astrocytoma (ASK) assay. In general, greater activity for 1—4 was observed with the human lung cancer (Lu 1) cell line, which vindicated the selection of this cell
line to monitor the fractionation of the Et,() and ,i-BuOEl plant extracts, and also was supportive of the notion that the cytotoxicity of B. brevituba stems was due mainly to cardenolides. It is known that cardenolides are generally cytotoxic for in vitro mammalian cells such as HeLa cells (13) and KB cells (14—17). and intensive efforts have been
made to study the structural relationships between their
antitumor and cardiotonic effects (13), and their cytotoxicity (15). Our results demonstrated that cardenolides 1—5 exhibited a similar order of cytotoxicity
against other human cell lines in comparison with published data on KB cells (14—17). Structural aspects of the cardenolides from B. brevituba stems, as related to cytotoxicity, may be summarized as follows: 1. Cytotoxicity was evident irrespective of whether or not the cardiac glycosides were glycosylated at C-3.
2. Activity reached a maximum with the mono-glycoside 3, and diminished with further saccharide substitution as in the triglycoside 4.
3. Cytotoxicity persisted with the introduction of a C-16 acetyl group in the aglycone, as in compound 2.
4. 16,17-Unsaturation in the aglycone, as in compound 5, led to the abrogation of cytotoxicity.
zenoid chromophore was observed from its UV (Amax 219,
271 nm) and IR (Vmax 1600, 1520cm') spectra. Enzymic hyrolysis of 6 gave a genin. which exhibited seven carbon
peaks (ô = 54.3, 71.8, 86.0, 102.6, 132.0, 134.3. and
In summary, while it is interesting to investigate the active sites and structural requirements of cardenolides or bufadienolides for in vitro cytotoxicity.
Table 1 Cytotoxic acti vity of compounds 1—4'. Compound
A-431
BC1'
Co12
HT'
KB'
KB-V1'
LNCaP'
Lul'
Me12'
ZR-75-1'
P-388'
1
0.37 0.25 0.14 8.4
0.031
0.27 0.17
0.13 0.15 0.034
1.9
1.6
1.8
1.4
0.16 0.077 2.9
0.083 0.027 0.0060 0.69
0.22 0.14 0.066 1.8
>20
0.056
0.067 0.12 0.039 1.4
0.10 0.086
0.068
0.13 0.17 0.030
0.21
0.20
0.03 0.4 0.06
0.1
0.01
0.004
0.5
0.03
0.005 0.02
0.03 0.04
0.04 0.02
3.0
0.05 0.1
0.03 0.03 0.02
0.06
0.03
0.01
0.3
0.05 0.02 0.02
2 3 4
Homo' harringtonine 0.02 Taxol 0.007 Vinblastine 0.05
0.01
0.031
>4
1.9
0.02
14
>20 >20
Results are expressed as ED50 values (pg/mi).
A-431, epidermoid carcinoma; BC1, breast; Co12, colon; NT, sarcoma; KB, nasopharyngeal carcinoma; KB-V1, drug-resistant KB; LNCaP, prostate; Lul, lung; Me12, melanoma; ZR75-1, breast; P-388, murine lymphocytic leukemia.
Downloaded by: National University of Singapore. Copyrighted material.
Cytotoxicity Assays
lignan. By comparison with reported data (5—7). the structure of 6 was identified as (—)-syringaresinol n-I)-
Planta Med. 58(1992) 431
Cytotoxic Activity of Cardenolides from Beaumontia brevituba Stems
their association with human toxicity limits development as useful anticancer agents.
References Kaneda. N.. Pezzuto, J. M.. Soejarto, D. D.. Kinghorn. A. D..
Acknowledgements
Farnsworth, N. H.. Santisuk, T., Tuchinda. P.. Udchachon. J.. Heutrakul,V. t1991)i. Nat. Prod. 54.196. Yamauchi. T.. Abe, F., Santisuk. T.(1990) Phytochemistry 29. 1961. Krasso, A. F., Weiss. F.., Reichstcin, T.(1964) Pharm. Acta llelv. 39, 168. Tori. K., Ishli, H.. Wolkowski. Z. W.. Chachaty. C.. Sangare. M.. Piriou, F., Lukacs, G.(1973) Tetrahedron Lett. 1077. Deyama, T., lkawa. T., Nishibe, S. 119851 Chem. l'harm. Bull. 33, 3651. Kobayashi, II.. Karasawa. H.. Miyase, 1., Fukushima. S. (1985) - Chem. Pharm. Hull. 33, 1452. Zhang. l..-G.. S'ligmann, (1., Jurcic. K.. Wagner. II. (1982) Planta 2
Health. Bethesda. Maryland, U.S.A. We acknowledge the NMR and
MS laboratories of the Research Resources Center. University of Illinois at Chicago. for expert assistance and the provision of spec-
troscopic equipment used in this study. We thank Mr. R. B.
Dvorak. of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. for recording the CI and FAB mass spectral data.
MPd. 45. 172. Arisawa. M., Pezzuto, .J. M, Bevelle, C.. Cordell. ft A. (1984) J. Nat. Prod. 47, 453.
Jayasuriya. II.. McCh'sney, .1. ft. Swanson, S. M.. Pezzuto, J. M. 1989) J. Nat. Prod. 52, 325. HlaskO, ft. Shiph, II. I... Pe/zuto, .1. M., Cordell. (i. A. (1989) J. Nat. Prod. 52, 1363. Swanson. S. M.. Jiang. J.-X.. de Souza. N. J.. Pezzuto, J. M. (1988)J.
Nat. Prod. 51, 929. Robien. W.. Kopp, B.. Schab). I)., Schwarz, H. (1987) Prog. Nun. Magn. Spectrosc. 19. 131. Shiratori, 0. (1967) Gann 58. 521. Hembree, J. A., Chang, C.-J.. McLaughlin. J. L.. Peck. G.. Cassady. J. M. (1979)J. Nat. Prod. 42, 293. Cassady. J. M., Suffness. M. (1980) in: Anticancer Agents Based on Natural Product Models, (Cassady, J. M., Douros, J. D., eds.), p. 201, Academic Press. New York. Koike. K.. Bevelle. C., Talapatra. S. K.. Cordell. G. A.. Farnsworth. N. - R. (1980) Chem. Pharm. Bull. 28. 401. Rovinski, J. M., 'l'ewalt, G. L., Sneden. A. 1'. )1987)J. Nat. Prod. 50, 211.
12
' ' '
Downloaded by: National University of Singapore. Copyrighted material.
This work was supported by grant RO1 CA33047
awarded by the National Cancer Institute. National Institutes of
Cytotoxic Activity of Cardenolides from Beaumon Cia brevituba Stems1 N. Kaneda2. H. Chai2. J. M. Pezzuto2, A. D. Kinghorn25. N. R. Farnsworth2, P. Tuchinda3, I Udchachon3. T. Santisuk4, and V. Reutrakul3 Plant Anticancer Agents, XLIX. For Part XLVIII. see Ref. (1) Program for Collaborative Research in the Pharmaceutical Sciences. Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago. Chicago, Illinois 60612. U.S.A. 'Department of Chemistry, Faculty of Science. Mahidol University. Bangkok 10900. Thailand The Forest Herbarium. Royal Forestry Department. Bangkok 10400. Thailand Address for correspondence
ferometer. NMR spectra ( ppm. J Hz) were obtained with TMS as
Abstract
internal standard, using Varian XL-300 (300MHz 'H-NMR, 75.6 MHz '3C-NMR spectra) and Nicolet NT-360 instruments
Five known cardenolides, digitoxigenin
(90.8MHz selective INEPT experiments). 1H-'H COSY and 'H-'3C HETCOR spectra were also obtained on the Varian XL-300 instrument, using standard Varian pulse sequences. Mass spectra were measured on a Varian MAT 112S double-focusing mass spectrometer (70eV). Cl-MS and FAB-MS spectra were recorded on a Finnegan MAT 90 instrument.
(1), oleandrigenin (2), digitoxigenin a-i.-cymaroside (3), digitoxigenin fi-gentiobiosyl-a-L-cymaroside (4). and '6-digitoxigenin $-o-glucosyl-a-i.-cymaroside (5). were isolated from the stems of Beaumontia brevituba Oliver
by cytotoxicity-directed fractionation monitored by a cultured human lung cancer cell line. The cytotoxic activ-
ity of these compounds was evaluated with a panel of twelve human and murine cancer cell lines. The lignan glycoside, syringaresinol f3-o-glucoside, was obtained for the first time in the form of its levo-enantiomer.
Key words
Plant material The stems of B. brevituba Oliver (Apocynaceae) were collected in December 1989, in Saraburi Province. Thailand. and identified by one of us (T.S.). A voucher specimen representing this collection has been deposited at the Herbarium of the Royal Forestry Department. Bangkok. Thailand.
Extraction and isolation
Beaumontia brevituba, Apocynaceae, cardenolides, cytotoxicity, lignan, structure elucidation.
Introduction The stems of Beau,nontia brevituba Oliver (Apocynaceae), a medium-sized tree native to South and Southeast Asia, were collected in Thailand as part of a continuing collaborative search for naturally occurring antineoplastic agents (1). The genus Beaumontia is known to contain cardenolides, and such compounds have been reported previously from the seeds, fruits and leaves of B. brevituba (2), the leaves of B. rnurtonii (2), and the seeds of B. grandflora (3). In this communication, we report the identification of five known cardenolides and a lignan, (—)syringaresinol fi-n-glucoside, which were isolated from the stems of B. brevituba. Four of the cardenolides exhibited a
general cytotoxic effect when evaluated with several human and murine canccr cell lines.
Materials and Methods
Air-dried powdered stems (14.8 kg) of B. brerituba were exhaustively extracted with MeOH (60 liters) at room temperature to afford 1.4kg of residue on removal of solvent under reduced pressure. This extract was taken up in MeOH (300 ml), and the resultant suspension formed by adding H2O (2 liters) was partitioned successively with Et20 (3 x 2 liters) and nBuOH (3 x 1.5 liters). The Et20 layer was concentrated to dryness, and a residue (260 g), which exhibited an ED50 of 0.7 zg/ml against
a human lung cancer cell line (Lul), was chromatographed batchwise over silica gel (Merck Silica gel 60. 70—2 30 mesh) with CHC13, toluene-acetone (10: 1). and CHCI3-acetone (5: 1), to afford
1 (340mg), 2 (250mg), and 3 (780mg). The n-BuOH layer was evaporated to dryness and the residue (140 g), which also showed cytotoxic activity against Lul (ED50, 0.4ig/ml). was subjected to silica gel column chromatography, using CHCI3-MeOH-H20 (40:8: 1) and EtOAc-MeOH-H20 (20:4: 1) as eluents, to afford 4 (13.2g), 5 (52mg), and 6 (280mg). Compounds 1—5 gave a greenish-blue or a purple color in daylight after spraying with 10%H2S04/H20(110°C, 10mm). Compounds 1—5 were identified, respectively, as
digitoxigenin, oleandrigenin, digitoxigenin a-L-cymaroside, digitoxigenin /3-gentiobiosyl-a-L-cymaroside, and z '-digitoxigenmn /3-o-glucosyl-a-L-cymaroside by comparison of their physical and spectroscopic data (m.p.. [a]0, IR. UV, 'H- and '3C-
NMR, MS) with those reported in the literature (2—4). (—)-
Melting points (uncorrected) were determined on a Kofler micro hot-stage. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. UV spectra were taken on a Beckman DU-7 spectrometer, and IR spectra on a Nicolet MX-1 inter-
Syringaresinol /3-o-glucoside (6) was crystallized from MeOH, and exhibited the following data: colorless prisms; m.p. 183—185°C; — 19.0° (c 0.20, MeOH); IR vcm: 3440, 1600, 1520, 1460, 1220, 1120, 1080, 820; UVAnm (log a): 219 (4.31), 236 (s/i)
Downloaded by: National University of Singapore. Copyrighted material.
Received: August 22, 1991
430 Planta Med. 58(1992)
N. Kaneda et al.
(4.18), 271 (3.41). 280(3.30); 'll-NMR, UCNMR and FAB-MS data
147.1 ppm) and one methoxy carbon peak (a = 56.3 ppm)
identical with the (+)-enantiomer (5. 6). Enzymic hydrolysis
in the '3C-NMR spectrum. Therefore, it was considered
(hesperidinase. Sigma Chemical Co.. St. Louis. Missouri. U.S.A.) of 6 gave a product identical (m.p.. lal,)) with (-)-syringaresinol (7).
that this aglycone was a 3,7-dioxabicyclol3.3.Oloctane type
The EtO and n-BuOFI extracts of B. bre,'ituba were tested for cytotoxic activity against eight cell lines: BCI (human breast cancer), Co12 (human colon cancer). llT-1080 (human fibrosarcoma), KB (human nasopharyngeal carcinoma). KB-V1 (vinblastine-resistant KB), Lul (human lung cancer), Me12 (human melanoma) and P-388 (murine lymphocytic leukemia), by procedures as described previously (8—10). The cytotoxic activity of chromatographic fractions obtained from the EtA) and BuOll extracts was monitored by l.u I cytotoxicity, for which the extracts showed the greatest potency. Pure compounds 1—6 isolated in this manner were tested for cytotoxicity against the above-mentioned
cell lines as well as three additional cell lines, A-431 (human epidermoid carcinoma), LNCaP (human prostate cancer) and ZR75-1 (human breast cancer). These evaluations were performed by
published procedures (9, 10). In addition, antimitotic potential was assessed using cultured ASK cells (11). Each test compound was evaluated in duplicate at seven concentrations (20.0, 4.0, 0.8, 0.16,0.032.0.0064, and 0.00128 pg/mI).
Results and Discussion
By cytotoxicity-directed fractionation, active compounds, 1—3 and 4 were isolated from the Et20 and n-BuOH extracts of B. brevituba stems, respectively. Two additional components of the n-BuOH extract. 5 and 6. were not active with any of the cancer cell lines available to this study. Analysis of the Cl-MS and 13C-NMR spectra of compounds 1 and 2 suggested them to be cardenolides, and they were individually identified, in turn, as digitoxigenin and oleandrigenin, by data comparison with
published values for these cardenolides (4, 12). The structures of the cardiac glycosides 3—5 were, in turn, con-
firmed by comparison with reported data (m.p.. [a]1), '1-land 13C-NMR, FAB-MS) as digitoxigenin a-i.-cymaroside, digitoxigenin /3-gentiobiosyl-a-i-cymaroside. and A 16-digitoxigenin fi-o-glucosyl-a-i.-cymaroside, which were obtained recently from the leaves and fruits of B. brevituba (2) while the present study was underway. For compound 6, a molecular formula of C25H36013 was consistent with its observed FAB-MS and 13C-NMR spectral data, suggesting it
to be a mono-glucoside. The presence of a simple ben-
glucoside. although this glycoside does not appear to have been obtained previously as the (—)-enantiomer.
The cytotoxic activity of isolates 1—6 was evaluated with ten human cancer cell lines and murine lymphocytic leukemia in cell culture (P-388), and was com-
pared with analogous data for three clinically used anticancer agents in Table 1. As summarized in the table, com-
pounds 1—4 were cytotoxic against most of the human cancer cell lines utilized, but not active against P-388. In contrast, A °'-digitoxigenin (-i-glucosyl-a-i.-cymaroside (5) and (—)-syringaresinol /3-n-glucoside (6) were inactive against all of the human cancer cell lines employed. None of the test compounds showed demonstrable antimitotic activity in the astrocytoma (ASK) assay. In general, greater activity for 1—4 was observed with the human lung cancer (Lu 1) cell line, which vindicated the selection of this cell
line to monitor the fractionation of the Et,() and ,i-BuOEl plant extracts, and also was supportive of the notion that the cytotoxicity of B. brevituba stems was due mainly to cardenolides. It is known that cardenolides are generally cytotoxic for in vitro mammalian cells such as HeLa cells (13) and KB cells (14—17). and intensive efforts have been
made to study the structural relationships between their
antitumor and cardiotonic effects (13), and their cytotoxicity (15). Our results demonstrated that cardenolides 1—5 exhibited a similar order of cytotoxicity
against other human cell lines in comparison with published data on KB cells (14—17). Structural aspects of the cardenolides from B. brevituba stems, as related to cytotoxicity, may be summarized as follows: 1. Cytotoxicity was evident irrespective of whether or not the cardiac glycosides were glycosylated at C-3.
2. Activity reached a maximum with the mono-glycoside 3, and diminished with further saccharide substitution as in the triglycoside 4.
3. Cytotoxicity persisted with the introduction of a C-16 acetyl group in the aglycone, as in compound 2.
4. 16,17-Unsaturation in the aglycone, as in compound 5, led to the abrogation of cytotoxicity.
zenoid chromophore was observed from its UV (Amax 219,
271 nm) and IR (Vmax 1600, 1520cm') spectra. Enzymic hyrolysis of 6 gave a genin. which exhibited seven carbon
peaks (ô = 54.3, 71.8, 86.0, 102.6, 132.0, 134.3. and
In summary, while it is interesting to investigate the active sites and structural requirements of cardenolides or bufadienolides for in vitro cytotoxicity.
Table 1 Cytotoxic acti vity of compounds 1—4'. Compound
A-431
BC1'
Co12
HT'
KB'
KB-V1'
LNCaP'
Lul'
Me12'
ZR-75-1'
P-388'
1
0.37 0.25 0.14 8.4
0.031
0.27 0.17
0.13 0.15 0.034
1.9
1.6
1.8
1.4
0.16 0.077 2.9
0.083 0.027 0.0060 0.69
0.22 0.14 0.066 1.8
>20
0.056
0.067 0.12 0.039 1.4
0.10 0.086
0.068
0.13 0.17 0.030
0.21
0.20
0.03 0.4 0.06
0.1
0.01
0.004
0.5
0.03
0.005 0.02
0.03 0.04
0.04 0.02
3.0
0.05 0.1
0.03 0.03 0.02
0.06
0.03
0.01
0.3
0.05 0.02 0.02
2 3 4
Homo' harringtonine 0.02 Taxol 0.007 Vinblastine 0.05
0.01
0.031
>4
1.9
0.02
14
>20 >20
Results are expressed as ED50 values (pg/mi).
A-431, epidermoid carcinoma; BC1, breast; Co12, colon; NT, sarcoma; KB, nasopharyngeal carcinoma; KB-V1, drug-resistant KB; LNCaP, prostate; Lul, lung; Me12, melanoma; ZR75-1, breast; P-388, murine lymphocytic leukemia.
Downloaded by: National University of Singapore. Copyrighted material.
Cytotoxicity Assays
lignan. By comparison with reported data (5—7). the structure of 6 was identified as (—)-syringaresinol n-I)-
Planta Med. 58(1992) 431
Cytotoxic Activity of Cardenolides from Beaumontia brevituba Stems
their association with human toxicity limits development as useful anticancer agents.
References Kaneda. N.. Pezzuto, J. M.. Soejarto, D. D.. Kinghorn. A. D..
Acknowledgements
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Dvorak. of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. for recording the CI and FAB mass spectral data.
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