Planta Med. 57 (1991) 437
Cytotoxic Activity of Ainaryllidaceae Alkaloids from Crinum augustum and Crinum bulbispermum M. A. Abd El HaJiz14, M. A. Ramadan1, M. L. Jung2, .1. P. Beck2, andR. Anton3 1
2
Pharmacognosy Department. Faculty of Pharmacy, Assiut University, Assiut, Egypt Laboratoire de Recherches en Immunologie, Universitd Louis Pasteur, F-67091 Strasbourg, France
Laboratoire de Pharmacognosie, Faculté de Pharmacie, B.P. 24, F-67401 Illkirch Cddex, France Address for correspondence
Abstract
CH3
0,-
H
The cytotoxic activity of five minor Amaryllidaceae alkaloids and one flavan isolated from Crinum augustum Rox and Crinum bulbispermum Mime were tested on human leukemic Molt 4 cells. Whereas the crinine-type alkaloids (6a-hydroxycrinine, powelline) and the new type augustamine did not even inhibit the growth of Molt 4 cells, the lycorine-type alkaloid (pratorinine) and the crinine-type alkaloid (6a-hydroxybuphanisine) showed a moderate cytotoxic activity and the flavan (4'-hydroxy-7-methoxyflavan) showed an
H
1 (Mr 254.00)
OH
2 (Mr 287.1165)
OCH3
O—.,.;:2
H OH
3
important cytotoxic effect.
(Mr 301.1331)
4 (Mr 301.00)
Key words
Crinurn augustum, Crinum bulbispermum, Amaryllidaceae alkaloids, cytotoxic effects, flavans.
H3CO
HOC 0
5 (Mr 265.00)
Introduction
6 (Mr 301.1281)
Fig. 1 Amaryllidaceae alkaloids 2—5 and the fiavan 1 isolated from Crinum augustum Rox. and Crinum bulbispermum M line.
The Amaryllidaceae alkaloids are known to exhibit different pharmacological and microbiological effects such as antiviral, antitumoral, and anticholinergic, one flavan [4'-hydroxy-7-methoxyflavan) (1)] (Fig. 1) isoetc. Some of them have been used in the treatment of myas- lated from C. augustum Rox and C. bulbispermum Milne thenia gravis, myopathy, and diseases of the nervous sys- (10—14). tem (1). The antiviral activity of lycorine, the most widespread Amaryllidaceae alkaloid, has been the subject of Materials and Methods several recent reports (2—5). Pseudolycorine and pretazetPlant materials and substance isolation tine are active against several leukemias and were reported Plant extracts were obtained through a classical to inhibit protein synthesis in eukaryotic cells by preventing peptide bond formation. On the other hand, galanthamine acid base extraction using ethyl acetate. The alkaloids were iso-
possesses analgesic, anticholinergic, and anticholinesterase activities, and has been used in the treatment of myasthenia gravis, myopathy, and also of Alzheimer's disease and other diseases of the nervous system (1).
In addition to our systematic chemical examination of the genus Crinum from Egypt (6—14), we report here the cytotoxic effects of five minor Amaryllidaceae
alkaloids [6a-hydroxycrinine (2), 6a-hydroxybuphanisine (3), augustamine (6), powelline (4), and pratorinine (511 and
lated by repeated column chromatography and identified on the basis of their spectral data. The alkaloids 6a-hydroxybuphanisine (3), 6a-hydroxycrinine (2), augustamine (6), and 4'-hydroxy-7methoxyflavan (1) have been isolated from Crinum bulbisperrnum Milne and Crinum augustum Rox, while the alkaloids powelline (4) and pratorinine (5) have been isolated from Crinum bulbispermum Milne.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Received: September 19, 1990
438 Planta Med. 57(1991)
M. A. AbdElHafiz et al.
Cytotoxicity tests 800
Cytotoxicity tests were performed on human
600
leukemic Molt 4 cells, derived from a child T-cell leukemia. Cells
were cultured in suspension in an RPMI 1640 medium supplemented with 10% v/v foetal calf serum (GIBCO) previously heated at 56°C for 30 minutes in 5% CO2 + 95% air, at 37°C. The assays were performed in multiwell plates; in each well were added successively the tested substance dissolved in 100 ii medium, then the cell suspension adjusted to 2 x cells/ml. After 24 hours of
400
io
200
incubation at 37°C in a C02/air atmosphere, tritiated thymidine (3HT: 1 .tg per well) was added. Cell growth or survival was mea-
sured through the incorporation of [3H]-thymidine. Thymidine being a precursor of the DNA, the amount of incorporated 3HT is a function of the cell multiplication. After 30 minutes of incubation at 37°C, the cells were collected on a cell harvester (TITERTEK®) and the radioactivity was measured in a scintillation counting system.
0 C,
Eioo
days
The results are expressed as the percentage of incorporation of 3HT by the treated cells compared to that of the control cells. The obtained results are related to the concentration of the tested substances expressed in g/ml. In addition, cell growth was measured every 24 hours by counting the cells with a Neubauer microcytometer and cell viability was evaluated using the trypan blue exclusion test.
20
Results and Discussion Amaryllidaceae alkaloids are of growing in-
terest, particularly concerning their pharmacological and microbiological potency (16, 17). In a continuing programme we are testing the cytotoxic effects of the five Amaryllidaceae alkaloids 2—6 and one flavan 1 (Fig. 1) on
Fig. 3 Effects of 4'-hydroxy-7-methoxyflavan (1) on the growth of Molt 4 cells. •: control; A: 16.8 .Lg/ml; 'v': 42 .tglml.
Molt 4 cells. These are the first evaluations on these types of compounds by this method. The percentage of incorporated
3HT by the cells in the presence of increasing concentrations of the different substances showed that the five alkaloids are unequally toxic to the Molt 4 cells. Thus the strongest effect occurred with the flavan (Fig. 2a). There was a 50% decrease of incorporation with less than 10 .tg/
ml of flavan. 6a-Hydroxybuphanisine (Fig. 2b) and
800
pratorinine (Fig. 2c) are less active, while the other three alkaloids had no activity at the higher concentration used.
600
a,
C,
a,
.0 C, >
0 a,
C
0 0, 0
C,
C,)
b
0
20
40 60 80 100 concentration (pg/mI)
120
Fig. 2 Effects of 4'-hydroxy-7-methoxyflavan (1) (a), 6a-hydroxy. buphanisine (3) (b), and pratorinine (5) (c), on the incorporation of [3H].thymidine by Molt 4 cells.
140
Fig. 4 Effects of 6a-hydroxybuphanisine (3) on the growth of Molt 4 cells.
•: control; A: 35 g/ml; ":71 ig/ml.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
50
Cytotoxic Activity ofAmaryllidaceae Alkaloids from Crinum augustum and Crinum bulbispermum
Planta Med. 57(1991) 439
References
The differences which appeared between the live alkaloids on Molt 4 cells could be due to their respec-
tive structural characteristics. Roughly, the relative cytotoxicities of these compounds could be schematized as follows: 4'-hydroxy-7-methoxyflavan (1)> 6a-hydroxybuphanisine (3) > pratorinine (5). Generally, the flavan compound 1 showed a high toxicity on human leukemic cells, the lycorine-type alkaloid 5 (pratorinine) and the crinine-type alkaloid 3 (6a-hydroxybuphanisine) showed a
comparatively lower cytotoxic effect, while the other crinine-type alkaloids 4 and 2 (powelline and 6a-hydroxycrinine) and the new type alkaloid 6 (augustamine) showed no activity.
2
Cordell, G. A. (1981) Introduction to Alkaloids. A Biogenetic Approach, John Wiley & Sons, New York. Leven, M., Totte, J., Van den Berghe, D. A., Vlietinck, A. J. (1978) Planta Med. 33, 284. Van den Berghe, D. A., Mortens, F., Vlietinck, A. J. (1978) J. Nat. Prod. 41,463. Leven, M., Van den Berghe, D. A., Vlietinck, A. J. (1979) Planta Med.
38, 254. Leven, M., Van den Berge, D. A., Vlietinck, A. J. (1983) Planta Med. 49, 109. 6 Abd El-Hafiz, M. A., Mesbah, M. K. (1984) Egypt. J. Pharm. Sci. 25, 171. Abd El-Hafiz, M. A., Mi, A. A., Ramadan, M. A. (1988) Bull. Pharm.
Sd. 11,273.
test with 3HT (flavan and buphanisine) were used for the trypan blue exclusion test. By counting the cells for 3 days, 4'-hydroxy-7-methoxyflavan (1) showed a strong cytotoxic effect at 42 .tg/ml (Fig. 3) whereas 6a-hydroxybuphanisine (3) had a strong activity at 71 .tg/ml (Fig. 4).
8 Abd El-Hafiz, M. A., Ramadan, M. A., Anton, H. (1990) J. Nat. Prod.
53, 1349. 10
12
13 14
16 17
Abd El-Hafiz, M. A. (1990) Phytochemistry 29, 3936. Alt, A. A., Kating, H., El-Moghazi, A. M., Ramadan, M. A. (1981) Phytochemistry 20, 1121. Mi, A. A., Kating, H., Fram, A. W. (1981) Phytochemistry 20, 1731. Fram, A. W., Mi, A. A., Kating, H. (1981) Phytochemistry 20, 1735. Mi, A. A., Hambloch, H., Fram, A. W. (1983) Phytochemistry 22. 283. El-Moghazi, A. M., Mi, A. A. (1976) Planta Med. 29, 156. Minowada, J., Ohnuma, T., Morre, G. E. (1972) J. Nat. Cancer Inst.
49,891. Mashkovskii, M.D. (1955) Farmakol. Toksikol. 18(4), 21. Medikyan, G. A., Vinkova, G. B. (1956) Farmakol. Toksilol. 18(5), 34.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Molt 4 cells represent a sensitive cell line for toxicity testing. Only the two most active compounds in the
Cytotoxic Activity of Ainaryllidaceae Alkaloids from Crinum augustum and Crinum bulbispermum M. A. Abd El HaJiz14, M. A. Ramadan1, M. L. Jung2, .1. P. Beck2, andR. Anton3 1
2
Pharmacognosy Department. Faculty of Pharmacy, Assiut University, Assiut, Egypt Laboratoire de Recherches en Immunologie, Universitd Louis Pasteur, F-67091 Strasbourg, France
Laboratoire de Pharmacognosie, Faculté de Pharmacie, B.P. 24, F-67401 Illkirch Cddex, France Address for correspondence
Abstract
CH3
0,-
H
The cytotoxic activity of five minor Amaryllidaceae alkaloids and one flavan isolated from Crinum augustum Rox and Crinum bulbispermum Mime were tested on human leukemic Molt 4 cells. Whereas the crinine-type alkaloids (6a-hydroxycrinine, powelline) and the new type augustamine did not even inhibit the growth of Molt 4 cells, the lycorine-type alkaloid (pratorinine) and the crinine-type alkaloid (6a-hydroxybuphanisine) showed a moderate cytotoxic activity and the flavan (4'-hydroxy-7-methoxyflavan) showed an
H
1 (Mr 254.00)
OH
2 (Mr 287.1165)
OCH3
O—.,.;:2
H OH
3
important cytotoxic effect.
(Mr 301.1331)
4 (Mr 301.00)
Key words
Crinurn augustum, Crinum bulbispermum, Amaryllidaceae alkaloids, cytotoxic effects, flavans.
H3CO
HOC 0
5 (Mr 265.00)
Introduction
6 (Mr 301.1281)
Fig. 1 Amaryllidaceae alkaloids 2—5 and the fiavan 1 isolated from Crinum augustum Rox. and Crinum bulbispermum M line.
The Amaryllidaceae alkaloids are known to exhibit different pharmacological and microbiological effects such as antiviral, antitumoral, and anticholinergic, one flavan [4'-hydroxy-7-methoxyflavan) (1)] (Fig. 1) isoetc. Some of them have been used in the treatment of myas- lated from C. augustum Rox and C. bulbispermum Milne thenia gravis, myopathy, and diseases of the nervous sys- (10—14). tem (1). The antiviral activity of lycorine, the most widespread Amaryllidaceae alkaloid, has been the subject of Materials and Methods several recent reports (2—5). Pseudolycorine and pretazetPlant materials and substance isolation tine are active against several leukemias and were reported Plant extracts were obtained through a classical to inhibit protein synthesis in eukaryotic cells by preventing peptide bond formation. On the other hand, galanthamine acid base extraction using ethyl acetate. The alkaloids were iso-
possesses analgesic, anticholinergic, and anticholinesterase activities, and has been used in the treatment of myasthenia gravis, myopathy, and also of Alzheimer's disease and other diseases of the nervous system (1).
In addition to our systematic chemical examination of the genus Crinum from Egypt (6—14), we report here the cytotoxic effects of five minor Amaryllidaceae
alkaloids [6a-hydroxycrinine (2), 6a-hydroxybuphanisine (3), augustamine (6), powelline (4), and pratorinine (511 and
lated by repeated column chromatography and identified on the basis of their spectral data. The alkaloids 6a-hydroxybuphanisine (3), 6a-hydroxycrinine (2), augustamine (6), and 4'-hydroxy-7methoxyflavan (1) have been isolated from Crinum bulbisperrnum Milne and Crinum augustum Rox, while the alkaloids powelline (4) and pratorinine (5) have been isolated from Crinum bulbispermum Milne.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Received: September 19, 1990
438 Planta Med. 57(1991)
M. A. AbdElHafiz et al.
Cytotoxicity tests 800
Cytotoxicity tests were performed on human
600
leukemic Molt 4 cells, derived from a child T-cell leukemia. Cells
were cultured in suspension in an RPMI 1640 medium supplemented with 10% v/v foetal calf serum (GIBCO) previously heated at 56°C for 30 minutes in 5% CO2 + 95% air, at 37°C. The assays were performed in multiwell plates; in each well were added successively the tested substance dissolved in 100 ii medium, then the cell suspension adjusted to 2 x cells/ml. After 24 hours of
400
io
200
incubation at 37°C in a C02/air atmosphere, tritiated thymidine (3HT: 1 .tg per well) was added. Cell growth or survival was mea-
sured through the incorporation of [3H]-thymidine. Thymidine being a precursor of the DNA, the amount of incorporated 3HT is a function of the cell multiplication. After 30 minutes of incubation at 37°C, the cells were collected on a cell harvester (TITERTEK®) and the radioactivity was measured in a scintillation counting system.
0 C,
Eioo
days
The results are expressed as the percentage of incorporation of 3HT by the treated cells compared to that of the control cells. The obtained results are related to the concentration of the tested substances expressed in g/ml. In addition, cell growth was measured every 24 hours by counting the cells with a Neubauer microcytometer and cell viability was evaluated using the trypan blue exclusion test.
20
Results and Discussion Amaryllidaceae alkaloids are of growing in-
terest, particularly concerning their pharmacological and microbiological potency (16, 17). In a continuing programme we are testing the cytotoxic effects of the five Amaryllidaceae alkaloids 2—6 and one flavan 1 (Fig. 1) on
Fig. 3 Effects of 4'-hydroxy-7-methoxyflavan (1) on the growth of Molt 4 cells. •: control; A: 16.8 .Lg/ml; 'v': 42 .tglml.
Molt 4 cells. These are the first evaluations on these types of compounds by this method. The percentage of incorporated
3HT by the cells in the presence of increasing concentrations of the different substances showed that the five alkaloids are unequally toxic to the Molt 4 cells. Thus the strongest effect occurred with the flavan (Fig. 2a). There was a 50% decrease of incorporation with less than 10 .tg/
ml of flavan. 6a-Hydroxybuphanisine (Fig. 2b) and
800
pratorinine (Fig. 2c) are less active, while the other three alkaloids had no activity at the higher concentration used.
600
a,
C,
a,
.0 C, >
0 a,
C
0 0, 0
C,
C,)
b
0
20
40 60 80 100 concentration (pg/mI)
120
Fig. 2 Effects of 4'-hydroxy-7-methoxyflavan (1) (a), 6a-hydroxy. buphanisine (3) (b), and pratorinine (5) (c), on the incorporation of [3H].thymidine by Molt 4 cells.
140
Fig. 4 Effects of 6a-hydroxybuphanisine (3) on the growth of Molt 4 cells.
•: control; A: 35 g/ml; ":71 ig/ml.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
50
Cytotoxic Activity ofAmaryllidaceae Alkaloids from Crinum augustum and Crinum bulbispermum
Planta Med. 57(1991) 439
References
The differences which appeared between the live alkaloids on Molt 4 cells could be due to their respec-
tive structural characteristics. Roughly, the relative cytotoxicities of these compounds could be schematized as follows: 4'-hydroxy-7-methoxyflavan (1)> 6a-hydroxybuphanisine (3) > pratorinine (5). Generally, the flavan compound 1 showed a high toxicity on human leukemic cells, the lycorine-type alkaloid 5 (pratorinine) and the crinine-type alkaloid 3 (6a-hydroxybuphanisine) showed a
comparatively lower cytotoxic effect, while the other crinine-type alkaloids 4 and 2 (powelline and 6a-hydroxycrinine) and the new type alkaloid 6 (augustamine) showed no activity.
2
Cordell, G. A. (1981) Introduction to Alkaloids. A Biogenetic Approach, John Wiley & Sons, New York. Leven, M., Totte, J., Van den Berghe, D. A., Vlietinck, A. J. (1978) Planta Med. 33, 284. Van den Berghe, D. A., Mortens, F., Vlietinck, A. J. (1978) J. Nat. Prod. 41,463. Leven, M., Van den Berghe, D. A., Vlietinck, A. J. (1979) Planta Med.
38, 254. Leven, M., Van den Berge, D. A., Vlietinck, A. J. (1983) Planta Med. 49, 109. 6 Abd El-Hafiz, M. A., Mesbah, M. K. (1984) Egypt. J. Pharm. Sci. 25, 171. Abd El-Hafiz, M. A., Mi, A. A., Ramadan, M. A. (1988) Bull. Pharm.
Sd. 11,273.
test with 3HT (flavan and buphanisine) were used for the trypan blue exclusion test. By counting the cells for 3 days, 4'-hydroxy-7-methoxyflavan (1) showed a strong cytotoxic effect at 42 .tg/ml (Fig. 3) whereas 6a-hydroxybuphanisine (3) had a strong activity at 71 .tg/ml (Fig. 4).
8 Abd El-Hafiz, M. A., Ramadan, M. A., Anton, H. (1990) J. Nat. Prod.
53, 1349. 10
12
13 14
16 17
Abd El-Hafiz, M. A. (1990) Phytochemistry 29, 3936. Alt, A. A., Kating, H., El-Moghazi, A. M., Ramadan, M. A. (1981) Phytochemistry 20, 1121. Mi, A. A., Kating, H., Fram, A. W. (1981) Phytochemistry 20, 1731. Fram, A. W., Mi, A. A., Kating, H. (1981) Phytochemistry 20, 1735. Mi, A. A., Hambloch, H., Fram, A. W. (1983) Phytochemistry 22. 283. El-Moghazi, A. M., Mi, A. A. (1976) Planta Med. 29, 156. Minowada, J., Ohnuma, T., Morre, G. E. (1972) J. Nat. Cancer Inst.
49,891. Mashkovskii, M.D. (1955) Farmakol. Toksikol. 18(4), 21. Medikyan, G. A., Vinkova, G. B. (1956) Farmakol. Toksilol. 18(5), 34.
Downloaded by: Chinese University of Hong Kong. Copyrighted material.
Molt 4 cells represent a sensitive cell line for toxicity testing. Only the two most active compounds in the
